Mechanisms of Cellular Signal Transduction

Return to The Medical Biochemistry Page

Return to The Medical Biochemistry Page

© 1996–2016, LLC | info @

Introduction to Mechanisms of Signal Transduction

Signal transduction at the cellular level refers to the movement of signals from outside the cell to inside. The movement of signals can be simple, like that associated with receptor molecules of the acetylcholine class: receptors that constitute channels which, upon ligand interaction, allow signals to be passed in the form of small ion movement, either into or out of the cell. These ion movements result in changes in the electrical potential of the cells that, in turn, propagates the signal along the cell. More complex signal transduction involves the coupling of ligand-receptor interactions to many intracellular events. These events include phosphorylations by tyrosine kinases and/or serine/threonine kinases. The human genome contains 90 genes that encode tyrosine kinase enzymes including both receptor type and non-receptor type enzymes. Protein phosphorylations change enzyme activities and protein conformations. The eventual outcome is an alteration in cellular activity and changes in the program of genes expressed within the responding cells.












Please refer to the page on Growth Factors for descriptions of the growth factors described in this page and the explanation of their abbreviations.

back to the top

Classifications of Signal Transducing Receptors

Signal transducing receptors are of three general classes:

1. Receptors that penetrate the plasma membrane and have intrinsic enzymatic activity. Receptors that have intrinsic enzymatic activities include those that are tyrosine kinases (e.g. PDGF, insulin, EGF and FGF receptors), tyrosine phosphatases (e.g. CD45 [cluster determinant-45] protein of T cells and macrophages), guanylate cyclases (e.g. natriuretic peptide receptors) and serine/threonine kinases (e.g. activin and TGF-β receptors). Receptors with intrinsic tyrosine kinase activity are capable of autophosphorylation as well as phosphorylation of other substrates. Additionally, several families of receptors lack intrinsic enzyme activity, yet are coupled to intracellular tyrosine kinases by direct protein-protein interactions (see below).

2. Receptors that are coupled, inside the cell, to GTP-binding and hydrolyzing proteins (termed G-proteins). Receptors of the class that interact with G-proteins all have a structure that is characterized by seven transmembrane spanning domains and as such are sometimes referred to as serpentine receptors. These receptors all belong the superfamily of G-protein coupled receptors, GPCRs. Examples of this class are the adrenergic receptors, odorant receptors, and certain hormone receptors (e.g. glucagon, angiotensin, vasopressin and bradykinin).

3. Receptors that are found intracellularly and upon ligand binding migrate to the nucleus where the ligand-receptor complex directly affects gene transcription. Because this class of receptors is intracellular and functions in the nucleus as transcription factors they are commonly referred to as the nuclear receptors. Receptors of this class include the large family of steroid and thyroid hormone receptors. Receptors in this class have a ligand-binding domain, a DNA-binding domain and a transcriptional activator domain.

back to the top

Receptor Tyrosine Kinases (RTKs)

The receptor tyrosine kinase (RTK) family of transmembrane ligand-binding proteins is comprised of 59 members in the human genome. Each of the RTKs exhibit similar structural and functional characteristics. Most RTKs are monomers, and their domain structure includes an extracellular ligand-binding domain, a transmembrane domain, and an intracellular domain possessing the tyrosine kinase activity. The insulin and insulin-like growth factor receptors are the most complex in the RTK family being disulfide linked heterotetramers.

The amino acid sequences of the tyrosine kinase domains of RTKs are highly conserved with those of cAMP-dependent protein kinase (PKA) within the ATP binding and substrate binding regions. Some RTKs have an insertion of non-kinase domain amino acids into the kinase domain termed the kinase insert. RTK proteins are classified into families based upon structural features in their extracellular portions (as well as the presence or absence of a kinase insert) which include the cysteine rich domains, immunoglobulin-like domains, leucine-rich domains, Kringle domains, cadherin domains, fibronectin type III repeats, discoidin I-like domains, acidic domains, and EGF-like domains. Based upon the presence of these various extracellular domains the RTKs have been sub-divided into at least 20 different families.

Characteristics of the Common Classes of RTKs

Class Family Name Examples Structural Features of Class
I ErbB Receptor Family EGF receptor (ERBB1); NEU/HER2 (ERBB2); HER3 (ERBB3); HER4 (ERBB4) cysteine-rich sequences; receptors dimerize in response to ligand binding; numerous ligands bind and activate the EGFR (ERBB1) including EGF, TGF-α, and amphiregulin
II Insulin Receptor Family insulin receptor (InsR); IGF-1 receptor (IGF1R); insulin receptor-related receptor (IRR) cysteine-rich sequences; characterized by disulfide-linked heterotetramers
III PDGF Receptor Family PDGF receptors (PDGFα and PDGFβ); c-Kit, CSF-1 receptor (CSFR); fms-related tyrosine kinase 3 (FLT3) contain 5 immunoglobulin-like domains; contain the kinase insert
IV VEGF Receptor Family vascular endothelial cell growth factor (VEGF) receptor-1 (VEGFR-1; also known as fms-related tyrosine kinase-1 (FLT1); kinase insert domain receptor (VEGFR-2); fms-related tyrosine kinase 4 (VEGFR-3) contain 7 immunoglobulin-like domains as well as the kinase insert domain
V FGF Receptor Family fibroblast growth factor receptor 1 (FGFR1); FGFR2; FGFR3; FGFR4 contain 3 immunoglobulin-like domains as well as the kinase insert; acidic domain
VII Neurotrophin Receptor Family neurotrophic tyrosine kinase receptor type 1 (TrkA); TrkB; TrkC contains several closely spaced leucine-rich regions (LRRs); one or two cysteine-rich domains; two immunoglobulin-like domains; no kinase insert
VIII ROR Family receptor tyrosine kinase-like orphan receptor type 1 (ROR1); ROR2  
X HGF Receptor Family met proto-oncogene (Met); macrophage stimulating receptor 1 (MST1R, also Ron) heterodimeric like the class II receptors except that one of the two protein subunits is completely extracellular. The HGF receptor is a proto-oncogene that was originally identified as the MET oncogene

schematic representation of various receptor tyrosine kinase (RTK) sub-types

Diagrammatic representation of several members of the receptor tyrosine kinase (RTK) family. Several members of each recepetor sub-family are indicated below each representative. The Roman numerals above the first seven sub-types correspond to those sub-types described in the Table above. These RTK sub-types do not represent the entire RTK family sub-types.

Many receptors that have intrinsic tyrosine kinase activity as well as the tyrosine kinases that are associated with cell surface receptors contain tyrosine residues, that upon phosphorylation, interact with other proteins of the signaling cascade. These other proteins contain a domain of amino acid sequences that are homologous to a domain first identified in the SRC proto-oncogene. These domains are termed SH2 domains (SRC homology domain 2). The typical SH2 domain is approximately 100 amino acids in length. Different SH2 domains recognize different tyrosine phosphorylated residues based upon the presence of the tyrosine phosphate as well as the amino acid sequences surrounding the tyrosine residue. These variable domains are, therefore, what determine the specificity of SH2 domain-containing protein binding. At least 110 different proteins are expressed in humans that contain SH2 domains. Another conserved protein-protein interaction domain identified in many signal transduction proteins is related to a third domain in SRC identified as the SH3 domain. Typical SH3 domains are composed of approximately 60 amino acid residues.

The interactions of SH2 domain-containing proteins with RTKs or receptor associated tyrosine kinases leads to tyrosine phosphorylation of the SH2 containing proteins. The result of the phosphorylation of SH2 containing proteins that have enzymatic activity is an alteration (either positively or negatively) in that activity. Several SH2 containing proteins that have intrinsic enzymatic activity include phospholipase Cγ (PLCγ, PLC-gamma), the proto-oncogene RAS associated GTPase activating protein (rasGAP), phosphatidylinositol-3-kinase (PI3K), protein phosphatase-1C (PTP1C), as well as members of the SRC family of protein tyrosine kinases (PTKs).

back to the top

Non-Receptor Protein Tyrosine Kinases (PTKs)

There are numerous intracellular PTKs that are responsible for phosphorylating a variety of intracellular proteins on tyrosine residues following activation of cellular growth and proliferation signals. There is now recognized two distinct families of non-receptor PTKs. The archetypal PTK family is related to the SRC protein. The SRC protein is a tyrosine kinase first identified as the transforming protein in Rous sarcoma virus. Subsequently, a cellular homolog was identified. Numerous proto-oncogenes were identified as the transforming proteins carried by retroviruses. The second family is related to the Janus kinase (JAK).

Most of the proteins of both families of non-receptor PTKs couple to cellular receptors that lack enzymatic activity themselves. This class of receptors includes all of the cytokine receptors (e.g. the interleukin-2 receptor, IL2R) as well as the CD4 and CD8 cell surface glycoproteins of T cells and the T cell antigen receptor (TCR). This mode of coupling receptors to intracellular PTKs suggests a split form of RTK.

Another example of receptor-signaling through protein interaction involves the insulin receptor (IR). This receptor has intrinsic tyrosine kinase activity but does not directly interact, following autophosphorylation, with enzymatically active proteins containing SH2 domains (e.g. PI3K). Instead, the principal IR substrate is a protein termed IRS-1. IRS-1 contains several motifs that resemble SH2 binding consensus sites for the catalytically active subunit of PI3K. These domains allow complexes to form between IRS-1 and PI3K. This model suggests that IRS-1 acts as a docking or adapter protein to couple the IR to SH2 containing signaling proteins.

Additional adapter proteins have been identified, the most commonly occurring being a protein termed growth factor receptor-binding protein 2, GRB2.

An example of an alteration in receptor activity in response to association with an intracellular PTK is the nicotinic acetylcholine receptor (AChR). These receptors comprise an ion channel consisting of five distinct subunits (alpha: α, beta: β, gamma: γ, delta: δ, and epsilon: ε). The β, γ, and δ subunits are tyrosine phosphorylated in response to acetylcholine binding which leads to an increase in the rate of desensitization to acetylcholine.

back to the top

Receptor Serine/Threonine Kinases (RSKs)

The receptors for the TGF-β (TGF-beta) superfamily of ligands have intrinsic serine/threonine kinase activity. A more complete description of the TGF-β signaling cascade can be found in the Signaling by Wnts and the TGFs-β/BMP Families page.

There are more than 30 multifunctional proteins of the TGF-β superfamily which also includes the activins, inhibins and the bone morphogenetic proteins (BMPs). This superfamily of proteins can induce and/or inhibit cellular proliferation or differentiation and regulate migration and adhesion of various cell types. The signaling pathways utilized by the TGF-β, activin and BMP receptors are different than those for receptors with intrinsic tyrosine kinase activity or that associate with intracellular tyrosine kinases.

At least 12 RSKs have been characterized as being expressed in humans. These receptors can be divided into two subfamilies identified as the type I and type II receptors. The type I RSKs are also known as activin receptors or activin receptor-like kinases, ALKs. Ligands first bind to the type II receptors which then leads to interaction with the type I receptors. The type II protein phosphorylates the kinase domain of the type I partner leading to displacement of proteins called subunit (or protein) partners. The displacement of these protein partners allows for the binding and phosphorylation of particular members of the Smad family. Once phosphorylated, the particular Smad will migrate to the nucleus and act as complexes that regulate the expression of specific target genes. One predominant effect of TGF-β is regulation of progression through the cell cycle. One nuclear protein involved in the responses of cells to TGF-β is the proto-oncogene, MYC which directly affects the expression of genes harboring MYC-binding elements.

Human Receptor Serine Threonine Kinases (RSKs)

Class Receptor Name Common Abbreviation Gene Symbol
I activin A receptor, type I ALK2 ACVR1
I activin A receptor, type IB ALK4 ACVR1B
I activin A receptor, type IC ALK7 ACVR1C
I activin A receptor type II-like 1 ALK1 ACVRL1
I bone morphogenetic protein (BMP) receptor, type IA BMPR1A BMPR1A
I bone morphogenetic protein (BMP) receptor, type IB BMPR1B BMPR1B
I transforming growth factor beta (TGF-β) receptor 1 TGFBR1 TGFBR1
II activin A receptor, type IIA ActR2 ACVR2A
II activin A receptor, type IIB ActR2B ACVR2B
II anti-Mullerian hormone receptor, type II MISR2 AMHR2
II bone morphogenetic protein (BMP) receptor, type II BMPR2 BMPR2
II transforming growth factor beta (TGF-β) receptor II TGFBR2 TGFBR2

back to the top

The Protein Kinase C (PKC) Family of Serine/Threonine Kinases

The protein kinase C (PKC) family of serine/threonine kinases are integrated into numerous signal transduction pathways elicited by a wide range of GPCRs and other growth factor-dependent cellular responses. The original PKC enzyme was demonstrated to be lipid- and calcium-sensitive. This lipid-sensitive family of kinases is known to be activated by growth factor receptors that stimulate phospholipase C (PLC) family member enzymes. The PLC enzymes (see below) hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP2) to generate membrane-bound diacylglycerol (DAG), which in turn activates PKC, and inositol trisphosphate (IP3), which mobilizes intracellular calcium. Expression of most of the PKC family members is observed in most tissues and many different PKC subfamilies are expressed in the same cell types. However, some of the PKC isoforms are expressed in a tissue-specific manner. Expression of PKCθ is primarily found in skeletal muscle and lymphoid/hematopoietic cells. Expression of PKCγ is limited to neuronal tissues.

PKC isoforms are members of the AGC (PKA, PKG, PKC) family of protein serine/threonine kinases that contain a highly conserved catalytic domain and a regulatory domain that maintains the enzyme in an inactive conformation. The PKC isoforms have been subdivided into three distinct subfamilies based upon differences in their N-terminal regulatory domain structure. These three subfamilies are referred to as the conventional PKC isoforms (cPKC), the novel PKC isoforms (nPKC), and the atypical PKC isoforms (aPKC). The regulatory domain of PKC isoforms resides in the N-terminus and contains an autoinhibitory pseudosubstrate domain. This pseudosubstrate domain contains a serine residue in place of the serine/threonine phosphoacceptor site, but otherwise resembles a natural PKC substrate. In addition to the catalytic and regulatory domains these enzymes contain two discrete membrane targeting modules, termed C1 and C2.

Conventional PKC Isoforms

The regulatory domains of cPKC isoforms (cPKCα: cPKC-alpha; cPKCβI: cPKC-beta I, cPKCβII: cPKC-beta II; and cPKCγ: cPKC-gamma) contain a C1 domain consisting of tandem ~50 amino acid long sequences termed C1A and C1B. The C1A and C1B subdomains each have six cysteines and two histidines that coordinate two Zn2+ ions. The cPKCβII enzyme is an alternatively spliced version of cPKCβI. The C1A/C1B motifs function as a DAG-/PMA-binding motif (PMA: phorbol myristic acid). The regulatory domains of the cPKC isoforms also contain a C2 domain that binds anionic phospholipids in a calcium-dependent manner. All the cPKC isoforms require DAG, Ca2+, and phospholipids for activation.

Conventional PKC Isoforms

Protein Name Gene Symbol Comments
PKCα PRKCA located on chromosome 17q24.2; composed of 21 exons; encodes a 672 amino acid protein; involved in events related to epithelial cell function, bile acid mediated effects in the liver, hypertrophic responses in the heart, and within the CNS related to memory
PKCβ1 PRKCB located on chromosome 16p11.2; composed of 17 exons; two alternatively spliced mRNAs; the PKCβ1 isoform is the shorter of the two isoforms; results from use of an alternative splice site at the 5'-end of the terminal exon; the PKCβ1 isoform has a different C-terminus than the PKCβ2 isoform; is a 671 amino acid protein; involved in B cell activation, intestinal carbohydrate absorption, endothelial cell proliferation, induction of apoptosis, and within the CNS the protein plays a role in stress responses
PKCβ2 PRKCB located on chromosome 16p11.2; composed of 17 exons; two alternatively spliced mRNAs; the PKCβ2 isoform is the longer of the two isoforms and is a 673 amino acid protein
PKCγ PRKCG located on chromosome 19q13.4; composed of 19 exons; two alternatively spliced mRNAs; isoform 1 encodes a 710 amino acid protein, isoform 2 encodes a 697 amino acid protein; expression limited to the brain and spinal cord; only expressed in neurons; required for long term potentiation (LTP) and long term depression (LTD) responses in the brain

Novel PKC Isoforms

The nPKC isoforms (nPKCδ/θ: nPKC-delta/theta and nPKCε/η: nPKC-epsilon/eta) also have twin C1 domains (C1A and C1B) and a C2 domain. However, the nPKC C2 domains lack the critical calcium-coordinating acidic residues. It is this structural difference between cPKC and nPKC isoforms that accounts for the distinct pharmacology of these two subclasses.

Atypical PKC Isoforms

The aPKCs (aPKCζ: aPKC-zeta and aPKCι/λ: aPKC-iota/lambda) are so-called because they contain an atypical C1 domain harboring only a single cysteine-rich membrane-targeting structure. In addition, the aPKC isoforms lack a calcium-sensitive C2 domain. The C1 domains of aPKC enzymes bind PIP3 or ceramide not DAG or PMA. The aPKC isoforms also contain a protein-protein interaction domain (PB1: Phox and Bem 1) that mediates interactions with other PB1-containing scaffolding proteins. The PBI-scaffolding proteins includes p62, partitioning defective-6 (PAR-6), and mitogen-activated protein kinase kinase 5 (MAPK/ERK kinase 5: MEK5). The activity of the aPKC isoforms is regulated via the PBI domain-mediated protein-protein interactions as well as via phosphoinositide-dependent kinase 1 (PDK1)-mediated phosphorylation.

Regulation of PKC Activities

Although the classic idea that PKCs act as generic kinases and achieve substrate specificity as a result of translocation events to the membrane, recent data indicates that this family of enzymes can also be regulated via phosphorylations on both serine/threonine and tyrosine residues. These phosphorylation events influence the stability, protease/phosphatase resistance, protein-protein interactions, subcellular localization, and substrate specificity and activity of the enzyme. Some PKC isoforms have also been shown to be substrates for caspase-mediated cleavage that generates a catalytically active kinase domain and a released regulatory domain fragment. In some cases the released catalytic domain exhibits altered substrate specificity relative to that of the intact enzymes. The released regulatory fragment can act both as an inhibitor of the full-length enzyme and as an activator of certain signaling responses. As indicated, above some PKC isoforms (e.g. the aPKCs) are activated by interaction with less traditional lipid cofactors such as ceramides. In addition, some PKCs can be activated via lipid-independent mechanisms that include oxidative modifications or tyrosine nitration.

Regulation of PKC activities have also been shown to be exerted by a family of proteins called receptors for activated C kinase (RACKs) which are a family of membrane-associated PKC anchoring proteins. RACKs serve as molecular scaffolds that localize individual PKCs to distinct membrane microdomains in close proximity to their allosteric activators and substrates. It has been proposed that cells express a unique RACK for each PKC isoform and that resultant PKC-RACK interactions are essential for isoform-specific cellular responses. RACK proteins have been identified for PKCβ (RACK1) and PKCε (RACK2 or β-COP).

back to the top

The MAP Kinase (MAPK) Cascades

The mitogen-activated protein (MAP) kinase (MAPK) family constitutes a large family of 13 genes that encode serine/threonine kinases that are involved in a wide range of signal transduction cascades. This large family of kinases has been organized into four distinct MAPK cascades named according to the MAPK component that is the central enzyme of each of the cascades. These four MAPK cascades are the extracellular signal-regulated kinase 1/2 (ERK1/2), the c-Jun N-terminal kinase (JNK), the p38, and the ERK5 cascades. Each of these four cascades is in turn comprised of a core component that consists of three tiers of protein kinases termed MAPK, MAPKK, and MAP3K (MAPKKK). In several cases the cascade contains two additional tiers consisting of an upstream MAP4K and a downstream MAPKAPK (MAP kinase-activated kinase; MAPKAPK). Signal transduction triggered by each cascade involves the sequential phosphorylation and activation of the components in the subsequent tiers. The MAPK signal transduction cascades involve the coordination of a variety of extracellular signals that are initiated to control diverse cellular processes such as proliferation, differentiation, survival, development, stress response, and apoptosis. The ERK1/2 cascade primarily plays a role in proliferation and differentiation, however, there are situations where this cascade participates in responses to stress and apoptosis. The JNK and p38 cascades are primarily activated in response to cellular stress, although the JNK kinases are known to mediate proliferation under certain conditions. Several of the components of the JNK and the p38 MAPK cascades are termed stress-activated protein kinases (SAPKs). The ERK5 cascade responds to both mitogenic signals and cellular stress signals. Of clinical significance is that defective regulation of the MAPK cascades often leads to diseases such as cancer and diabetes. The entirety of the MAPK systems involves nearly 70 individual genes which, due to alternative splicing events, generates a highly complex system of signaling molecules that includes over 200 proteins.

The MAPK signaling cascades are most often initiated by receptor-mediated activation of members of the small monomeric G protein family (see next section below), such as Ras, Rac or Rho. In addition, the MAPK cascades can activate upstream components via their interactions with adaptor proteins. The initial signals are then propagated to downstream proteins of the three to five tiers of the four major MAPK cascades. The kinases in each tier phosphorylate and activate the kinases located in the next tier downstream. This process is repeated from tier to tier allowing for rapid and regulated transmission of the original initiating signal. As indicated above, the MAPK, MAPKK and MAP3K tiers are core components of all MAPK cascades. The upstream (MAP4K) or the downstream (MAPKAPK) tiers are not always necessary for signaling through the MAPK cascades.

Human MAP Kinase Family

Protein Name Gene Symbol Other Names Comments
mitogen-activated protein kinase-1 MAPK1 ERK, ERK2, p41mapk, MAPK2, ERT1, PRKM1, PRKM2 located on chromosome 22q11.2; composed of 9 exons; generates two alternatively spliced mRNAs encoding the same 360 amino acid protein
mitogen-activated protein kinase-3 MAPK3 ERK1, p44mapk, p44ERK1, ERT2, PRKM3 located on chromosome 16p11.2; composed of 10 exons; three alternatively spliced mRNAs encoding three isoforms; isoform 1 is a 379 amino acid protein, isoform 2 is a 357 amino acid protein, isoform 3 is a 335 amino acid protein
mitogen-activated protein kinase-4 MAPK4 ERK3-related, ERK4, PRKM4, p63MAPK located on chromosome 18q21.1; composed of 12 exons; three alternatively spliced mRNAsencoding three isoforms; isoform 1 is a 587 amino acid protein, isoform 2 is a 376 amino acid protein, isoform 3 is a 233 amino acid protein
mitogen-activated protein kinase-6 MAPK6 ERK3, p97MAPK, PRKM6 located on chromosome 15q21; composed of 9 exons; encodes a 721 amino acid protein, protein is localized to the nucleus
mitogen-activated protein kinase-7 MAPK7 ERK4, ERK5, BMK1, PRKM7 located on chromosome 17p11.2; composed of 9 exons; four alternatively spliced mRNAs, three of which encode the same protein; isoform 1 is a 816 amino acid protein; isoform 2 is a 677 amino acid protein
mitogen-activated protein kinase-8 MAPK8 JNK, JNK1, SAPK1, PRKM8 located on chromosome 10q11.22; composed of 15 exons; five alternatively spliced mRNAs encoding five protein isoforms identified as α1, α2, β1, β2, and 5; isoform α1 encodes a 384 amino acid protein; isoform α2 encodes a 427 amino acid protein; isoform β1 encodes a 384 amino acid protein, isoform β2 encodes a 427 amino acid protein; isoform 5 encodes a 308 amino acid protein; although the α2 and β2 isoforms are the same number of amino acids they differ by several amino acids in the internal region of the proteins
mitogen-activated protein kinase-9 MAPK9 JNK2, JNK55, SAPK, PRKM9 located on chromosome 5q35; composed of 16 exons; six alternatively spliced mRNAs encoding six protein isoforms identified as α1, α2, β1, β2, γ, and γ2
mitogen-activated protein kinase-10 MAPK10 JNK3, PRKM10, SAPK1b, p54bSAPK located on chromosome 4q22.1-q23; composed of 22 exons; five alternatively spliced mRNAs encodong six different isoforms identified as 1, 1x, 2, 3, 5, and 6; isoform 1 represents the dominant form of the MAPK10 encoded proteins; isoform 1x results from read-through of the UGA-stop codon utilized in the generation of isoform 1; isoform 5 is composed of the same number of amino acids as isoform 1 but due to altrenative splicing has a different N-terminus comprising 23 different amino acid; isoform 6 initiates translation from an in-frame downstream start codon
mitogen-activated protein kinase-11 MAPK11 SAPK2, SAPK2B, p38β, PRKM11 located on chromosome 22q13.33; composed of 13 exons; two alternatively spliced mRNAs; only one protein isoform is generated from transcription of the MAPK11 gene and is a 364 amino acid protein; the other mRNA is a non-coding variant
mitogen-activated protein kinase-12 MAPK12 ERK3, ERK6, SAPK3, PRKM12, p38γ located on chromosome 22q13.33; composed of 12 exons; two alternatively spliced mRNAs; isoform 1 encodes a 367 amino acid protein, isoform 2 encodes a 357 amino acid protein; protein functions in the differentiation of myoblasts
mitogen-activated protein kinase-13 MAPK13 SAPK4, p38δ, PRKM13 located on chromosome 6p21.31; composed of 12 exons; two alternatively spliced mRNAs; only one protein isoform is generated from transcription of the MAPK13 gene and is a 365 amino acid protein; the other mRNA is a non-coding variant
mitogen-activated protein kinase-14 MAPK14 p38, PRKM14, PRKM15, SAPK2A, CSBP, EXIP, Mxi2 located on chromosome 6p21.3-p21.2; composed of 19 exons; four alternatively spliced mRNAs encoding four distinct protein isoforms; isoform 1 encodes a 360 amino acid protein, isoform 2 encodes a 360 amino acid protein; isoform 3 encodes a 297 amino acid protein; isoform 4 encodes a 307 amino acid protein; although isoforms 1 and 2 have the same number of amino acids they differ at an internal segment of the proteins
mitogen-activated protein kinase-15 MAPK15 ERK7, ERK8 located on chromosome 8q24.3; composed of 15 exons encoding a 544 amino acid protein

The ERK Cascade

The key proteins that comprise the ERK cascade are encoded by the MAPK1, MAPK3, MAPK4, and MAPK6. The proteins of the ERK cascade are activated by a variety of extracellular agents, such as growth factors and hormones, leading to the induction of, primarily, proliferation and differentiation. However, as pointed out above, some conditions such as cellular stress involve the ERK cascade. The extracellular signals are relayed to the ERK cascade via the activation of GPCRs, receptors with intrinsic tyrosine kinase activity (RTKs), and ion channels. The extracellular signal transmission to ERK cascade kinases often involves adaptor proteins such as Shc or Grb2 (growth factor receptor-bound protein 2). These adapter proteins are recruited to the signaling receptor and then in turn activate guanine nucleotide exchange in membrane-bound monomeric G-proteins, such as Ras, rendering these G-proteins active. This in turn allows transmission of the signal to components of the MAP3K tier of the ERK cascade. The primary MAP3K tier proteins are members of the Raf kinase family (Raf-1, A-Raf, B-Raf), but can also include tumor progression 1, TPL2 (formally called MAP3K8 but also known as MEKK8) and the stress-activated kinases MEKK1 and leucine zipper- and sterile alpha motif-containing kinase (ZAK; also called MLK-like mitogen-activated triple kinase: MLTK). Although MOS is another MAP3K of the ERK cascade, its primary function is in the reproductive system and has a distinct mode of regulation. Subsequent to activation of proteins in the MAP3K tier, the signal is transmitted down the cascade to the MAPKK components. The proteins in this tier are called the MAPK/ERK kinases 1 and 2 (MEK1/2). The substrates for ERKs are regulatory proteins that includes one or more the MAPKAPK tier proteins. The MAPKAPK tier includes the ribosomal S6 kinase (RSK), the MAPK/SAPK-activated kinase (MSK), MAPK signal-interacting kinases 1 and 2 (MNK1/2), and MAPKAPK3/5. The important regulatory kinases, glycogen synthase kinase 3 (GSK3) and serine threonine kinase 11 (STK11; also called LKB1 or Peutz-Jeghers syndrome kinase: PJS) are known substrates for MAPKAPKs, but these latter kinases are not usually considered as integral components of the MAPK cascades.

The p38 Cascade

The key proteins that comprise the p38 cascade are encoded by the MAPK11, MAPK12, MAPK13, and MAPK14 genes. The p38 MAPK cascade is primarily functional when cells respond to various stressful stimuli but is also known to participate in immune responses and inflammation. Activation of the p38 MAPK cascade occurs in response to various stress factors as well as ligands that activate GPCRs, RTKs, and apoptosis-related receptors. In addition to receptor-mediated activation of the p38 MAPK cascade, physical stresses such as osmotic shock or heat shock, strongly activate the cascade via receptor-independent processes that includes changes in membrane fluidity. The primary inducing signals are then transmitted to monomeric G-proteins, similarly to the similar process of the ERK cascade, but involves other members of the monomeric G-protein family such as Rac. The subsequent steps in the p38 MAPK cascade involve activation of either the MAP4K tier or directly the MAP3K tier. At least 20 distinct kinase encoding genes are known to express kinases that participate in the MAP3K tier of the p38 MAPK cascade. Additionally, many of these kinase genes express multiple splice variants leading to even more complexity to the MAP3K tier. The MAP3K components in the p38 MAPK cascade are many of the same kinases in the JNK cascade. Characterizing the various p38 isoforms, based upon their differential sensitivity to various inhibitors and their unique sequences, allows them to be subdivided into two groups, p38α/p38β and p38γ/p38δ. Following their activation the p38 kinases then transmit their activation signals to the MAPKAPK tier components MAPKAPK2, MAPKAPK3, MNK1/2, MSK1/2, and MK5/PARK. Alternatively, activated p38 kinases phosphorylate regulatory molecules such as PLA2, transcription factors such as ATF2, ELK1, CHOP, MEF2C, and various heat shock proteins. The p38 kinases can undergo bidirectional redistribution between the nucleus and cytosol upon their activation. Similar to the processes by which the ERK cascade activated MAPKAPKs can phosphorylate additional kinases such as STK11, so too can the p38-activated MAPKAPKs.

The JNK Cascade

The key proteins that comprise the JNK cascade are encoded by the MAPK8, MAPK9, and MAPK10 genes. Like the p38 MAPK cascade, the JNK cascade plays an important role in the response to cellular stress by inducing apoptosis. Given the similarities in activation triggers between the JNK and p38 cascades, it is apparent that the JNK cascade is responsive to the activation of stress/apoptosis-related receptors, GPCRs, RTKs, and receptor-independent physical stresses. Following activation of the JNK kinases they transmit their signals to adapter that in turn activate the kinases in the MAP4K tier, and on occasion the MAP3K tier, of the JNK cascade. An additional activation scheme of the JNK cascade involves a network of interacting proteins that either induces changes in the activity of adapter proteins, such as members of the TRAF (TNF receptor-associated factor) family, or the activation of monomeric G-proteins such as Rac. Both of these activation processes then transmits the signal by activating MAP4K tier kinases, or sometimes directly activating MAP3K tier kinases. The kinases in the MAP4K tier of the JNK cascade includes MAP4K2 (also called germinal center kinase, GCK), MAP4K3 (also called germinal center-like kinase, GLK), MAP4K1 (also called hematopoietic progenitor kinase 1, HPK1), and other Sterile 20-like (Ste20-like) kinases. Each of these can, in turn phosphorylate and activate kinases in the MAP3K tier. Most of the MAP3K tier kinases of the JNK cascade are the same as those in the p38 MAPK cascade. However, several other MAP3K tier kinase are unique to the JNK cascade such as ASK2, LZK1, MLK1, and MEKK4. Following activation of the MAP3K kinases the signal is transmitted to kinases at the MAPKK level which are primarily MKK4 and MKK7 but may also include MKK3/6. The principal terminal proteins of the JNK cascade are the JNK proteins themselves. A total of 17 JNK proteins are translated from the three different JNK genes that undergo alternative splicing. The JNK cascade is a major regulator of transcription and involves migration of the JNK proteins to the nucleus where they interact with and activate transcription factor targets such as c-Jun, ATF2, and ELK1.

The ERK5 (MAPK7) Cascade

The fourth MAPK cascade was originally identified as the ERK5 cascade. This cascade comprises the protein isoforms encoded by the alternatively spliced mRNAs generated from the MAPK7 gene. This cascade is the least studied of the four. The ERK5 cascade was originally identified as being activated in response to stress stimuli, such as oxidative stress and hyperosmolarity, but was subsequently shown to also be activated mitogens. Activation of this cascade can include protein Y-kinases that transmit their signals to the adapter proteins Lad1 or WNK1 (protein kinase, leucine deficient 1). These adapter proteins appear to play the role of the MAP4K tier in this cascade. These adapters then activate the MAP3K kinases MEKK2/3, as well as ZAK and TPL2. The MAP3K tier kinases of the ERK5 cascade then phosphorylate and activate the two alternatively spliced MAPKK isoforms MEK5a and MEK5b. The MEK5s then phosphorylate and activate MAPK7 proteins. MAPK7 proteins can be localized to the cytoplasm and be translocated to the nucleus upon stimulation. However, in some cells MAPK7 proteins reside in the nucleus where they are activated by nuclear MEK5. Several transcription factors, such as FOS, MYC, and MEF2 family members, are targets for activated MAPK7 proteins. Additionally, activated MAPK7 proteins can phosphorylate the serum and glucocorticoid-activated kinase (SGK), which may serve as a MAPKAPK of ERK5 cascade. Unique to this cascade is the fact that MAPK7 proteins can influence transcription through either direct protein–protein interactions or via its intrinsic transcriptional activity. Thus, MAPK7 proteins are unique dual activity proteins that, unlike other MAPKs, catalyze two independent activities.

MAPK Regulation

Regulation and specificity of the four MAPK cascades is complex given that the consensus phosphorylation sites and the protein–protein interaction domains are shared by all MAPKs. Adding to this regulatory complexity is the fact that the MAPKs induce phosphorylation of a large number of proteins. Indeed, ERK1/2 has been shown to have at least 160 different substrates and the number of substrates for p38 and JNK kinases is likely to be similarly high. Adding to regulatory complexity is the fact that the distinct MAPK cascades utilize proteins that are shared between the MAP4K and MAP3K tiers of the four cascades. Five mechanisms for determination of MAPK specificity have been proposed. These include pathway specific strength and duration of the signals; interaction with various scaffold proteins that control the localization of MAPK kinases to distinct components and substrates of the cascade; interactions between the various MAPK cascades or interactions with other signaling pathways; compartmentalization of components and their targets to subcellular regions of organelles; the presence of multiple components with distinct specificities in each level of a given cascade.

back to the top

Cyclic Nucleotides in Signal Transduction

Cyclic nucleotides represent a family of naturally occurring nucleotide analogs that act as second messengers in hormone and ion channel signal transduction processes. Structurally the cyclic nucleotides are single phosphate nucleotides that harbor a "cyclic" bond between the phosphate and both the 3'-OH and the 5'-OH of the ribose portion of the nucleotide. The major cyclic nucleotides are derived from the purines, adenosine and guanosine forming 3',5'-cyclic adenosine monophosphate (cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP), respectively. However, the existence of the cyclic pyrimidines (cCMP and cUMP) has been documented in human cells and the degradation of cUMP has been demonstrated to be catalyzed by one of the known cyclic nucleotide phosphodiesterases. The production and utilization of cAMP and cGMP is paramount for the proper regulation of numerous biochemical and physiological processes. The primary effects of cAMP and cGMP are exerted through the activation of their respective cyclic nucleotide dependent kinases, PKA and PKG. However, the cyclic nucleotides, in particular cGMP, play important roles directly by modulating the activity of ligand-gated ion channels. Details of the synthesis and signal transduction pathways regulated by cAMP and cGMP are covered in the following two section. Once produced, both cAMP and cGMP (as well as the cyclic pyrimidines) must be degraded to limit the extent of the activities. The degradation of cyclic nucleotides is catalyzed by cyclic nucleotide-specific phosphodiesterases which are covered below.

back to the top

Adenylate Cyclases

The adenylate cyclases (adenylyl cyclases) are a family of enzymes that generate the second messenger molecule, cyclic adenosine-3',5'-monophosphate (cAMP) from ATP. Adenylate cyclases (AC) belong to the family of nucleotidyl cyclase enzymes that is divided into six broad classes identified as Class I–Class VI. All of the adenylate cyclase enzymes expressed in humans belong to the class III family. The human adenylate cyclases are further divided into two distinct sub-types, the transmembrane adenylate cyclases (tmAC) and the soluble adenylate cyclase (sAC). Humans express ten adenylate cyclase genes with nine of the genes (ADCY1–ADCY9) encoding tmAC isoforms and one gene (ADCY10) encoding a sAC isoform.

The PKA Family of Kinases

Once produced by the action of an adenylate cyclase, cAMP binds to the regulatory subunits of cAMP-dependent protein kinase (PKA). In the absence of cAMP, PKA exists as an inactive heterotetrameric complex composed of two regulatory and two catalytic subunits (see Enzyme Kinetics page). Two molecules of cAMP bind to each of the two regulatory subunits of PKA in order for the two catalytic subunits to be released. Once released the catalytic subunits phosphorylate serine and/or threonine residues in numerous target proteins. The activity of PKA is self-limiting due to the fact that one of the substrates for the enzyme is a member of the cyclic nucleotide phosphodiesterase (PDE) family (see section below for details on PDEs). Humans express four different regulatory subunit genes, PRKAR1A, PRKAR1B, PRKAR2A, and PRKAR2B. These four genes encode four different proteins that exhibit tissue-specific patterns of expression and exert distinct roles in cell differentiation and growth control. Humans express three different catalytic subunit genes: PRKACA encoding the alpha (α) catalytic subunit protein, PRKACB encoding the beta (β) catalytic subunit protein, and PRKACG encoding the gamma (γ) catalytic subunit protein.

All of the tmAC enzymes are activated by the alpha (α) subunit of Gs-type heterotrimerc G-proteins (see below). The transmembrane localized enzymes possess two sets of six transmembrane segments separated by two cytoplasmic segments, designated C1 and C2 (C1 and C2). Theses cytoplasmic domains are commonly referred to as cyclase homology domains (CHD). The two cytoplasmic domains associate with each other forming a heterodimeric catalytic core of the enzyme. Adenylate cyclase isoforms have been characterized that result from alternative splicing events and which contain a single CHD. The CHD in these isoforms homodimerizes to form the catalytic core. Calcium ions influence the activity of several of the tmAC either positively (AC1, AC3, and AC8) or negatively (AC5 and AC6). The activation of tmAC activity by Ca2+ involves the calcium-binding regulatory subunit, calmodulin. The mechanism by which Ca2+ inhibits tmAC activity is by inducing the displacement of the Mg2+ cofactor.

The soluble adenylate cyclase is not localized to the cytosol but is actually distributed within discrete sub-domains within the cell such as the nucleus, mitochondria, mitotic spindle, and cilia. The discrete localization allows for, what are referred to as, microdomains of cAMP production and action to be established. Soluble AC is tethered to these sites by being anchored to PKA or scaffold proteins called A-kinase anchoring proteins (AKAP). Like the tmAC enzymes, the sAC enzyme also possesses two domains (also identified as C1 and C2) that associate together to form the catalytic core of the enzyme. Unlike the tmACs, sAC activity is not regulated by heterotrimeric G-proteins. The activity of sAC is enhanced by both bicarbonate (HCO3) and Ca2+. The effect of bicarbonate is to enhance substrate (ATP) turnover and reduce substrate inhibition, while Ca2+ enhances substrate binding. the sAC isoform can also be activated by the presence of another divalent cation, Mg2+ similarly to the role of this divalent cation in the regulation of several of the tmAC isoforms. The ability of HCO3 to regulate sAC activity allows for the enzyme to serve as a sensor for the CO2/HCO3 ratio and thus, indirectly as a pH sensor. Human sAC plays important roles in airway epithelial cells, the exocrine pancreas, renal epithelium, skin, eye, and the nervous system. Within the nervous system, sAC is involved as a modulator of chemoreceptor sensation in the carotid and aortic bodies and the brain stem that regulate respiratory rate.

Adenylate Cyclases Expressed in Humans

Adenylate Cyclase Gene Location/
AC1 ADCY1 7p12.3: 22 exons primarily expressed in the brain, retina, and the adrenal medulla; stimulated by Ca2+ (via calmodulin) and PKA-mediated phosphorylation; inhibited by Gαi and by Gβγ dimers; involved in regulation of circadian rhythms likely by modulating cAMP production in the retina; mutations in ADCY1 associated with autosomal recessive deafness type 44 (DFNB44)
AC2 ADCY2 5p15.3: 25 exons highest levels of expression seen in brain, skeletal muscle, and lung; stimulated by Gβγ dimers following Gαs activation; activity enhanced by PKA phosphorylation
AC3 ADCY3 2p23.3: 26 exons highest expression seen in pancreas, olfactory epithelium, brain, brown adipose tissue (BAT), also expressed in lung, heart, brain, kidney, and liver; activated by Ca2+ (via calmodulin) and PKA; inhibited by Gαi
AC4 ADCY4 14q12: 26 exons highest expression seen in brain but also widely expressed; stimulated by Gβγ dimers and by PKA
AC5 ADCY5 3q21.1: 29 exons highest levels of expression in heart and brain striatum; inhibited by Gαi, Gβγ, Ca2+, and PKA-mediated phosphorylation; mutations in the ADCY5 gene associated with familial dyskinesia with facial myokmia (FDFM)
AC6 ADCY6 12q12–q13: 23 exons widely expressed; inhibited by Gαi, Gβγ, PKA-mediated phosphorylation, Ca2+, and NO
AC7 ADCY7 16q12.1: 31 exons highest expression in brain and platelets but also widely expressed; stimulated by Gβγ dimers and by PKA-mediated phosphorylation
AC8 ADCY8 8q24: 18 exons highest levels of expression seen in brain and lung; inhibited by Gαi; stimulated by Ca2+ (via calmodulin); plays a role in learning, memory, and drug dependence
AC9 ADCY9 16p13.3: 12 exons highest expression in airway smooth muscle, also expressed in lung, pituitary and skeletal muscle; inhibited by Gαi; stimulated by β-adrenergic receptors; may be a membrane docking site for calcineurin
AC10 ADC10 1q24: 36 exons soluble enzyme (sAC); expressed at highest levels in testes but also widely expressed; insensitive to G-protein regulation; regulated by bicarbonate ion and Ca2+; alternative mRNA splicing yields at least three distinct sAC isoforms, two of the alternatively spliced mRNAs generate isoforms of sAC that contain only one (C2 only) of the two catalytic domains

back to the top

Guanylate Cyclases and PKG

The guanylate cyclases are a family of enzymes that generate the second messenger molecule, cyclic guanosine-3',5'-monophosphate (cGMP) from GTP. Like cAMP, cGMP acts as a second messenger molecule in a variety of signal transduction events. Intracellular cGMP has been shown to play a critical role in the modulation of lipolysis, platelet aggregation, blood pressure, gut peristalsis, intestinal fluid secretion, neurotransmission, vision, sexual arousal, long bone growth, cardiac myocyte growth, and oocyte maturation. The guanylate cyclases are regulated by a broad spectrum of agents that includes nitric oxide (NO), bicarbonate ion (HCO3), the natriuretic peptides (ANP, BNP, and CNP), and the guanylyl cyclase activating proteins (GCAPs).

The PKG Family of Kinases

Like the role of cAMP in modulating the activity of PKA, cGMP modulates the activity of the cGMP-dependent protein kinases, PKGs. Humans express three distinct isoforms of PKG identified as PKGIα, PKGIβ, and PKGII. Both PKGIα and PKGIβ are soluble enzymes and both are derived from the same gene (PRKG1) as a result of alternative splicing. The PKGII enzyme is a membrane-bound version of the PKG kinase family and is encoded by a separate gene, PRKG2. Unlike the subunit structure of PKA, PKG enzymes do not possess separate catalytic and regulatory subunits. Active PKG isoforms are homodimers and are activated by direct binding of cGMP to each subunit in the homodimer. The PKGI isoforms are most highly expressed in vascular smooth muscle cells and in platelets. PKGII is most highly expressed in retinal cells, zona glomerulosa cells in the adrenal cortex, intestinal mucosal cells, and pancreatic duct cells.

The guanylate cyclase (GC) enzymes, like the adenylate cyclase enzymes, belong to the class III family of nucleotidyl cyclases. There are two distinct types of guanylate cyclases expressed in humans, the soluble enzymes (sGC) and the sub-family of single transmembrane-spanning guanylate cyclases. The sGC family members all function as heterodimers of one α-subunit and one β-subunit. The heme moiety, to which NO binds, is attached to the N-terminus of the β-subunits. The catalytic activity of the sGC enzymes is created by C-terminal domains in both subunits. The human α1 subunit (also identified as α3) is encoded by the GUCY1A3 gene located on chromosome 4q32.1 and is composed of 15 exons that generate seven alternatively spliced mRNAs, five of which encode the same protein identified as isoform A (690 amino acids). The α1 isoform B is 455 amino acids and isoform D is 624 amino acids. The α2 protein is encoded by the GUCY1A2 gene located on chromosome 11q21–q22 and is composed of 9 exons that generate two alternatively spliced mRNAs encoding isoform 1 (763 amino acids) and isoform 2 (732 amino acids). The α2 subunit of sGC heterodimers may actually direct this isoform of sGC to membranes. The highest concentrations of the α2/β1 isoform of sGC is in the brain with the α1/β1 isoform predominating in all other tissues. The human genome contains two β-subunit genes but one (GUCY1B2) is a pseudogene. The β1 subunit (also identifies as β3) is encoded by the GUCY1B3 gene located on chromosome 4q31.3–q33 and is composed of 17 exons that generate six alternatively spliced mRNAs that encode six different isoforms of the protein. In addition to the classic role of sGC in the formation of cGMP, recent evidence has shown the the α1β1 enzyme can form cyclic pyrimidine nucleotides (cCMP and cUMP) in the presence of Mn2+, whereas in the presence of Mg2+ only cGMP is formed. As yet, the proteins to which the cyclic pyrimidines bind have not been identified.

Although there are seven transmembrane-spanning guanylate cyclase genes in mammals, humans express only five isoforms designated as GC-A, GC-B, GC-C, GC-E, and GC-F. The GC-D and GC-G encoding genes in humans are pseudogenes. The transmembrane-spanning GC isoforms all serve as receptors for specific ligands. The GC-A and GC-B isoforms are more commonly referred to as the natriuretic peptide receptor 1 (NPR1 or NPR-A) and natriuretic peptide receptor 2 (NPR2 or NPR-B) isoforms. NPR1 binds both ANP and BNP, whereas, NPR2 binds CNP. All of the membrane-spanning GC isoforms function as homodimers. NPR1 is highly expressed in kidney, lung, adrenal glands, vasculature tissues, brain, liver, endothelial cells, and adipose tissues with lower levels of expression observed in cardiac tissues. When the NPR1 gene is knocked out in mice they develop cardiac hypertrophy, ventricular fibrosis, and hypertension. The activity of the NPR1 protein is regulated by its state of intracellular phosphorylation. The receptor is unable to respond to ligand binding in the dephosphorylated state, thus, requiring phosphorylation for activity. Guanylate cyclase C (GC-C, encoded by the GUCY2C gene) is a receptor for several endogenous and exogenous peptides. Pathogenic bacteria produce a heat-stable enterotoxin that binds and activates the GC-C protein. When the bacterial entertoxin binds to the GUCY2C-encoded guanylate cyclase, the increased production of intracellular cGMP results in activation of a cGMP-dependent protein kinase, PKG (specifically PKGII). Under these conditions, PKGII phosphorylates the cystic fibrosis transmembrane conductance regulator (CFTR) resulting in increased Cl secretion in the gut. As the intestinal ion secretion increases there is concomitant loss of water to the lumen of the gut. The loss of intestinal water leads to severe dehydration and the increased water in the lumen leads to severe diarrhea. Guanylin and uroguanylin are endogenous peptide ligands for the GUCY2C-encoded guanylate cyclase (see below). Humans express two transmembrane guanylate cyclases in the retina, both of which are involved in the processess of phototransduction. These two enzymes are GC-E (more commonly called retinal guanylate cyclase 1, RETGC-1) and GC-F (more commonly called RETGC-2). The activities of the two RETGC enzymes are regulated, not by ligand binding, but through the action of a GCAP (details below the following Table).

All of the sGC enzymes function as heterodimers. The sGC isoforms possess a heme-binding domain in the N-terminal region of the proteins that is required for activation. The primary endogenous activator of the sGC enzymes is nitric oxide (NO), but carbon monoxide (CO) can also bind to the heme prosthetic group. The soluble GC enzymes are expressed in most human tissues with enriched expression seen in brain, kidney, vascular tissues, and lung. Although there are four different sGC isoforms that can form heterodimers, the most well characterized forms in human tissues are the α11 and α21 forms. Within these heterodimers it is the β-subunit that contains a His residue in its N-terminal domain that coordinates the ferrous (Fe2+) iron within the heme prosthetic group. Activation of sGC isoforms, by NO, requires the presence of the ferrous iron-heme prosthetic group. There is, however, complex interplay between the α-subunits and the β-subunits in sGC heterodimers that results in activity specificity. For example, disruption of the α1-subunit abolishes NO-dependent platelet aggregation yet there remains a normal vasorelaxation response. Conversely, disruption of the β1-subunit results in loss of both responses.

Single Transmembrane-Spanning Guanylate Cyclases Expressed in Humans

GC Designation Gene Location/
GC-A NPR1 1q21–q22: 22 exons also identifed as GUCY2A and ANPRA; receptor for ANP and BNP; expression in lung, kidney, adrenal gland, vascular smooth muscle, endothelium, heart, adipose tissue
GC-B NPR2 9p21–p12: 27 exons also identified as GUCY2B and ANPRB; receptor for CNP; expression in bone, vascular smooth muscle, lung, brain, heart, liver, uterus, and follicle
GC-C GUCY2C 12p12: 27 exons receptor for the gut-derived peptides, guanylin and uroguanylin; originally identified as the receptor for bacterial heat-stable enterotoxin; highest expression is in apical membrane of intestinal epithelial cells
GC-E GUCY2D 17p13.1: 20 exons expressed in the retina; also called retinal guanylate cyclase 1 (RETGC-1) or just guanylate cyclase 1 (GC1); regulates phototransduction in the dark; no ligand, activated by guanylate cyclase activating protein 1 (GCAP1) and GCAP2; mutations in gene are the cause of Leber congenital amaurosis and autosomal dominant cone rod dystrophy 6 (CRD6)
GC-F GUCY2F Xq22: 22 exons expressed in the retina; also called retinal guanylate cyclase 2 (RETGC-2) or just guanylate cyclase 2 (GC2); regulates phototransduction in the dark; no ligand, activated by guanylate cyclase activating protein 1 (GCAP1) and GCAP2; mutations in gene are the causes of X-linked retinitis pigmentosa

Gyanylate Cyclase Activating Proteins, GCAP

Guanylate cyclase activator proteins (GCAPs) are a family of proteins that regulate the activity of several members of the single transmembrane-spanning guanylate cyclases. Humans express three GCAP proteins that function in the regulation of retinal cone and rod cell guanylate cyclases. The retinal cell guanylate cyclases are called RetGC-1 (often just simply referred to as GC1) and RetGC-2 (often just simply referred to as GC2). Humans also express two additional genes encoding proteins of the GCAP family. However, in the case of these latter two genes the encoded proteins (guanylin and uroguanylin: see below) are the ligands for the intestinal transmembrane guanylate cyclase encoded by the GUCY2C gene.

Retinal cells of the eye can adjust their sensitivity to photons as a means to allow for phototransduction over a wide range of light intensities. These adjustments to sensitivity are brought about through the effect that calcium ions (Ca2+) exert on the activity of the retinal cell membrane guanylate cyclases which produce cGMP. For more details on the role of cGMP in phototransduction go the the Vitamins page. The effects of Ca2+ on retinal guanylate cyclase activity is due to regulated function, not of the guanylate cyclases directly, but of guanylate cyclase activating proteins, GCAPs. Humans express three GCAP genes, whose encoded proteins are identifed as GCAP1, GCAP2, and GCAP3, that function in the regulaiton of cGMP-mediated phototransduction. Both GCAP1 and GCAP2 bind Ca2+ and have similar dependence and cooperativity for guanylate cyclase activation by Ca2+. The GCAP1 protein is encoded by the GUCA1A gene, the GCAP2 protein is encoded by the GUCA1B gene, and the GCAP3 protein is encoded by the GUCA1C gene.

Guanylin and Uroguanylin Peptides

The guanylin family of intestinal peptides was originally characterized due to attempts to understand how ingested sodium led to increased renal electrolyte and water secretion. These original observations predicted the existence of intestinal natriuretic-like hormones. Following their full characterization these intestinal hormones were called guanylins. Humans express two intestinal guanylin family peptides identified as guanylin and uroguanylin. Guanylin is a 15 amino acid peptide that contains two intramolecular disulfide bonds, whereas, uroguanylin is a 19 amino acid peptide whose disulfide bonds are conserved relative to those in guanylin. The guanylin peptide is encoded by the GUCA2A gene and uroguanylin is encoded by the GUCA2B. Both guanylin and uroguanylin have been called guanylate cyclase activating proteins (GCAPs) with guanylin identified as GCAP2A and uroguanylin identified as GCAP2B. The GUCA2A gene is located on chromosome 1p35–p34 and is composed of 3 exons that encode a 115 amino acid preproprotein. The GUCA2B gene is located very close to the GUCA2A gene on chromosome 1 (1p34–p33) and is composed of 3 exons that encode a 112 amino acid preproprotein. In addition to guanylin and uroguanylin, there are two additional guanylin peptide family members identified as lymphoguanylin and renoguanylin. However, as yet a human isoform of renoguanylin remains to be identified.

Following the ingestion of a salty meal, intestinal cells secrete guanylin and uroguanylin into the intestinal lumen where they bind to the GUCY2C-encoded transmembrane guanylate cyclase. Guanylin producing cells of the intestine are goblet and epithelial cells of the colonic mucosa, whereas, uroguanylin producing cells are enterochromaffin cells of the small intestine. Activation of cGMP production by the intestinal GUCY2C encoded enzyme results in inhibition of sodium absorption from intestinal lumen by inhibiting a Na+/H+ exchange (NHE) transporter (specifically NHE2 encoded by the SLC9A2 gene), increased bicarbonate (HCO3) and Cl secretion, inhibited water absorption, and increased renal Na+ and K+ secretion. These effects of guanylin and uroguanylin, exerted as a result of increased intracellular cGMP production, are exerted both at the level of increased PKG and PKA activity. The increased PKG (specifically PKGII) activity is a direct result of cGMP activating this kinase. PKA activity is also enhanced because the increased cGMP leads to inhibition of phosphodiesterase 3, PDE3, family (see next section) enzyme activity. PDE3 enzymes normally hydrolyze cAMP leading to reduced levels of active PKA. However, with cGMP-mediated inhibition of PDE3 the level of active PKA remains high. One of the targets for intestinal PKGII is the CFTR protein. Phosphorylation of CFTR by PKGII results in increased Cl transport into the lumen of the gut. PKA can also phosphorylate the CFTR protein and also phosphorylates an apical membrane HCO3/Cl antiporter. The inhibition of the NHE2 transporter occurs directly via cGMP binding to the transporter.

In addition to guanylin peptide secretion into the intestinal lumen, intestinal cells transport guanylin and uroguanylin into the blood. The secretion of these peptides into the blood allows them to interact with their receptor guanylate cyclase in renal tubular cells where the response is natriuresis, diuresis, and kaliuresis. In addition to the intestine, the guanylin and uroguanylin genes are expressed in the pancreas, adrenal glands, lung, and reproductive systems where these pepotides exert regulation on membrane transport systems.

back to the top

Cyclic Nucleotide Phosphodiesterases: PDEs

Phosphodiesterases are enzymes that catalyze the hydrolysis of a phosphodiester bond. Humans express a large number of phosphodiesterases, with the cyclic nucleotide phosphodiesterases (PDEs) being the class of enzymes most commonly associated with the term, phosphodiesterase. Other mammalian phosphodiesterases include several members of the phospholipase family of enzymes such as the phospholipase D (PLD) enzymes and the phospholipase C (PLC) enzymes. Ribonucleases (RNases) and deoxyribonucleases (DNases) are also members of the phosphodiesterase superfamily of enzymes.

The PDEs expressed in human tissues hydrolyze cAMP to AMP, and/or cGMP to GMP. Certain members of the human PDE family are specific for one cyclic nucleotide or the other while some do not exhibit substrate specificity and can hydrolyze either cAMP or cGMP. Humans express 12 PDE gene families identified as PDE1–PDE12. Within each gene family there may be several distinct genes and many of these genes generate alternatively spliced mRNAs encoding variant isoforms of the enzyme. The cAMP-specific phosphodiesterases are encoded by the PDE4, PDE7, and PDE8 gene families. The cGMP-specific phosphodiesterases are encoded by the PDE5, PDE6, and PDE9 gene families. The dual-specificity phosphodiesterases are encoded by the PDE1, PDE2, PDE3, PDE10, and PDE11 gene families. The activity of PDE12 is unique in that is hydrolyzes cAMP as well as oliogadenylate molecules. The designation for a particular phosphodiesterase gene includes the PDE term followed by an Arabic numeral for the gene family, followed by a capital letter to designate the particular gene in that family. If a gene generates splice variants then there is another Arabic number added to the enzyme nomenclature to designate the specific splice variant. For example, the phosphodiesterase designated PDE1B4 is derived from the fourth splice variant of the PDE1B gene, where the PDE1B gene is one of several family 1 PDE genes.

Mammalian Cyclic Nucleotide Phosphodiesterases (PDEs)

PDE Gene Family Family Members Comments
1 PDE1A, PDE1B, PDE1C all members of the PDE1 family are calmodulin-regulated enzymes; PDE1A produces five splice variant isoforms; PDE1B produces four splice variant isoforms; although dual-specific the PDE1B isoforms show preference for cGMP as substrate
2 PDE2A generates four splice variants
3 PDE3A, PDE3B PDE3 genes are members of the cGMP-inhibited cyclic nucleotide phosphodiesterase (cGI-PDE) family; PDE3A expresses two splice variants; PDE3B involved in insulin-mediated signal transduction events that result in reduced levels of active PKA
4 PDE4A, PDE4B, PDE4C, PDE4D PDE4A produces five splice variant isoforms; PDE4B produces six splice variant isoforms; altered activity of PDE4B isoforms are associated with bipolar disorder and schizophrenia; PDE4C produces three splice variant isoforms; PDE4D produces nine spice variant isoforms
5 PDE5A PDE5A produces three splice variant isoforms, although PDE5A1 and PDE5A2 only isoforms for which activity has been demonstrated; is critical enzyme involved in smooth muscle cell relaxation in the cardiovascular system; is the target of the PDE5 inhibitor class of drugs used to treat erectile dysfunction and pulmonary hypertension
6 PDE6A, PDE6B, PDE6C, PDE6D PDE6A and PDE6B encode the alpha (α) and beta (β) subunits, respectively, of the catalytic core of the rod outer segment phosphodiesterase 6 holoenzyme; the rod PDE6 enzyme is a heterotetrameric complex composed of one α-subunit, one β-subunit, and two γ-subunits (inhibitory subunits); mutations in PDE6A cause a form of autosomal dominant retinitis pigmentosa; PDE6B produces three splice variant isoforms; mutations in PDE6B are associated with retinitis pigmentosa and autosomal dominant congenital stationary night blindness; PDE6C encodes the alpha prime (α') subunit of the cone cell phosphodiesterase 6 which is a complex composed of two α'-subunits and three additional small proteins of 11kDa, 13kDa, and 15kDa; mutations in PDE6C cause cone dystrophy type 4 (COD4); PDE6D encodes the delta (δ) subunit of a retinal rod phosphodiesterase; PDE6D produces two splice variant isoforms; most PDE5 inhibitors also inhibit rod PDE6 which has significance for contraindication of that class of drug in certain individuals
7 PDE7A, PDE7B PDE7A produces two splice variant isoforms; inhibitors of PDE7 isoforms are being tested for efficacy in inflammatory airway disorders and inflammatory disorders in the CNS
8 PDE8A, PDE8B PDE8A produces three splice variant isoforms; PDE8B produces five splice variant isoforms; PDE8A1 isoform is involved in activated lymphocyte chemotaxis; mutations in PDE8B result in autosomal dominant striatal degeneration (ADSD)
9 PDE9A PDE9A produces fifteen splice variant isoforms; pharmacologic inhibition of PDE9A may promote cognitive function in patients with Alzheimer disease
10 PDE10A PDE10A produces two splice variant isoforms; pharmacologic inhibition of PDE10A is being investigated for the treatment of schizophrenia and Huntington disease
11 PDE11A PDE11A produces four splice variant isoforms; mutations in PDE11A associated with Cushing disease and adrenocortical hyperplasia
12 PDE12 originally identified as 2',5'-phosphodiesterase 12

back to the top

Purine and Pyrimidine Nucleotide Signaling

The intracellular signaling pathways that involve nucleotides as effectors are quite broad and the details of the intracellular cyclic nucleotides in signal transduction processes are described in the preceding sections. Extracellularly nucleotides also exert critically important processes of signal transduction. Although numerous extracellular nucleotides are known to exert effects, it is predominantly ATP that is the major initiator. The mechanisms by which extracellular nucleotides exert their effects include binding to ligand-gated ion channel (ionotropic) receptors and to metabotropic G-protein coupled receptors. The ionotropic receptors to which nucleotides bind are identified as the P2X receptors and the metabotropic receptors are identified as the P2Y receptors. In addition to nucleotide receptor-mediated signal transduction mechanisms occurring directly within target cells, the signaling cascades activated by P2X and P2Y receptors result in intercellular communication through the stimulated release of other extracellular messenger substances. These concerted effects result in the activation of additional signal transduction pathways via the receptors to which the secondary messengers bind. The signaling substances that are induced to be released following nucleotide receptor activation include neurotransmitters, hormones, growth factors, lipid mediators, nitric oxide (NO), numerous proteins (e.g. enzymes and cytokines), and reactive oxygen species (ROS). Of considerable significance to the overall effects of nucleotide receptor activation is that nucleotides also activate or co-activate numerous growth factor receptors.

back to the top

P2X Receptors: Ligand-Gated Ion Channels

The P2X family of receptors are ATP-gated non-selective ion channels (ligand-gated ion channels). Each of the P2X receptors are homo- or heterotrimeric channels that primarily transport Na+, K+, or Ca2+ ions in response to the binding of extracellular ATP. There are seven subtypes of P2X receptors in humans identified as P2X1–P2X7. Each of the seven receptors are known to form homotrimeric receptor channels. Heterotrimeric P2X receptor channels are predominantly composed of subunits from two different P2X receptor genes and include the P2X1/P2X2, P2X1/P2X4, P2X1/P2X5, P2X2/P2X3, P2X2/P2X6, and P2X4/P2X6 heterotrimers. However, a heterotrimeric P2X receptor composed of P2X2/P2X4/P2X6 has been identified in Leydig cells. The genes that encode these receptor proteins are identified as P2RX genes (P2RX1–P2RX7). These genes are expressed in a wide array of cell types including both excitatory and non-excitatory cells. The signal transduction cascades initiated by activation of P2X receptors include neuronal synaptic transmission, nociception (sensation of pain), taste, modulation of inflammatory processes, modulation of cardiovascular processes, and tumorigenesis. As might be expected, the loss of function of several P2X encoding genes has been associated with numerous pathological conditions.

The P2X receptors are organized within the plasma membrane where both the N-terminus and C-terminus are located intracellularly, there are two transmembrane spanning domains (TM1 and TM2), and there is a large extracellular domain defined as the ectodomain. The initial response to ATP binding to the P2X receptors is Chanel opening and ion flow. Following prolonged exposure to ATP the receptor becomes desensitized such that ion influx terminates even in the presence of bound ATP. Following ATP dissociation the receptor returns to a sensitized state capable of responding again to ATP binding.

In addition to channel activation by ATP binding, the P2X receptors are also regulated by numerous allosteric effectors such as pH and heavy metals. At pH levels below 7, ATP effects on P2X1, P2X3, and P2X4 are attenuated, whereas, at the P2X1 channel ion currents are inhibited. Zinc ions (Zn2+), which represents the second most abundant trace metal in the body (second to iron) has been shown to have important P2X modulating effects. At low concentration zinc can potentiate the ion current through the P2X2, P2X3, and P2X4 channels following ATP binding, but at higher concentration zinc exerts an inhibitory effect on the channel. Intracellularly phospholipids exert increased ion flux through the channel through interactions with the C-terminus of the protein. The effects of phospholipids has been shown to be exerted on the P2X1, P2X2, and P2X4 homotrimeric receptors and the P2X1/P2X5 and P2X2/P2X3 heterotrimeric receptors.

The P2RX1 gene is located on chromosome 17p13.2 and is composed of 13 exons that encode a 399 amino acid protein. The P2RX2 gene is located on chromosome 12q24.33 (near the location of the P2RX4 and P2RX7 genes) and is composed of 10 exons that generate eight alternatively spliced mRNAs encoding eight distinct protein isoforms. The P2RX3 gene is located on chromosome 11q12.1 and is composed of 16 exons that encode a 397 amino acid protein. The P2RX4 gene is located on chromosome 12q24.31 and is composed of 14 exons that generate four alternatively spliced mRNAs each of which encode a distinct protein isoform. Additional non-coding alternative transcripts have been shown to be derived from the P2RX4 gene. The P2RX5 gene is located on chromosome 17p13.2 and is composed of 12 exons that generate four alternatively spliced mRNAs each of which encodes a distinct protein isoform. There is also a read-through transcript that includes the downstream Tax1 binding protein 3 (TAX1BP3) gene. The P2RX6 gene is located on chromosome 22q11.21and is composed of 14 exons that generate two alternatively spliced mRNAs encoding two distinct protein isoforms. The P2RX7 gene is located on chromosome 12q24.31 and is composed of 14 exons that encode a 595 amino acid protein. Additional P2XR7 transcripts are generated but do not encode functional proteins and are degraded by the nonsense-mediated decay pathway.

The P2X1 receptor protein (identified as P2X1R) is primarily expressed in smooth muscle, platelets and the CNS. Within smooth muscle cells the P2X1R is involved in the vasoconstrictive actions in the arteries and arterioles, as well as in the vas deferens, induced by sympathetic innervation. Function of the P2X1R is essential during the development and functioning of the male reproductive system. The P2X2 receptor protein (P2X2R) is expressed in the CNS, autonomic and sensory ganglia, smooth muscle, and the pancreas. The P2X3 receptor protein (P2X3R) is expressed the CNS, sympathetic neurons, and in nociceptive (pain sensation) sensory neurons. The P2X4 receptor protein (P2X4R) is expressed in the CNS, microglial cells, testis, colon, and endothelial cells. The P2X5 receptor protein (P2X5R) is expressed in the skin, skeletal muscle, and epithelial cells. The P2X6 receptor protein (P2X6R) is expressed in the CNS and in peripheral nerves. The P2X7 receptor protein (P2X7R) is expressed in microglial cells, oligodendrocytes, macrophages, mast cells, immune cells, and in the pancreas and skin. The P2X7 receptor (P2X7R) is unique among the seven P2X receptors in that it does not significantly function at normal physiological concentrations of extracellular ATP indicating that it most likely functions in the mediation of potential pathophysiological states such as those associated with inflammation and stress. In addition, activation of P2X7R can trigger signal transduction cascades that involve the activation of caspase 1, phospholipases A2 and D (PLA2 and PLD), the MAP kinases (MAPK), PKC, SRC, and glycogen synthase kinase-3 (GSK-3), as well as several phosphatases.

back to the top

P2Y Receptors: G-Protein Coupled Receptors, GPCR

Extracellular purine and pyrimidine nucleotides bind to and activate a class of metabotropic G-protein coupled receptors (GPCR) termed the P2Y receptor family. Humans express ten P2Y receptor genes encoding the P2Y1, P2Y2, P2Y4, P2Y6, P2Y8, and P2Y10–P2Y14 receptors. The genes that encode the P2Y receptors are identified by P2RY with the corresponding identifying number. Although purine nucleotides, primarily ADP but in some cases ATP also, are the preferred ligands for the P2Y receptors, the P2Y4 and P2Y6 receptors are only activated by the pyrimidines, UTP and UDP, respectively. The P2Y2 receptor can be activated by both ATP and UTP while the P2Y11 receptor is only activated by ATP. In addition to free nucleotides, the P2Y14 receptor is known as the UDP-glucose receptor. Indeed, this member of the P2Y family of nucleotide activated receptors was originally isolated and characterized as a GPCR responding to UDP-glucose binding as well as to several other nucleotide-coupled sugars.

The P2Y1, P2Y2, P2Y4, and P2Y6 receptors activate associated Gq-type G-proteins leading to the production of the second messengers, DAG and IP3. The P2Y12, P2Y13, and P2Y14 receptors activate associated Gi-type G-proteins resulting in the inhibition of adenylate cyclase and, therefore, reduced levels of cAMP and consequent decreases in PKA activity. The P2Y11 receptor activates both Gq- and Gs-type G-proteins. In addition to the activation of the associated Gα-subunits, several P2Y receptor-associated G-proteins release functional Gβγ-subunits following Gα-subunit release and activation. The Gβγ subunits of P2Y receptors have been shown to be involved in the activation of numerous signaling cascades that involve phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), inwardly rectifying K+ (GIRK) channels, MAP kinase, monomeric G-proteins of the RHO family, and G-protein receptor kinases (GRKs).

The P2RY1 gene is an intronless gene located on chromosome 3q25.2 that encodes a 373 amino acid protein. The P2RY2 gene is located on chromosome 11q13.43 is composed of 12 exons that generate three alternatively spliced mRNAs each of which encode the same 377 amino acid protein. The P2RY4 gene is an intronless gene located on the X chromosome (Xq13.1) that encodes a 365 amino acid protein. The P2RY6 gene is located on chromosome 11q13.4 and is composed of 7 exons that generate eight alternatively spliced mRNAs that collectively encode two proteins, isoform 1 is a 328 amino acid protein while isoform 2 is 429 amino acid protein. The P2RY8 gene is located on both the X chromosome (Xp22.33) and the Y chromosome (Yp11.2) both of which are composed of 6 exons that encode the same 359 amino acid proteins. The P2RY10 gene is located on the X chromosome (Xq21.1) and is composed of 5 exons that generate five alternatively spliced mRNAs encoding three distinct protein isoforms. The P2RY11 gene is located on chromosome 19p13.2 and is composed of 2 exons that encode a 374 amino acid protein. A read-through transcription product is generated from the P2RY11 gene and the immediate upstream gene that encodes a human homolog (PPAN) of the Drosophila peter pan gene. This read-through transcript encodes a fusion protein that exhibits functionality of both individual gene products. The P2RY12 gene is located on chromosome 3 (3q25.1) near the location of the P2RY1 gene and is composed of 4 exons that generate two alternatively spliced mRNAs that both encode the same 342 amino acid protein. The P2RY13 gene is also located in the same region of chromosome 3 (3q25.1) as the P2RY12 gene and is composed of 2 exons that encode a 354 amino acid protein. The P2RY14 gene is also located in the same 3q25.1 region as the P2RY12 and P2RY13 genes and is composed of 6 exons that generate two alternatively spliced mRNAs both of which encode the same 338 amino acid protein.

The P2RY genes are expressed in numerous tissues with various members having selective significance in specific tissues. For example, the P2RY12 receptor (and likely the P2Y1 receptor) is critical in the involvement of platelet function in blood coagulation. The P2RY8 gene is highly expressed in lymphocyte precursor cells. A chromosomal abnormality that involves the P2RY8 gene has been found to be associated 5-7% of pediatric B-cell progenitor acute lymphoblastic leukemia (B-ALL) and also with >50% of Down syndrome-associated acute lymphoblastic leukemia. This chromosomal abnormality results from an interstitial deletion that places the promoter region of the P2RY8 gene upstream of the cytokine receptor-like factor 2 (CRLF2) gene. This chromosomal disruption results in high level expression of full-length function CRLF2 leading to the overgrowth of the B-progenitor cells.

back to the top


G-proteins are so-called because their activities are regulated by binding and hydrolyzing GTP. When a G-protein is bound to GTP it is in the active ("on") state and when the GTP is hydrolyzed to GDP the protein is in the inactive ("off") state. The G-proteins possess intrinsic GTPase activity that is regulated in conjunction with interaction with membrane-associated signal transducing receptors (termed G-protein coupled receptors, GPCRs; see next section) or with intracellular effector proteins. There are two major classes of G-protein: those that are composed of three distinct subunits (α, β and γ) and the monomeric class that are related to the archetypal member Ras (originally identified as an oncogene causing sarcomas in rats). The monomeric class of G-protein is also referred to as the Ras superfamily or the small GTPase family of G-proteins. The structure and function of the monomeric G-proteins is similar to that of the α-subunit of the trimeric G-proteins.

All known cell surface receptors that are of the G-protein coupled receptor class interact with trimeric G-proteins. The α-subunit of the trimeric class of G-proteins is responsible for the binding of GDP/GTP. When G-proteins are activated by receptors or intracellular effector proteins there is an exchange of GDP for GTP turning on the G-protein which enables it to transmit the original activating signal to downstream effector proteins. In the trimeric class of G-protein, when associated receptor activation stimulates the GDP/GTP exchange in the α-subunit, the protein complex dissociates into separate α and βγ activated complexes. The released and activated βγ complex serves as a docking site for interaction with downstream effectors of the signal transduction cascade. Once the α-subunit hydrolyzes the bound GTP to GDP it re-associates with the βγ complex thereby terminating its activity.

ligand-receptor interaction-mediated activation of associated G-proteins

Diagrammatic representation of the activation of trimeric G-proteins upon ligand binding to typical G-protein coupled receptors. Upon ligand binding to a GPCR there is an activated exchange of GDP bound to the α-subunit for GTP catalyzed by an associated guanine nucleotide exchange factor (GEF). The resultant GTP-associated α-subunit can then activate downstream effectors proteins. In some cases G-protein βγ-subunits also regulate the activity of downstream effectors. Hydrolysis of GTP to GDP during the G-protein activation of effectors as a result of the action of GTPase activating proteins (GAPs) results in termination of the activity of the α-subunit.

The GTPase activity of G-proteins is augmented by GTPase activating proteins (GAPs) and the GDP/GTP exchange reaction is catalyzed by guanine nucleotide exchange factors (GEFs). Often the GEF activity is associated with the GPCR itself. Within the small GTPase family of G-proteins there are guanosine nucleotide dissociation inhibitors (GDIs) that maintain the G-protein in its inactive state.

Gα Subtypes and Functions

The Gα subunits are a family of 39–52 kDa proteins that share 45%–80% amino acid similarity. There are 15 Gα subunit genes in the human genome, with several genes expressing splice variants. The distinction of the different types of α-subunits found in heterotrimeric G-proteins is based upon the downstream signaling responses activated or inhibited as a result of G-protein activation. These classifications allow the Gα subunits to be divided into four subtypes include Gs (since it is the α-subunit these designations are also written Gαs), Gi, Gq, and G12.

The Gs family of G-proteins is comprised of Gαs and Gαolf. These α-subunits stimulate the activity of adenylate cyclase resulting in increased production of cAMP from ATP. Increased production of cAMP results in the activation of PKA. Gαolf is so-called due to the fact that it was originally identified as being involved in olfaction.

The Gi class is comprised of Gαi, Gαo, Gαz, Gαt, and Gαgust. These α-subunits either inhibit adenylate cyclase thereby, inhibiting the production of cAMP (Gαi, Gαo, Gαz) or activate phosphodiesterases leading to increased hydrolysis of cGMP (Gαt) or cAMP (Gαgust). The βγ subunits that are associated with Gαi and Gαo function to open K+ channels. The designation Gαt defines this α-subunit as being in transducin which is the G-protein activated by the visual receptor rhodopsin. The designation Gαgust defines this α-subunit as being in gustducin which is a G-protein involved in the gustatory system which is the sensory system for taste.

The Gq class (comprised of Gαq, Gα11, Gα14, Gα15, and Gα16) activates membrane-associated PLCβ resulting in increased production of the intracellular messengers IP3 and DAG. This class of G-protein is associated with adrenergic (specifically α1-adrenergic), muscarinic, serotonin, and histamine receptors.

The G12 family is comprised of Gα12 and Gα13. The Gα12 family of G-proteins is involved in the activation of the Rho family of monomeric G-protein.

Gβγ Subtypes and Functions

The primary function that was originally proposed for the βγ-subunits (Gβγ dimer) was solely that of an inactivator of Gα subunits. In this capacity it was proposed that the Gβγ dimer facilitated the re-association of the inactive heterotrimer with the receptor for subsequent rounds of signaling. In this capacity it was viewed that the Gβγ dimer was a negative regulator of Gα signaling. However, in 1987 it was shown that Gβγ dimers were able to activate a cardiac muscarinic-gated inwardly rectifying potassium channel normally activated by acetylcholine. Subsequent to this initial observation it was found that the Gβγ subunits could modulate many other effectors via direct interactions. Indeed, many of the effectors are those that are also regulated by Gα subunits such as phospholipase Cβ (PLCβ), several adenylate cyclase (AC) isoforms, phosphoinositide-3 kinases (PI3Ks), and voltage-gated calcium channels. In addition to these membrane-associated targets activated by Gβγ subunits, the dimers have also been shown to effect modulation of numerous other proteins located throughout the cell. These include proteins in the cytosol, nucleus, endosomes, mitochondria, ER, Golgi apparatus, and cytoskeleton. It is, however, not fully understood as yet, whether all of these intracellular events require an initial event triggered through a GPCR and/or whether Gα subunits are also involved in the overall Gβγ activation process.

A total of five Gβ (β1–5) and twelve Gγ genes (γ1–5, 7–13) are expressed in humans. The Gβ1-4 subunits share extensive amino acid sequence homology of between 79% and 90%. The Gβ5 subunit is only approximately 52% identical to the other four Gβ subunits. In addition, there are two Gβ3 isoforms (β3 and β3S) and two Gβ5 isoforms (β5 and β5L). It is likely that the Gβ subunit genes evolved from a common ancestor into two subfamilies with one consisting of the Gβ1-4 subtypes and another consisting of Gβ5 subtypes. The twelve known Gγ subunits are much more diverse, exhibiting amino acid similarities of between 26% and 76%. The Gγ subunit genes diverged from each other into five classes designated I through V. The class I group includes Gγ7 and Gγ12. Class II contains Gγ2, Gγ3, Gγ4, and Gγ8. Class III contains Gγ5 and Gγ10. Class IV contains Gγ1, Gγ9, and Gγ11. Class V contains Gγ13. Given the large diversity in resultant Gβγ dimer composition it is very likely that widely varied functional roles for the various dimers also exists.

Although the canonical adenylate cyclase (AC) enzyme has long been known to be activated by Gαs-type G-proteins and inhibited by the Gαi-type, there are several known isoforms of AC in human tissues (see section above). All of the AC isoforms are transmembrane proteins consisting of two sets of six transmembrane segments separated by cytoplasmic (C1 and C2) domains. Certain isoforms of AC are regulated by direct interaction with Gβγ subunits. The consequences of Gβγ binding is isoform specific with some forms being activated and others inhibited by the interaction with Gβγ dimers. In all of the AC isoforms activated by Gβγ dimers (AC2, AC4, and AC7), the site of interaction has been shown to contain a motif consisting of the amino acids: PFAHL. This motif is absent in the AC isoforms that are not activated by Gβγ dimers.

There are 13 phospholipase C (PLC) genes in the human genome (see below discussion) and the first subfamily demonstrated to be regulated by Gβγ was PLCβ. Indeed, each of the four PLCβ isoforms is regulated by both Gαq and Gβγ resulting in increased phospholipase activity. However, the regulation involves distinct binding sites on the PLCβ protein. The binding of Gαq occurs within a domain at the C-terminus whereas the Gβγ dimers bind to a domain at the N-terminus. In addition to the PLCβ isoforms, the Gβγ dimers regulate the activities of the PLC epsilon (PLCε) and PLC eta (PLCη) isoforms.

The cardiac muscarinic-gated inwardly rectifying potassium channel is comprised of a heterotetramer of Kir3.1 and Kir3.4 subunit proteins. These two proteins are members of the larger family of Kir3 channels that are comprised of four distinct subunits encoded on separate genes identified as Kir3.1 through Kir3.4. All Kir3 channels can be regulated by direct binding of Gβγ dimers.

Voltage-dependent Ca2+ channels (Cav) are responsible for calcium ion flux across plasma membranes. The main pore-forming protein of these channels, the α1 subunit, is classified into three groups: Cav1, Cav2, and Cav3. In addition to the pore-forming α-subunits, Cav channels contain a cytoplasmic Cavβ subunit and a membrane-associated α2δ subunit. The α1 subunits harbor the Gβγ binding sites and all three classes of channel are regulated by dimer binding.

Monomeric G-proteins

The RAS superfamily of monomeric G-proteins comprises well over 100 different proteins. This superfamily is divided into eight main families with each of these major families being comprised of several subfamilies. The eight major families are RAS (31 members), RHO (20 members), RAB (65 members), RAP (5 members), RAN, RHEB (2 members), RAD, RIT (2 members), and ARF (31 members). A recent addition to the RAS superfamily is the Miro family (for mitochondrial RHO) of monomeric G-proteins that is composed of two members (RHOT1 and RHOT2) involved in mitochondrial transport processes.

The RAS family is primarily responsible for regulation of events of cell proliferation. The RHO family is involved in the regulation of cell morphology through control of cytoskeletal dynamics. The RAB family is involved in membrane trafficking events. The RAP family is involved in control of cell adhesion. The RAN protein is involved in regulation of nuclear transport. The RHEB family get its name from the original member identified as RAS homolog expressed in brain. The RHEB proteins are involved in the regulation of mTOR (mammalian target of rapamycin, see the Insulin Function page for more information on the activities of mTOR). The ARF family is involved in intracellular vesicle transport.

back to the top

G-Protein Regulators

The activity state of G-proteins is regulated both by the rate of GTP exchange for GDP and by the rate at which the GTP is hydrolyzed to GDP. The former process is catalyzed by guanine nucleotide exchange factors (GEFs). With respect to GPCRs, the receptor itself acts as the GEF upon ligand binding and receptor activation. The activity of G-proteins with respect to GTP hydrolysis is regulated by a family of proteins termed GTPase activating proteins, GAPs. Both of these G-protein regulatory protein classes are termed regulators of G-protein signaling, RGS. Another related family of proteins are termed the RGS-like proteins.

The proto-oncogenic protein, Ras, is a G-protein involved in the genesis of numerous forms of cancer (when the protein sustains specific mutations). Of particular clinical significance is the fact that oncogenic activation of Ras occurs with higher frequency than any other gene in the development of colorectal cancers. Regulation of Ras GTPase activity is controlled by RasGAP. There are several other GAP proteins besides RasGAP that are important in signal transduction. There are two clinically important proteins of the GAP family of proteins. One is the gene product of the neurofibromatosis type-1 (NF1) susceptibility locus. The NF1 gene is a tumor suppressor gene and the protein encoded is called neurofibromin. The second is the protein encoded by the BCR locus (break point cluster region gene). The BCR locus is rearranged in the Philadelphia+ chromosome (Ph+) observed with high frequency in chronic myelogenous leukemias (CMLs) and acute lymphocytic leukemias (ALLs).

back to the top

G-Protein Coupled Receptors

There are several different classifications of receptors that couple signal transduction to G-proteins. These classes of receptor are termed G-protein coupled receptors, GPCRs. All GPCRs are composed of a similar structure that includes seven membrane-spanning helices connected by three intracellular loops and three extracellular loops with an extracellular amino terminus and an intracellular carboxy terminus. The GCPR superfamily of transmembrane proteins all belong to the type IV-B multipass transmembrane protein family. There are at least 1409 identified GPCR genes in the human genome, although not all are protein coding as several hundred are pseudogenes. In addition, many of the protein coding GPCR genes encode proteins for whom the ligand(s) is unknown and are, therefore, referred to as orphan GPCRs. The GPCR superfamily consists of seven defined families or classes. These seven families are the class A rhodopsin-like receptors, the class B secretin-like receptors, the class C metabotropic glutamate/pheromone receptors, the class F frizzled receptors, the taste receptors, the vomeronasal receptors, and the 7-transmembrane (7TM) orphan receptors. The last family, the 7TM orphan, receptors are a separate family that do not constitute members of the class A nor class C orphan receptors.

G-protein coupled receptor (GPCR) structure

Diagrammatic representation of a typical member of the serpentine class of G-protein coupled receptor. White, red, blue, and green spheres represent amino acids. Serpentine receptors are so-called because they pass through the plasma membrane seven times. Structural characteristics include the three extracellular loops (EL-1, EL-2, EL-3) and three intracellular loops (IL-1, IL-2, IL-3). Most GPCRs are modified by carbohydrate attachment to the extracellular portion of the protein. Shown is typical N-linked carbohydrate attachment. The different colored spheres are involved in ligand-binding and associated G-protein binding as indicated in the legend.

The vast majority of G-proteins to which GPCRs are coupled are members of the heterotrimeric G-protein family discussed in the previous section. All trimeric G-proteins, whether or not they are coupled to receptor-mediated signal transduction cascades, are composed of three subunits: α, β, and γ. The α-subunit is responsible for the activity of the G-protein and the βγ subunits are regulatory and are involved in binding GTP. All GPCRs act as guanine nucleotide exchange factors (GEFs) and when they are activated by ligand binding, they catalyze exchange of GDP tightly bound to the α-subunit of heterotrimeric G-proteins for GTP.

Class A GPCR Superfamily: The class A GPCR superfamily is referred to as the rhodopsin-like family. Class A contains the largest number of members compiled into at least 21 classes (families). Class A GPCRs include opsins, the vast majority of the odorant (olfactory) receptors, and receptors for monoamines, purines, opioids, chemokines, some small peptide hormones, and the large glycoprotein hormones that consist of thyroid stimulating hormone (TSH), luteinizing hormone (LH), and follicle-stimulating hormone (FSH).

Class A Superfamily Family Members/Genes Comments
Amine receptors six subfamilies of receptors that together includes 45 genes (4 of which are pseudogenes) the six subfamilies are the 5-hydroxytryptamine (5-HT, serotonin) receptors (13 genes), the dopamine receptors (5 genes), the histamine receptors (4 genes), the muscarinic acetylcholine (mAChR) receptors (5 genes), the adreno receptors (9 genes), and the trace amine receptors (9 genes, 4 of which are pseudogenes)
Chemokine receptors five subfamilies of receptors CXC motif receptors: six family member genes CXCR1–CXCR6
CC motif receptors: ten family member genes CCR1–CCR10
CX3C motif receptor: CX3CR1
XC motif receptor: XCR1
Atypical chemokine receptors: ACKR1, ACKR2, ACKR3, ACKR4, CCRL2, PITPNM3
Olfactory receptors 18 families in this superfamily of receptors many of which have numerous subfamlilies; collectively there are 861 identified genes in this superfamily, many are suspected to be pseudogens or are in fact pseudogenes the superfamily includes subfamilies 1–14, 51, 52, 55, and 56
Complement component GPCRs C3AR1, C5AR1, C5AR2  
Formyl peptide recptors FPR1, FPR2, FPR3 chemotaxis receptors that bind N-formylated peptides derived from the degradation of bacteria
Glycoprotein hormone receptors FSHR, LHCGR, TSHR see the Peptide Hormones page for more detailed information on the glycoprotein hormones and their receptors
Hydroxycarboxylic acid receptors HCAR1, HCAR2, HCAR3 HCAR1 encoded proteins is HCA1 and is also known as GPR81; HCAR2 encoded proteins is HCA2 and also is known as GPR109A; HCAR3 encoded proteins is HCA3 and is also known as GPR109B; details on the hydroxycarboxylic acid receptors are in the Bioactive Lipids page
Lipid-like receptors seven subfamilies of receptors cannabinoid receptors: CNR1 gene encodes the CB1 protein, CNR2 encodes the CB2 protein
Free fatty acid receptors: FFAR1 (GPR40), FFAR2 (GPR43), FFAR3 (GPR41), FFAR4 (GPR120)
Leukotriene receptors: CYSLTR1, CYSLTR2, FPR2, LTB4R, LTB4R2, OXER1
Prostaglandin receptors: PTGDR, PTGDR2, PTGER1, PTGER2, PTGER3, PTGER4, PTGFR, PTGIR, TBXA2R
Sphingosine-1-phosphate (S1P) receptors: S1PR1–S1PR5
Lysophosphatidic acid (LPA) receptors: LPAR1–LPAR6
Platelet activating factor (PAF) receptor: PTAFR
Melatonin receptors MTNR1A, MTNR1B melatonin is derived from the amino acid tryptophan and is synthesis and function are discusses in the Amino Acid Derivatives page
Nucletide-like receptors adenosine receptors: A1 (ADORA1), A2A (ADORA2A), A2B (ADORA2B), A3 (ADORA3)

P2Y receptors: P2RY1, P2RY2, P2RY4, P2RY6, P2RY8, P2RY10–P2RY14
adenosine acts as an inhibitory neurotransmitter in the CNS, exerts effects in the periphery on the heart and the lungs, and also exerts anti-inflammatory effect; A1 and A3 are coupled to Gi-type G-proteins; A2A is coupled to a Gs-type G-protein; A2B is coupled to both Gs- and Gq-type G-proteins; activation of A1 results in bronchoconstriction and decreased heart rate; activation of A2A results in reduced neural excitation in the CNS, decreased dopaminergic stimulation, and coronary artery vasodilation; activation of A2B results in bronchoconstriction; activation of A3 relaxes cardiac muscle, casues smooth muscle contraction, and inhibits neutrophil degranulation

the P2Y receptors are discussed above
Opsin receptors RHO, OPN1LW, OPN1MW, OPN1MW2, OPN1MW3, OPN1SW, OPN3, OPN4, OPN5, RGR, RRH all opsins are light-sensitive GPCRs with rhodopsin (encocoded by the RHO gene) being the light receptor in rod cells in the retina; the opsins (OPN genes) are primarily localized to the cone cells in the retina
Prokineticin receptors PROKR1, PROKR2 prokineticins are gastrointestinal (GI) hormones that act to induce smooth muscle contraction in the GI; these hormones are also involved in nociception (sensation of pain), angiogenesis, and neurogenesis
Relaxin family peptide receptors RXFP1, RXFP2, RXFP3, RXFP4 the relaxins are a family of peptide hormones that belong to the insulin superfamily of hormones; these hormones are structurally most closely related to the insulin-like (INSL) peptides
Chemerin receptor CMKLR1 chemerin is an immune system modulating cytokine produced and secreted by adipose tissue; regulates adipocyte development and exerts effects on glucose metabolism in bith the liver and skeletal muscles
F2R receptors F2R, F2RL1, F2RL2, F2RL3 these genes encode the protease-activated receptor (PAR) family proteins; F2R encoded protein is PAR1 which is a receptor for the coagulation factor thrombin; F2RL1 encodes PAR2, F2RL2 encodes PAR3, F2RL3 encodes PAR4
Peptide receptors 16 subfamilies of receptors this superfamily represents the receptors for the opioids, angiotensin, bradykinin, cholecystokinin, somatostatin, oxytocin and arginine vasopressin, melanocortins, melanin-concentrating hormone, endothelins, galanin, gonadotropin releasing hormone, hypocretins, tachykinins, neurotensins, neuromedin U, and the neuropeptides
the neuropeptides include neuropeptide Y (NPY), neuropeptide B (NPB), neuropeptide W (NPW), and neuropeptide FF (NPFF)
G-protein coupled bile acid receptor GPBAR1 originallly identifed as TGR5 and is also known as GPR131; bile acids also bind and activate the farnesoid X receptors (FXR) which belong to the nuclear receptor superfamily
G-protein coupled estrogen receptor GPER1 estrogens predominantly bind to receptors that are members of the nuclear receptor superfamily; this receptor binds the estrogen 17-β-estradiol (E2); activation of GPER1 results in modulation of cardiovascular, endocrine, reproductive, immune and central nervous system functions
Oxoglutarate receptor OXGR1 is also knonwn as the cyteinyl leukotriene receptor E (CysLTE); originally identifed as GPR99; the receptor is also activated by leukotriene E4 (LTE4); receptor is closely related to the P2Y family of purinergic receptors; the drug montelucast, which is used to treat asthma, allergic rhinitis, and urticaria (hives), was originally targeting for inhibition of the cysteinyl leukotriene receptor 1 (CysLT1) has been shown to inhibit the activation of OXGR1 as well,
Succinate receptor SUCNR1 was originally identifed as GPR91; related the the P2Y family of purinergic receptors; is expressed in various blood cells, in adipose tissue, liver, retina, and kidney; mediates cellular responses to ischemia, hypoxia, toxicity, and hyperglycemia
Class A orphan GPCRs large subfamily composed of 78 genes subfamily includes MAS1 protooncogene which is a Gq-coupled receptor for angiotensin(1-7); also includes nine MAS related proteins and three leucine-rich repeat containing GPCRs

back to the top

Class B GPCR Superfamily: The class B GPCR superfamily is referred to as the secretin-like receptor class. Class B is comprised of 6 classes (families) the include the receptors for calcitonin, parathyroid hormone, corticotropin releasing hormone, glucagon, and vasoactive intestinal peptide in addition to the large adhesion receptors family. The adhesion receptor family contains nine subfamilies identified as A, B, C, D, E, F, G, L, and V.

Class B Superfamily Family Members/Genes Comments
Adhesion receptors 9 adhesion receptor subfamilies adhesion receptor subfamily A: ADGRA1, ADGRA2, ADGRA3
adhesion receptor subfamily B: ADGRB1, ADGRB2, ADGRB3
adhesion receptor subfamily C (cadherin receptors): CELSR1, CELSR2, CELSR3
adhesion receptor subfamily D: ADGRD1, ADGRD2
adhesion receptor subfamily E: ADGRE1, ADGRE2, ADGRE3, ADGRE5
adhesion receptor subfamily F: ADGRF1–ADGRF5
adhesion receptor subfamily G: ADGRG1–ADGRG7
adhesion receptor subfamily L: ADGRL1–ADGRL4
adhesion receptor subfamily H: ADGRV1
Calcitonin receptors CALCR, CALCRL see the Peptide Hormones page
Corticotropin releasing hormone (CRH) receptors CRHR1, CRHR2 see the Peptide Hormones page
Glucagon receptors GCGR, GHRHR, GIPR, GLP1R, GLP2R, SCTR GCGR encodes the glucagon receptor
GHRHR encodes the growth hormone releasing hormone (GHRH) receptor
GIPR encodes the glucose-dependent insulinotropic peptide (GIP) receptor; GIP also called gastric inhibitory peptide
GLP1R encodes the glucagon-like peptide 1 (GLP-1) receptor
GLP2R encodes the glucagon-like peptide 2 (GLP-2) receptor
SCTR encodes the secretin receptor
Parathyroid hormone receptors PTH1R, PTH2R see the Peptide Hormones page
Vasoactive intestinal peptide (VIP) receptors VIPR1, VIPR2, ADCYAP1R1 VIPR1 and VIPR2 encode the two receptors for vasoactive intestinal peptide (VIP); VIPR2 is 100% identical to VIPR1 except that it has a novel 67 amino acid N-terminus; VIP is produced in the gut and in the hypothalamus; VIP functions to induce smooth muscle relaxation leading to reductions in blood pressure, also stimulates cardiac contractility; within the gut VIP inhibits parietal cell acid production and chief cell release pf pepsinogen
ADCYAP1R1 encodes the type 1 (pituitary) adenylate cyclase activating polypeptide (PACAP) receptor; PACAP is encoded by the ADCYAP1 gene; PACAP can also bind the the VIPR1 and VIPR2 encoded receptors; PACAP is a hormone that induces activity in the pituitary and also functions as a neurotransmitter

back to the top

Class C GPCR Superfamily: The class C superfamily is referred to as the metabotropic glutamate/pheromone receptor family. Class C is comprised of 4 classes (families) that include the metabotropic glutamate receptors (mGluR), extracellular Ca2+-sensing receptors, the GABA-B type receptors, as well as the class C orphan receptors.

Class C Superfamily Family Members/Genes Comments
Calcium sensing receptors CASR, GPRC6A the CASR encoded protein (identified as CaSR) is expressed in the parathyroid glands, renal tubule cells, bone marrow, thyroid gland C-cells, gastrin-secreting cells in the stomach, several areas of the brain, as well as in several other tissues; CaSR was the first receptor shown to be activated by an ion ligand
the GPRC6A gene encodes a receptor for amino acids (preferably basic amino acids) as well as extracellular Ca2+ and the osteocalcin; osteocalcin is produced exclusively by osteoblasts and its function is regulated by vitamin K-dependent carboxylation
Gamma aminobutyrate type B (GABA-B) receptors GABBR1, GABBR2 the GABA-B receptors are metabotropic GABA receptors that are coupled to the activation of K+ channels; for more details see the Biochemistry of Nerve Transmission page
Metabotropic glutamate receptors GRM1–GRM8 see the Biochemistry of Nerve Transmission page
Class C orphan GPCRs GPRC5A–GPRC5D, GPR156, GPR158, GPR179  

Other Members of the GPCR Superfamily

The Frizzled receptor subfamily of GPCRs is composed of 11 proteins encoded by the smoothened gene (SMO) and the FZD1–FZD10 genes. The taste (gustatory) receptor family is divided into the taste 1 receptor and taste 2 receptor subfamilies. There are three taste 1 receptor family members encoded by the TAS1R1–TAS1R3 genes. There are 39 genes of the taste 2 receptor family, 29 of which encode functional proteins, 10 of which encode pseudogenes. The vomeronasal (pheromone) receptor family is composed of 129 genes with only three (VN1R1, VN1R2, and VN1R4) encoding functional proteins. There are also seven genes in the 7TM orphan receptor family (orphan receptors whose structures do not place them in the class A nor class C orphan receptor family).

back to the top

GPCR Desensitization

A characteristic feature of GPCR activity following ligand binding is a progressive loss of receptor-mediated signal transduction. This process is referred to as desensitization or adaptation. The events that reflect desensitization of a G-protein coupled signaling system can involve the receptor itself, the G-protein associated with the receptor, and/or the downstream effector(s). In the majority of cases it is impairment of the receptor’s ability to activate its G-protein that accounts for most desensitization, especially within minutes of agonist stimulation. Within milliseconds to minutes of ligand binding, cells can diminish or virtually eliminate the receptor-mediated responses. This process involves phosphorylation of the GPCRs on one or more intracellular domains. On a longer time scale (several hours after ligand binding) the short-term desensitization is augmented by receptor down-regulation which involves the loss of membrane-associated receptor through a combination of protein degradation, transcriptional, and posttranscriptional mechanisms.

Heterologous desensitization involves phosphorylation of GPCRs by second-messenger-dependent kinases, such as PKA and PKC. Receptor phosphorylation by these kinases, as an isolated event, substantially impairs the ability of GPCRs to stimulate their G-proteins. Homologous desensitization of GPCRs involves a family of kinases termed G-protein coupled receptor kinases (GRKs). The GRKs constitute a family of six mammalian serine/threonine kinases that phosphorylate ligand-activated GPCRs as their primary substrates, hence the designation of the process as homologous desensitization. These six kinases are identified as GRK1 (originally called rhodopsin kinase); GRK2 (originally called β-adrenergic receptor kinase-1, βARK1); GRK3 (originally called β-adrenergic receptor kinase-2, βARK2); GRK4 (originally called IT-11); GRK5; and GRK6. Expression of GRK1 is almost exclusive to the retina and GRK4 expression is observed at significant levels only in testes. The remaining GRKs are found ubiquitously expressed. These kinases preferentially phosphorylate ligand (agonist) bound and activated receptor rather than inactive or antagonist-occupied GPCR substrates. Interaction of GRKs with their activated receptor substrates potently activates these enzymes. GRK-mediated GPCR phosphorylation requires the participation of regulatory mechanisms responsible for the membrane localization and receptor targeting of these enzymes.

The discovery of GRKs was the result of experiments designed to understand the mechanisms responsible for short-term, homologous desensitization of the β2-adrenergic receptor (β2AR) and rhodopsin. With respect to the β2AR, agonist-induced receptor phosphorylation associated with homologous desensitization was found to occur even in cells genetically lacking PKA. The enzyme responsible for this activity was subsequently purified and named β-adrenergic receptor kinase (now GRK1). Rhodopsin kinase (GRK1) was identified as the enzyme responsible for phosphorylating light bleached (agonist-activated) rhodopsin in rod outer segments. Subsequently, GRK1-mediated phosphorylation of rhodopsin was associated with desensitization of the rhodopsin/GT/cGMP phosphodiesterase system. The GRK family of serine/threonine kinases shares the unusual feature of phosphorylating specifically the agonist-occupied, or activated, conformation of GPCRs.

The model proposed to describe the mode of action of GRKs suggests that a receptor phosphorylated by a GRK can subsequently bind stoichiometrically to one of a family of cytoplasmic inhibitory proteins that have been termed the arrestins. In the rhodopsin system this inhibitory protein is referred to as arrestin. In non-retinal tissues there are two related inhibitory proteins known as β-arrestin-1 and β-arrestin-2. As a result of arrestin or β-arrestin binding, the GPCR is prevented from activating its G-protein and, therefore, its downstream effector(s). This two-step process of GRK-initiated desensitization can reduce by as much as 70%–80% the ability of fully activated β2ARs or rhodopsin to activate their respective G-proteins. Furthermore, the binding of β-arrestins to GRK-phosphorylated GPCRs is believed to initiate GPCR endocytosis, or sequestration, into recycling endosomes. Within the endosome a GRK-phosphorylated GPCR can be dephosphorylated by a membrane associated phosphatase. This latter process allows for resensitization of the GPCR prior to it being recycled back to the plasma membrane where it can once again respond to ligand binding.

back to the top

Diseases/Disorders Associated with GPCR Defects

Disease Affected Receptor Comments
Blomstrand chondrodysplasia parathyroid hormone receptor 1, PTHR1 manifests with remarkably advanced skeletal maturation at birth, loss-of-function mutation, autosomal recessive inheritance
central hypogonadism gonadotropin releasing hormone receptor, GNRHR impairment of pubertal maturation and reproductive function; loss-of-function mutation, autosomal recessive inheritance
central hypothyroidism thyrotropin releasing hormone receptor, TRHR characterized by insufficient TSH secretion resulting in low levels of thyroid hormones; loss-of-function mutation, autosomal recessive inheritance
color blindness cone opsins loss-of-function mutation, autosomal recessive inheritance, X-linked
congenital hypothyroidism thyroid stimulating hormone receptor, TSHR increased levels of plasma TSH and low levels of thyroid hormone; loss-of-function mutation, autosomal recessive inheritance
congenital night blindness rhodopsin congenital stationary night blindness (CSNB), loss-of-function mutation, autosomal dominant inheritance; numerous additional forms of CSNB are known and are characterized by impaired night vision, decreased visual acuity, nystagmus, myopia, and strabismus
familial ACTH resistance adrenocorticotropic hormone
loss-of-function mutation, autosomal recessive inheritance
familial hypocalcemia Ca2+ sensing receptor; CASR characterized by hypocalcemia and hyperphosphatemia; can manifest with mild neuromuscular irritability, calcification of the basal ganglia, extrapyramidal disorders, cataracts, alopecia, abnormal dentition, coarse brittle hair, mental retardation, or personality disorders; gain-of-function mutation, autosomal dominant inheritance
familial hypocalciuric hypercalcemia Ca2+ sensing receptor; CASR hypercalcemic onset before age 10 years (unlkie primary hyperparathyroidism), not accompanied by urinary stone or renal damage, pancreatitis and chondrocalcinosis are common, parathyroid hyperplasia is present in most patients and will persist after parathyroidectomy, loss-of-function mutation, autosomal dominant inheritance; the gain-of-function mutations in the CASR gene cause familial hypocalcemia
familial male precocious puberty luteinizing hormone receptor, LHR gain-of-function mutation, autosomal dominant inheritance
familial non-autoimmune hyperthyroidism thyroid stimulating hormone receptor, TSHR gain-of-function mutation, autosomal dominant inheritance
growth hormone deficiency growth hormone releasing hormone receptor, GHRH loss-of-function mutation, codominant inheritance
Hirschsprung disease susceptibility type 2 endothelin receptor type B classic Hirschsprung disease (type 1) is caused by defects in the RET gene which encodes a receptor tyrosine kinase receptor; type 2 Hirschsprung is also known as Waardenburg syndrome type 4A; loss-of-function mutation, complex mode of inheritance
ovarian dysgenesis 1 (ODG1), also called hypergonadotropic ovarian failure follicle stimulating hormone receptor, FSHR lack of menstruation accompanied by severe osteoporosis, gonadal dysgenesis, often with somatic abnormalities; loss-of-function mutation, autosomal recessive inheritance
Jansen metaphyseal chondrodysplasia parathyroid hormone hormone receptor 1, PTHR1 also known as metaphyseal dystosis, presents with extreme disorganization of the metaphyses of the long bones and of the metacarpal and metatarsal bones; gain-of-function mutation, autosomal dominant inheritance
male pseudohermaphroditism luteinizing hormone/choriogonadotropin receptor, LHCGR loss-of-function mutation, autosomal recessive inheritance
morbid obesity melanocortin 4 receptor, MC4R mutations in this gene are the most frequent genetic cause of severe obesity; MC4R binds α-melanocyte stimulating hormone (α-MSH); loss-of-function mutation, codominant inheritance
neonatal hyperparathyroidism Ca2+ sensing receptor, CASR manifests in the first 6 months of life with severe hypercalcemia, bone demineralization, and failure to thrive; loss-of-function mutation, autosomal recessive inheritance
nephrogenic diabetes insipidus vasopressin V2 receptor, AVPR2 symptoms include vomiting and anorexia, failure to thrive, fever, and constipation, caused by the inability of the renal collecting ducts to absorb water in response to antidiuretic hormone (ADH) which is also known as arginine vasopression (AVP); loss-of-function mutation, X-linked inheritance
retinitis pigmentosa rhodopsin loss-of-function mutation, autosomal dominant and recessive modes of inheritance
sporadic hyperfunctional thyroid adenomas thyroid stimulating hormone receptor, TSHR gain-of-function mutation, somatic inheritance
sporadic Leydig cell tumors luteinizing hormone/choriogonadotropin receptor, LHCGR gain-of-function mutation, somatic inheritance

back to the top

Intracellular Hormone Receptors

The steroid/thyroid hormone receptor superfamily [e.g. glucocorticoid (GR), vitamin D (VDR), retinoic acid (RAR) and thyroid hormone TR) receptors] is a class of proteins that reside in the cytoplasm, or the nucleus, and bind their lipophilic hormone ligands in these locations since the hormones are capable of freely penetrating the hydrophobic plasma membrane. Because these receptors bind ligand intracellularly and then interact with DNA directly they are more commonly called the nuclear receptors (NR). In addition to binding hormone, all receptors of this class are capable of directly activating gene transcription. Upon binding ligand the cytoplasmic hormone-receptor complex translocates to the nucleus and binds to specific DNA sequences termed hormone response elements (HREs). The binding of the complex to an HRE results in altered transcription rates of the associated gene. An important exception are the thyroid hormone receptors (TR) and retinoic acid receptors (RAR) which are constitutively present in the nucleus bound to their target genes in the absence of their cognate hormones. These two receptor families exhibit potent transcriptional repression function in the absence of hormones and the repressor function is mapped to the domain that is responsible for binding ligand.

Analysis of the human genome has revealed 48 nuclear receptor genes that can be classified into seven defined subfamilies. Many of these genes are capable of yielding more than one receptor isoform. The nuclear receptors all contain a ligand-binding domain (LBD), a DNA-binding domain (DBD) and, in most cases, two activation function domains (identified as AF-1 and AF-2). The activity of the AF-1 domain is independent of the presence of ligand bound to the LBD, whereas the activity of the AF-2 domain is dependent upon ligand being bound to the LBD. Based upon the sequences of these two domains the nuclear receptor family is divided into six sub-families. Some members of the family bind to DNA as homodimers such as is the case for subfamily III receptors which comprises the steroid receptors such as the estrogen receptor (ER), mineralocorticoid receptor (MR), progesterone receptor (PR), androgen receptor (AR), and the glucocorticoid receptor (GR). Other family members (such as all subfamily I members) bind to DNA as heterodimers through interactions with the retinoid X receptors (RXRs, see below). In addition to the steroid hormone and thyroid hormone receptors there are numerous additional family members that bind lipophilic ligands. These include the retinoid X receptors (RXRs), the liver X receptors (LXRs), the farnesoid X receptors (FXRs) and the peroxisome proliferator-activated receptors (PPARs).

Nuclear Receptor Families

Receptor Nomenclature Receptor Common Name Human Gene Name
Type 1A: Thyroid Hormone Receptors
NR1A1 thyroid hormone receptor-α THRA
NR1A2 thyroid hormone receptor-β THRB
Type 1B: Retinoic Acid Receptors (RAR)
NR1B1 retinoic acid receptor-α (RARα) RARA
NR1B2 retinoic acid receptor-β (RARβ) RARB
NR1B3 retinoic acid receptor-γ (RARγ) RARG
Type 1C: Peroxisome Proliferator-Activated Receptors (PPAR)
NR1C1 peroxisome proliferator-activated receptor-α (PPARα) PPARA
NR1C2 peroxisome proliferator-activated receptor-β/δ (PPARβ/δ) PPARD
NR1C3 peroxisome proliferator-activated receptor-γ (PPARγ) PPARG
Type 1D: Reverse ERBA Receptors
NR1D1 Thyroid hormone receptor, α-1-like (Rev-erbα) THRAL
NR1D2 Rev-erbα-related receptor (Rev-erbγ) RVR
Type 1F: RAR-Related Orphan Receptors
NR1F1 RAR-related orphan receptor-α RORA
NR1F2 RAR-related orphan receptor-β RORB
NR1F3 RAR-related orphan receptor-γ RORC
Type 1H: Liver X Receptor-Like Receptors
NR1H2 liver X receptor-β (LXRβ) NR1H2
NR1H3 liver X receptor-α (LXRα) NR1H3
NR1H4 farnesoid X receptor (FXR) NR1H4
NR1H5 farnesoid X receptor-β (FXRβ) NR1H5P
Type 1I: Vitamin D Receptor-Like Receptors
NR1I1 vitamin D receptor (VDR) VDR
NR1I2 pregnane X receptor (PXR) NR1I2
NR1I3 constitutive androstane receptor (CAR) NR1I3
Type 2A: Hepatocyte Nuclear Factor-4 (HNF4) Receptors
NR2A1 hepatocyte nuclear factor-4-α (HNF-4α) HNF4A
NR2A2 hepatocyte nuclear factor-4-γ (HNF-4γ) HNF4G
Type 2B: Retinoid X Receptors (RXR)
NR2B1 retinoid X receptor-α (RXRα) RXRA
NR2B2 retinoid X receptor-β (RXRβ) RXRB
NR2B3 retinoid X receptor-γ (RXRγ) RXRG
Type 2C: Testis Receptors
NR2C1 testes receptor 2 TR2
NR2C2 testicular nuclear receptor 4 TR4, TAK1
Type 2E: Orphan Ligand Receptors
NR2E1 homolog of Drosophila tailless TLX
NR2E3 photoreceptor-specific nuclear receptor PNR
Type 2F: Chicken Ovalbumin Upstream Promoter Transcription Factor-Related Receptors
NR2F1 chicken ovalbumin upstream promoter transcription factor 1 TFCOUPI
NR2F2 chicken ovalbumin upstream promoter transcription factor 2 TFCOUPII
NR2F6 ERBA-related 2 EAR2
Type 3A: Estrogen Receptors
NR3A1 estrogen receptor-α ESR1
NR3A2 estrogen receptor-β ESR2
Type 3B: Estrogen Receptor-Related Receptors
NR3B1 estrogen receptor-relatedα ESRRA
NR3B2 estrogen receptor-relatedβ ESRRB
NR3B3 estrogen receptor-relatedγ ESRG
Type 3C: Steroid Receptors
NR3C1 glucocorticoid receptor GCCR
NR3C2 mineralocorticoid receptor MR
NR3C3 progesterone receptor PGR
NR3C4 androgen receptor AR
Type 4A: Orphan Ligand Receptors
NR4A1 nerve growth factor (NGF)-induced factor B NGFI-B
NR4A2 nuclear receptor-related 1 NURR1
NR4A3 neuron-derived orphan receptor 1 NOR1
Type 5A: Orphan Ligand Receptors
NR5A1 steroidogenic factor 1 SF1
NR4A2 liver receptor homolog 1 LRH-1
Type 6A: Orphan Ligand Receptors
NR6A1 germ cell nuclear factor GCNF
Type 0B: DAX-Like Receptors
NR0B2 small heterodimer partner, SHP NR0B2

Although all members of the NR family possess activation function domains that are responsible for the regulation of transcription of target genes, the regulation of transcription is much more complex due to the association of numerous coregulatory proteins. These coregulatory proteins are of two distinct classes: those that function to co-activate the NR and those that function to co-repress the receptor complex. Co-activators are found to be associated with ligand-bound NR and thereby induce gene expression. Co-repressors selectively repress gene expression through interaction with NR that are ligand free or bound to antagonists. In addition, coregulators can be classified into two main groups: one that modifies histones (e.g, by acetylation/deacetylation or methylation/demethylation) and the other that includes ATP-dependent chromatin remodeling factors. these remodeling factors modulate promoter accessibility to other transcription factors as well as to the basal transcriptional machinery. The properties of several members of each class of coregulator are discussed below.

RXRs: The RXRs represent a class of receptors that bind the retinoid 9-cis-retinoic acid. There are three isotypes of the RXRs: RXRα, RXRβ, and RXRγ and each isotype is composed of several isoforms. The RXRs serve as obligatory heterodimeric partners for numerous members of the nuclear receptor family including those discussed below (PPARs, LXRs, and FXRs). In the absence of a heterodimeric binding partner the RXRs are bound to hormone response elements (HREs) in DNA and are complexed with co-repressor proteins (see below for details) that include histone deacetylases (HDACs) and nuclear receptor corepressor 1 (NCoR1), or silencing mediator of retinoid and thyroid hormone receptor (SMRT; also called NCoR2).

RXRα is widely expressed with highest levels liver, kidney, spleen, placenta, and skin. The critical role for RXRα in development is demonstrated by the fact that null mice are embryonic lethals. RXRβ is important for spermatogenesis and RXRγ has a restricted expression in the brain and muscle.

PPARs: The PPAR family is composed of three family members: PPARα, PPARβ/δ, and PPARγ. Each of these receptors forms a heterodimer with the RXRs. For more detailed information on the PPARs visit the PPAR page.

The first family member identified was PPARα and it was found by virtue of it binding to the fibrate class of anti-hyperlipidemic drugs and resulting in the proliferation of peroxisomes in hepatocytes, hence the derivation of the name of the protein. Although PPARγ and PPARδ are related to PPARα they do not stimulate peroxisome proliferation. Subsequently it was shown that PPARα is the endogenous receptor for polyunsaturated fatty acids. PPARα is highly expressed in the liver, skeletal muscle, heart, and kidney. Its function in the liver is to induce hepatic peroxisomal fatty acid oxidation during periods of fasting. Expression of PPARα is also seen in macrophage foam cells and vascular endothelium. Its role in these cells is thought to be the activation of anti-inflammatory and anti-atherogenic effects.

PPARγ is a master regulator of adipogenesis and is most abundantly expressed in adipose tissue. Low levels of expression are also observed in liver and skeletal muscle. PPARγ was identified as the target of the thiazolidinedione (TZD) class of insulin-sensitizing drugs. The mechanism of action of the TZDs is a function of the activation of PPARγ activity and the consequent activation of adipocytes leading to increased fat storage and secretion of insulin-sensitizing adipocytokines such as adiponectin.

PPARδ is expressed in most tissues and is involved in the promotion of mitochondrial fatty acid oxidation, energy consumption, and thermogenesis. PPARδ serves as the receptor for polyunsaturated fatty acids and VLDLs. Current pharmacologic targeting of PPARδ is aimed at increasing HDL levels in humans since experiments in animals have shown that increased PPARδ levels result in increased HDL and reduced levels of serum triglycerides.

LXRs: There are two forms of the LXRs: LXRα and LXRβ. The LXRs form heterodimers with the RXRs and as such can regulate gene expression either upon binding oxysterols (e.g. 22R-hydroxycholesterol) or 9-cis-retinoic acid. Because the LXRs bind oxysterols they are important in the regulation of whole body cholesterol levels. The function of LXRs in the liver is to mediate cholesterol metabolism by inducing the expression of SREBP-1c. SREBP-1c is a transcription factor involved in the control of the expression of numerous genes including several involved in cholesterol synthesis. For more detailed information on the LXRs visit the LXR page.

FXRs: There are two genes encoding FXRs identified as FXRα and FXRβ. In humans at least four FXR isoforms have been identified as being derived from the FXRα gene as a result of activation from different promoters and the use of alternative splicing; FXRα1, FXRα2, FXRα3, and FXRα4. The FXR gene is also known as the NR1H4 gene (for nuclear receptor subfamily 1, group H, member 4). The FXR genes are expressed at highest levels in the intestine and liver. FXR forms a heterodimer with members of the RXR family. Following heterodimer formation the complex binds to specific sequences in target genes resulting in regulated expression. One major target of FXR is the small heterodimer partner (SHP) gene. Activation of SHP expression by FXR results in inhibition of transcription of SHP target genes. Of significance to bile acid synthesis, SHP represses the expression of the cholesterol 7-hydroxylase gene (CYP7A1). CYP7A1 is the rate-limiting enzyme in the synthesis of bile acids from cholesterol. The FXRs were originally identified by their ability to bind farensol metabolites. However, subsequent research has demonstrated that FXRs are receptors for bile acids which is the primary mechanism by which bile acids negatively regulate their own expression. In addition to binding bile acids, FXRs have been shown to bind polyunsaturated fatty acids (PUFAs) such as the omega-3 PUFAs docashexaenoic acid (DHA) and α-linolenic acid (ALA). Most recently, FXR has been shown to bind the androgen hormone, androsterone, derived via testosterone metabolism. For more detailed information on the FXRs visit the FXR page.

PXR: A particular receptor of this family that has been shown to bind numerous structurally unrelated chemicals was originally identified as the pregnane X receptor (PXR). PXR is highly expressed in the liver and is involved in mediating drug-induced multi-drug clearance. For this reason PXR is  important in protecting the body from harmful metabolites. An additional physiologically significant function of PXR is in the regulation of bile acid synthesis. PXR is a recognized receptor for lithocholic acid and other bile acid precursors. PXR activation leads to repression of bile acid synthesis due to its physical association with hepatocyte nuclear factor 4α (HNF-4α) causing this transcription factor to no longer be able to associate with the transcriptional co-activator PGC-1α (PPARγ co-activator 1α) which ultimately leads to loss of transcription factor activation of the rate-limiting enzyme of bile acid synthesis CYP7A1 which, as described above, is also the target of FXR action. In addition to regulation of bile acid metabolism, PXR represses the expression of the gluconeogenic enzyme PEPCK.

In addition to the nuclear receptors discussed here additional members are being identified all the time such at the estrogen related receptors (ERRβ and ERRγ), the retinoid-related orphan receptor (RORα), and the constitutive androstane receptor (CAR).

Nuclear Receptor Coactivators

The first nuclear receptor coactivator to be identified was steroid receptor coactivator-1 (SRC-1). To date, more than 400 coregulators (both coactivators and corepressors) have been identified. There are now known to exist three SRC gene families. SRC-1 (encoded by the NCOA1 gene), SRC-2 (also known as GRIP1 for glucocorticoid receptor-interacting protein 1 and TIF2 for transcriptional intermediary factor 2) encoded by the NCOA2 gene, and SRC-3 (also known as AIB1 for amplified in breast cancer 1 and TRAM-1 for thyroid hormone receptor activator molecule 1) encoded by the NCOA3 gene. The three members of the SRC family contain homologous domains and share between 50% and 54% amino acid sequence similarity. There is also a diverse family of enzymes that interact with and modify SRCs which includes histone acetyltransferases (HATs), histone methyltransferases (HMTs), kinases, phosphatases, ubiquitin ligases, and small ubiquitin-related modifier (SUMO) ligases.

Peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (PGC-1α) is another critical NR coregulator. PGC-1α has been shown be involved in the regulation of metabolism and energy homeostasis. Indeed, expression levels of PGC-1α have been associated with genetic diseases associated with impaired mitochondrial function, including type 2 diabetes and obesity. Another important coactivator is cAMP response-element binding protein (CREB)-binding protein (CBP), also identified as CBP-p300. CBP-p300 possesses intrinsic histone acetyltransferase (HAT) activity that leads to relaxation of the chromatin structure near a NR target gene. Other chromatin remodeling complexes, such as coactivator-associated arginine methyltransferase 1 (CARM1), can also stimulate gene transcription by NRs as well as other transcription factors in combination with the SRC family of coactivators.

In addition to acting a coactivators for NRs, the SRC family proteins also interact with many different types of transcription factors and potentiate their transcriptional activity. These include p53, signal transducers and activators of transcription (STATs), nuclear factor-κB (NF-κB), hypoxia-inducible factor 1 (HIF1), and hepatocyte nuclear factor-4 (HNF4) to name just a few. Several extracellular stimuli, such as growth factors and cytokines, that activate membrane-spanning signal transducing receptors, generating phosphorylation codes on SRCs that lead to increased coactivator affinity for the androgen receptor (AR), estrogen receptor-alpha (ERα), and progesterone receptor (PR).

model of nuclear receptor (NR) coactivator complex assembly at a target gene

Model for NR interactions with coactivators: An example of the transcription factor complexes associated with both the RXR and PPAR heterodimeric transcription factor complex at an HRE, and several basal transcription factors associated with RNA pol II at a target gene transcriptional start site. Binding of ligand to a particular PPAR results in assembly of the complete coregulatory (in this case coactivator) complex. Formation of the complex induces histone modifications (such as acetylation, Ac; and methylation, Me) that in turn alter chromatin structure allowing entry of the basal transcriptional machinery including RNA pol II. The complete assembly then leads to activation of target gene transcription.

Nuclear Receptor Corepressors

As a general rule it has been established that when nuclear receptors are free of activating ligand they preferentially interact with corepressor complexes to mediate transcriptional repression. Nuclear receptor corepressor 1 (NCoR1) and silencing mediator of retinoic and thyroid receptors (SMRT) are the most well-characterized NR corepressor complexes. The core NCoR/SMRT protein complex consists of NCoR/SMRT, transducin β-like 1/related 1 (TBL1/TBLR1), histone deacetylase 3 (HDAC3), and G-protein pathway suppressor 2 (GPS2). NCoR and SMRT serve as the docking sites for corepressor complex assembly. NCoR/SMRT bind NRs and associate with each of the other complex subunits.

As discussed above, when the NR interacts with ligand, transcriptional activation results due to the ability of the NR-ligand complex to recruit coactivator proteins and displace corepressor proteins. Nuclear receptor corepressors can inhibit the transcriptional activity of most members of the NR superfamily. As always in biology, there are a few exceptions to the general rule of  unliganded NR binding corepressors. These exceptions include LCoR (ligand-dependent nuclear-receptor corepressor), RIP140 (receptor-interacting protein-140) and REA (repressor of estrogen-receptor activity). These repressors bind to NR in a ligand-dependent manner and compete with coactivators by displacing them. In addition, there are several coregulatory factors, such as the ATP-dependent chromatin remodeling complexes SWI/SNF (switching of mating type/sucrose non-fermenting, chromatin remodeling complex), which have been shown to be involved in the regulation of both transcriptional activation and repression.

model of nuclear receptor (NR) corepressor complex assembly at a target gene

Model for NR interactions with corepressors: An example of the transcription corepressor complexes associated with both the RXR and RAR heterodimeric transcription factor complex at an HRE, and several basal transcription factors associated with RNA pol II at a target gene transcriptional start site. The presence of histone deacetylases (e.g. HDAC3) leads to removal of any chromatin activating histone acetylation sites causing formation of transcriptionally repressed chromatin structure.

back to the top

Phospholipases and Phospholipids in Signal Transduction

Phospholipases and phospholipids are involved in the processes of transmitting ligand-receptor induced signals from the plasma membrane to intracellular proteins. The locations at which the various phospholipases hydrolyze phospholipids is shown in a Figure in the Fatty Acid, Triglyceride, and Phospholipid Synthesis page. The primary enzymes whose activities are modulated as a consequence of plasma membrane receptor activation are the members of the phospholipase C (PLC) family (see below). Once a PLC enzyme is activated a chain of events occurs leading to subsequent activation of the kinase, PKC. PKC is maximally active in the presence of calcium ion (Ca2+) and DAG. Activation of PLC enzymes result in the hydrolysis of membrane phospholipids, primarily phosphatidylinositol-4,5-bisphosphate (PIP2) leading to an increase in intracellular DAG and inositol trisphosphate (IP3). The released IP3 interacts with intracellular membrane receptors leading to an increased release of stored calcium ions. Together, the increased DAG and intracellular free calcium ion concentrations lead to increased activity of PKC.

Humans express three distinct IP3 receptors encoded by the ITPR1, ITPR2, and ITPR3 genes. The ITPR1 gene which is located on chromosome 3p26.1 and is composed of 63 exons that generate three alternatively spliced mRNAs encoding three distinct isoforms of the receptor. ITPR1 isoform 1 is a 2710 amino acid protein, isoform 2 is a 2695 amino acid protein, and isoform 3 is a 2743 amino acid protein. The ITPR2 gene is located on chromosome 12p11 and is composed of 60 exons that encode a 2701 amino acid protein. The ITPR3 gene is located on chromosome 6p21 and is composed of 61 exons that encode a 2671 amino acid protein. Each of the IP3 receptors possesses a cytoplasmic N-terminal ligand-binding domain and is comprised of six membrane-spanning helices that forms the core of the ion pore.

transmembrane receptor activation of PLC isoforms

Receptor-mediated activation of PLC: There are 13 members of the PLC family of phospholipases with the PLCβ and PLCγ members being the most well characterized with respect to their role in signal transduction cascades. The PLCβ enzymes are activated by GPCRs coupled to Gq-type G-proteins while the PLCγ enzymes are activated when their SH2 domains dock with a phosphotyrosine in a receptor with intrinsic tyrosine kinase activity or receptors that activate associated tyrosine kinases. Both pathways ultimately activate the kinase PKC, leading to numerous changes within the activated cell.

Recent evidence indicates that phospholipases D and A2 (PLD and PLA2) also are involved in the sustained activation of PKC through their hydrolysis of membrane phosphatidylcholine (PC). PLD action on PC leads to the release of phosphatidic acid which in turn is converted to DAG by a specific phosphatidic acid phosphomonoesterase. PLA2 hydrolyzes PC to yield free fatty acids and lysoPC both of which have been shown to potentiate the DAG mediated activation of PKC. Of medical significance is the ability of phorbol ester tumor promoters to activate PKC directly. This leads to elevated and unregulated activation of PKC and the consequent disruption in normal cellular growth and proliferation control leading, ultimately, to neoplasia.

Phospholipase C (PLC) Family

Members of the phospholipase C (PLC) family play crucial roles in the regulation of signal transduction in a wide array of systems. The members of the PLC family hydrolyze membrane-associated phosphatidylinositol-4,5-bis-phosphate PIP2 resulting in the generation of two second messengers, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), in response to activation of receptors by hormones, growth factors, and neurotransmitters. As indicated above, these second messengers in turn activate the kinase, PKC.

Thus far, a total of 13 PLC genes have been identified in the human genome. The proteins encoded by these 13 genes have been assigned to six subclasses of enzyme defined on the basis of structure and regulatory activation mechanisms. These six subfamilies are referred to as PLC-beta (PLCβ1–β4), PLC-gamma (PLCγ1 and PLCγ2), PLC-delta (PLCδ1, δ3, and δ4), PLC-epsilon (PLCε), PLC-zeta (PLCζ), and PLC-eta (PLCη1 and PLCη2). The most recently characterized family members are the two PLCη enzymes. All of the PLC enzymes contain catalytic X and Y domains in addition to subtype-specific domains and domains conserved across multiple members. Each enzyme also contains several regulatory domains, including the C2 domain, EF-hand motif, and the pleckstrin homology (PH) domain. Activation of the PLCβ family members occurs via association with GPCRs that are coupled to the Gq class of G-protein. The PLCγ enzymes contain an SH2 domain that allows them to interact with phosphotyrosine residues present on receptors with intrinsic tyrosine kinase activity or with receptor-associated tyrosine kinases. The PLCε family member is activated by GPCRs that are associated with members of the G12/13 G-protein family or by receptors that activate members of the RAS and RHO family of monomeric G-proteins.

back to the top

Phospholipase A (PLA) Family

The PLA family of lipases consists of the PLA1 and PLA2 subfamilies. The designation of PLA1 or PLA2 relates to the target of the enzyme. PLA1 enzymes catalyze hydrolysis of fatty acids from the sn-1 position of glycerophospholipids generating 2-acyl-lysophospholipids and free fatty acids. PLA2 enzymes catalyze hydrolysis of the sn-2 position of glycerophospholipids releasing free fatty acids and 1-acyl-lysophospholipids.

Mammals express several different extracellular enzymes that exhibit PLA1 activity all of which belong to the pancreatic lipase gene family. These enzymes include phosphatidylserine (PS)-specific PLA1 (PS-PLA1), two membrane-associated phosphatidic acid (PA)-selective PLA1 (mPA-PLA1α and mPA-PLA1β), hepatic lipase (HL, encoded by the LIPC gene, also commonly called hepatic triglyceride lipase, HTGL), endothelial cell-derived lipase (EDL, encoded by the LIPG gene) and pancreatic lipase-related protein 2 (PLRP2). Due to differences in substrate specificities, structural features and gene organizations, PS-PLA1, mPA-PLA1α and mPA-PLA1β form a subfamily in the pancreatic lipase gene family. In addition, PS-PLA1, mPA-PLA1α and mPA-PLA1β exhibit only PLA1 activity as well as exhibiting preference for certain phospholipids such as phosphatidylserine (PS) and phosphatidic acid (PA). In contrast, HL, EDL and PLRP2 possess triacylglyceride-hydrolyzing activity in addition to PLA1 activity. In addition to the above described enzymes, the pancreatic lipase family of enzymes includes pancreatic lipase (PL) and lipoprotein lipase (LPL) both of which exhibit specificity toward triglycerides.

PS-PLA1 preferentially hydrolyzes phosphatidylserine (PS) hence the naming of this enzyme. The products of PS-PLA1 are a fatty acid and lysoPS. LysoPS has been implicated in several biological processes that include suppression of T-cell proliferation, activation of mast cells, induction of fibroblast and glioma cell chemotaxis, and the promotion of neurite outgrowth. The recently characterized receptor for lysoPS is GPR34. Activation of GPR34 by lysoPS is greatest when there is a fatty acid in the sn-2 position.

The mammalian genome contains more than 30 genes encoding PLA2 and PLA2-related enzymes. All of these genes are subdivided into several classes that includes low-molecular-weight secreted PLA2s (sPLA2s), Ca2+-dependent cytosolic PLA2s (cPLA2s), Ca2+-independent PLA2s (iPLA2s), platelet-activating factor acetylhydrolases (PAF-AHs), lysosomal PLA2s, and a recently identified adipose-specific PLA2 (AdPLA). The intracellular cPLA2 and iPLA2 families and the extracellular sPLA2 family are recognized as the most significant PLA2 enzyme families. For more details on the PLA family of enzyme go to the Bioactive Lipids page.

The sPLA2 family contains ten identified enzymes. The sPLA2 family affects various biological events by modulating the extracellular phospholipid environment. The cPLA2 family contains six members. The cPLA2 enzymes all contain an N-terminal domain that is required for calcium-binding and association with membranes. cPLA2α (the prototypic cPLA2) plays a major role in the initiation of arachidonic acid metabolism. The iPLA2 family is composed of nine enzymes and is also referred to as the patatin-like phospholipase domain-containing lipase (PNPLA) family. The patatin domain was originally discovered in lipid hydrolases of certain plants and named after the most abundant protein of the potato tuber, patatin. One member of this PNPLA family is adipose triglyceride lipase (ATGL, less commonly PNPLA2) which is described in detail in the Lipolysis and Fatty Acid Oxidation page. The PAF-AH family contains four members each of which has substrate specificity for PAF and/or oxidized phospholipids.

back to the top

Phospholipase D (PLD) Family

Humans express two major PLD isoforms identified as PLD1 and PLD2. These two enzymes share 50% amino acid sequence homology, the highest of which is in the catalytic domain. Although other PLD isoforms have been characterized, the majority of observable PLD activity can be attributed to these two enzymes. Both PLD1 and PLD2, as well as several splice variants, hydrolyze phospholipids such that the polar head group, attached to the phosphate, is released with the other resulting product from this type of reaction being phosphatidic acid. Both PLD1 and PLD2 are capable of hydrolyzing PC, PE, PS, lysophosphatidylcholine (LPC), and lysophosphatidylserine (LPS). However, these two isoforms are not capable of hydrolyzing PI, PG, or cardiolipin. When the substrate is LPC or LPS the product of PLD action is lysophosphatidic acid (LPA).

In addition to PLD1 and PLD2, two other mammalian enzymes of the PLD family have been identified. PLD3, which is also called Hu-K4, is highly similar to the amino acid sequence of viral PLD enzymes. PLD3 contains an N-terminal type II transmembrane domain that facilitates the insertion of the enzyme into the ER membrane. Although this protein has PLD sequence identity it is has not been conclusively demonstrated that is harbors phospholipid hydrolytic activity. The other PLD family member is called PLD6, which is also called mitoPLD. PLD6 is associated with mitochondria (hence the mitoPLD nomenclature) where it exhibits substrate specificity for cardiolipin, an abundant mitochondrial lipid. The reaction catalyzed by PLD6 occurs at the surface of the mitochondria and the product, PA, facilitates mitochondrial fusion events.

LysoPLD is an enzyme which hydrolyzes lysophospholipids (lysoPL) to produce lysophosphatidic acid (LPA). LysoPLD exhibits the highest substrate preference for lysophosphatidylcholine (lysoPC). Two major forms of lysoPLD have been characterized, a microsomal and an extracellular form. The microsomal lysoPLD exhibits a substrate preference for alkylphospholipids and as such is important in the metabolism of platelet-activating factor (PAF). The extracellular lysoPLD turned out to be identical to another previously characterized protein defined as a tumor autocrine motility factor and called autotaxin (ATX). ATX possess 5'-nucleotide pyrophosphatase and phosphodiesterase (PDE) activities. Because of this demonstrated enzymatic activity, the approved nomenclature for lysoPLD/ATX is ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2). The ENPP2 gene generates three splice variants that were identified as ATX-alpha (ATX-α, composed of 915 amino acids, ATX-beta (ATX-β, composed of 863 amino acids), and ATX-gamma (ATX-γ, composed of 889 amino acids).

back to the top

Phosphatidylinositol-3-Kinase (PI3K) Family

Phosphatidylinositol-4,5-bisphosphate (PIP2) is not only a substrate for the PLC family enzymes, whose actions lead to the release of DAG and IP3, but is also a substrate for a family of kinases that phosphorylate the membrane lipid on the 3-OH to generate phosphatidylinositol-3,4,5-trisphosphate, PIP3. These kinases are referred to as the phosphatidylinositol-3-kinases, PI3K. The PI3K enzymes represent a family of both receptor-activated and non-receptor-activated phosphatidylinositide kinases. Initial characterization of PI3K activity found that it was associated with two protein subunits, one of 85 kDa (p85) and the other of 110 kDa (p110). Further work demonstrated that the p85 subunit had no intrinsic PI3K activity but that it contained a protei-protein interaction domain of the SRC homology domain 3 (SH3) type and two phosphotyrosine-binding sites of the SRC homology domain 2 (SH2) type. The presence of the SH3 domain allows p85 and p110 to form a heterodimer and the presence of the SH2 domains allows the heterodimer to bind to phosphorylated tyrosine docking sites on growth factor receptors harboring intrinsic tyrosine kinase activity such as the epidermal growth factor receptor (EGFR). Characterization of the p110 protein demonstrated that it showed significant homology to the yeast vacuolar protein sorting-associated protein 34 (Vps34). In yeast the Vps34 protein is involved in endosomal sorting of proteins towards the vacuole which is the yeast equivalent of the mammalian lysosome. Indeed, the yeast Vps34 protein harbors PI3K activity. Additional studies demonstrated that mammalian cells also contain a PI3K activity that is not activated by receptor-mediated signaling and that this kinase has a role in vesicular trafficking.

These studies, and many others, identified several different p110 and p85 subunit genes resulting in the charaterization of PI3K enzymes as a large family of enzymes. The receptor-activated PI3K members include both receptor tyrosine kinase (RTK) and G-protein coupled receptor (GPCR) activated isoforms. The GPCR activated PI3K enzymes consist of the p110 catalytic subunit and a different regulatory subunit identified as p101. The p101 subunit facilitates interaction of the catalytic p110 subunit with the βγ-subunits that are released from the heterotrimeric G-proteins activated by GPCR stimulation. Another class of PI3K enzymes were characterized that function as monomeric enzymes. These PI3K harbor a C-terminal C2 domain (a membrane targeting domain that binds Ca2+ ions) and are, therefore, referred to as PI3K-C2 kinases. The result of this large body of work was the classification of PI3K enzymes into three distinct groups based upon structural and biochemical characteristics. The three groups are designated class I, class II, and class III.

Class I PI3K enzymes are receptor-regulated PIP2 kinases. Mammals express two subgroups of the class I PI3K isoforms, with the IA subgroup representing the RTK-activated enzymes and the IB subgroup representing the GPCR-activated enzymes. Within the class IA subgroup there are four catalytic subunit encoding genes and two regulatory subunit encoding genes. The catalytic subunits of class IA PI3K enzymes are designated p110α (PIK3CA gene), p110β (PIK3CB gene), p110δ (PIK3CD gene), and p110γ (PIK3CG). The two class IA regulatory subunits are designated p85α (PIK3R1 gene) and p85β (PIK3R2 gene). The class IB PI3K enzyme is a heterodimer composed of only one catalytic subunit (the p110γ protein) and one of two regulatory subunits. The two regulatory subunits are the original p101 subunit (encoded by the PIK3R5 gene) and the protein encoded by the PIK3R6 gene (originally identified as p87PIKAP). Class II PI3K enzymes are the PI3K-C2 kinases. Humans express three class II PI3K isoforms identified as PI3K-C2α, PI3K-C2β, and PI3KJ-C2γ. The PI3K-C2α enzyme is encoded by the PIK3C2A gene, the PI3K-C2β enzyme is encoded by the PIK3C2B gene, and the PI3K-C2γ enzyme is encoded by the PIK3C2G gene. The class III PI3K enzyme (there is only a single class III enzyme) is the phosphatidylinositol-specific enzyme (human homolog of yeast Vps34) which is encoded by the PIK3C3 gene. The human PIK3C3 protein forms a heterodimer with a regulatory subunit that is designated p150 (Vps15 in yeast) which is encoded by the PIK3R4 gene.

The enzymes of the PI3K family harbor domains, distinct from the catalytic domain, that are critical to their function. These domains are also found in numerous other signal transduction proteins. One important domain in PI3K enzymes is the pleckstrin homology (PH) domain, a loosely conserved modular domain of about 120 amino acids. The PH domain is found in all the class I PI3K enzymes. The significance of the PH domain is that it binds to PIP2 as well as to PIP3 and to phosphatidylinositol-3,4-bisphosphate. A critical regulator of cell metabolism, growth, and proliferation, that is coupled to the PI3K signaling pathway, is the Ser/Thr kinase PKB, also known as AKT. There are three members of the PKB/AKT family of serine/threonine kinases identified as AKT1 (PKB, also PKBα), AKT2 (PKBβ), and AKT3 (PKBγ). PKB/AKT also contains a PH domain allowing it to be directly associated with the PI3K signal transduction pathway. Activation of PKB/AKT occurs via phosphorylation which is catalyzed by another upstream kinase which is itself activated by phosphoinositides. Specifically this PKB/AKT kinase is called phosphoinositide-dependent kinase 1, PDK1. The phosphoinositides that activate PDK1 are PIP3 generated via the PI3K enzymes. The significance of PI3K activity in the regulation of PKB/AKT is evident from the fact that numerous PKB/AKT substrates are involved in the regulation of metabolism, cell growth, apoptosis, and autophagy. As an examples PKB/AKT phosphorylates the tumor suppressor knonwn as tuberous sclerosis 2 (TSC2) which in turn regulates the activity of the Ser/Thr kinase mTOR (mammalian target of rapamycin) which is in the mTOR complexes (mTORC1 and mTORC2) that control metabolism, cell growth, and autophagy. PKB/AKT also phosphorylates glycogen synthase kinase 3 (GSK3) in the insulin signaling pathway, the apoptosis promoting protein BAD (BCL-2-associated agonist of cell death), and the cell cycle regulator p21CIP. Indeed more than 100 PKB/AKT substrates have been identified.

back to the top


Lysophospholipids (LPLs) are minor lipid components compared to the major membrane phospholipids such as phosphatidylcholline (PC), phosphatidylethanolamine (PE), and sphingomyelin. The LPLs were originally presumed to be simple metabolic intermediates in the de novo biosynthesis of phospholipids. However, subsequent studies demonstrated that the LPLs exhibited biological properties resembling those of extracellular growth factors or signaling molecules. The most biologically significant LPLs are lysophosphatidic acid (LPA), lysophosphatidylcholine (LPC), lysophosphatidylinositol (LPI), sphingosine 1-phosphate (S1P), and sphingosylphosphorylcholine (SPC). Each of these LPLs functions via interaction with specific G-protein coupled receptors (GPCRs) leading to autocrine or paracrine effects. The first LPL receptor identified was called LPA1 because it bound LPA. The first GPCR shown to bind S1P was called S1P1. For more information on the activities of S1P please go to the Sphingolipids page. For more detailed information on the activities of the lysophospholipids please go to the Bioactive Lipids page.

Currently there are fifteen characterized LPL receptors. Because several of the LPL receptors were independently identified in unrelated assays, there are several different names for some members of this receptor family. In particular, there is a group of genes that were originally identified as GPCRs and called endothelial differentiation genes (EDGs) that were later found to be the same as several of the LPL receptors. Thus LPA1 is also known as EDG-2, LPA2 as EDG-4, and LPA3 as EDG-7. S1P1 is also known as EDG-1, S1P2 as EDG-5, S1P3 as EDG-3, S1P4 as EDG-6, and S1P5 as EDG-8. Activation of the LPA receptors triggers several different downstream signaling cascades. These include activation of MAP kinase (MAPK), activation of PLC, PKB/AKT activation, calcium moblization, release of arachidonic acid, inhibition or activation of adenylate cyclase, and activation of several small GTPases such as Ras, Rho, and Rac. The LPs exert a wide-range of biochemical and physiological responses including platelet activation, smooth muscle contraction, cell growth, and fibroblast proliferation.

LPA is produced by activated platelets, activated adipocytes, neuronal cells, as well as several other cell types. The mode(s) of LPA synthesis intracellularly remains to be fully elucidated. LPA is produced in the serum through the action of several different enzymes including monoacylglyceride kinase (MGL), phospholipase A1 (PLA1), secretory phospholipase A2 (sPLA2), and lysophospholipase D (lysoPLD). LysoPLD is also called autotaxin (ATX) as discussed above. Degradation of LPA occurs via lysophospholipase, lipid phosphate phosphatase, or LPA acyl transferase (also called endophilin).

S1P is stored in platelets and released upon platelet activation. Synthesis of S1P occurs exclusively from sphingosine via the action of sphingosine kinases. Degradation of S1P occurs through the action of S1P lyases or S1P phosphatases.

back to the top

Phosphatases in Signal Transduction

Substantial evidence links both tyrosine and serine/threonine phosphorylation with increased cellular growth, proliferation and differentiation. Removal of the incorporated phosphates must be a necessary event in order to turn off the proliferative signals. This suggests that phosphatases may function as anti-oncogenes or growth suppressor genes. The loss of a functional phosphatase involved in regulating growth promoting signals could lead to neoplasia. However, examples are known where dephosphorylation is required for promotion of cell growth. This is particularly true of specialized kinases that are directly involved in regulating cell cycle progression. Therefore, it is difficult to envision all phosphatases as being tumor suppressor genes.

Protein Tyrosine Phosphatases

There are two broad classes of protein tyrosine phosphatases (PTPs) divided into four families. There are at least 107 genes in the huuman genome that encode enzymes that belong to one or the other of the broad classes of PTP. One broad class are single pass transmembrane-spanning (transmembrane receptor-like) enzymes that contain the phosphatase activity domain in the intracellular portion of the protein. The transmembrane PTPs are commonly called the receptor (R) class and designated RTP. The other broad class is intracellularly localized enzymes and are referred to as NT PTPs (for non-transmembrane). The first transmembrane PTP characterized was the leukocyte common antigen protein, CD45. This protein was shown to have homology to the intracellular PTP, PTP1B. Humans express 20 expressed members of the RTP family. These enzymes are designate by the RTP abbreviation and an additional letter of the alphabet (e.g RTPA and RTPB). There are 17 expressed human genes that encode what are referred to as classical type I non-transmembrane PTPs. The designation for non-receptor tyrosine phosphatases is the abbreviation PTPN followed by an Arabic numeral (e.g. PTPN1 which was originally identified as PTP1B). The rest of the PTP genes are defined as non-classical PTPs and includes the dual-specificity phosphatases (DUSPs: 18 human genes). As the name implies, DUSPs can dephosphorylate tyrosine phosphate as well as serine and threonine phosphates.

The clearest studies of a role for transmembrane PTPs in signal transduction have involved the CD45 protein. These studies have shown that CD45 is involved in the regulation of the tyrosine kinase activity of LCK in T cells. As indicated above LCK is associated with T cell antigens CD4 and CD8 generating a split-RTK involved in T cell activation. It is suspected that CD45 dephosphorylates a regulatory tyrosine phosphorylation site in the C-terminus of LCK, thereby, increasing the activity of LCK towards its substrate(s).

Human Receptor Tyrosine Phosphatases (RTPs)

Symbol Enzyme Name Other Common Abbreviations
PTPRA protein tyrosine phosphatase, receptor type, A LRP, PTPA, PTPRL2
PTPRB protein tyrosine phosphatase, receptor type, B PTPB
PTPRC protein tyrosine phosphatase, receptor type, C CD45
PTPRD protein tyrosine phosphatase, receptor type, D PTPD
PTPRE protein tyrosine phosphatase, receptor type, E PTPE
PTPRF protein tyrosine phosphatase, receptor type, F LAR
PTPRG protein tyrosine phosphatase, receptor type, G PTPG
PTPRH protein tyrosine phosphatase, receptor type, H SAP-1
PTPRJ protein tyrosine phosphatase, receptor type, J CD148, DEP1
PTPRK protein tyrosine phosphatase, receptor type, K R-PTP-kappa
PTPRM protein tyrosine phosphatase, receptor type, M PTPRL1
PTPRN protein tyrosine phosphatase, receptor type, N IA-2
PTPRN2 protein tyrosine phosphatase, receptor type, N polypeptide 2 IA-2β, phogrin
PTPRO protein tyrosine phosphatase, receptor type, O PTPU2
PTPRQ protein tyrosine phosphatase, receptor type, Q DFNB84
PTPRR protein tyrosine phosphatase, receptor type, R PTPRQ, PTPBR7
PTPRS protein tyrosine phosphatase, receptor type, S  
PTPRT protein tyrosine phosphatase, receptor type, T RPTPrho
PTPRU protein tyrosine phosphatase, receptor type, U PTPRO
PTPRZ1 protein tyrosine phosphatase, receptor type, Z polypeptide 1 PTPZ, PTP18, phosphacan

The second class of PTPs are the intracellular proteins. The C-terminal residues of most if not all intracellular PTPs are very hydrophobic and suggest these sites are membrane attachment domains of these proteins. One role of intracellular PTPs is in the maturation of Xenopus oocytes in response to hormone. Over expression of PTPN1 in oocytes resulted in a marked retardation in the rate of insulin- and progesterone-induced maturation. These results suggest a role for PTPN1 in countering the signals leading to cellular activation.

Classical Non-Receptor Tyrosine Phosphatases (PTPNs)

Symbol Enzyme Name Other Common Abbreviations
PTPN1 protein tyrosine phosphatase, nonreceptor type 1 PTP1B
PTPN2 protein tyrosine phosphatase, nonreceptor type 2 PTPT, TCPTP
PTPN3 protein tyrosine phosphatase, nonreceptor type 3 PTPH1
PTPN4 protein tyrosine phosphatase, nonreceptor type 4 (megakaryocyte) PTPMEG
PTPN5 protein tyrosine phosphatase, nonreceptor type 5 (striatum-enriched) STEP
PTPN6 protein tyrosine phosphatase, nonreceptor type 6 PTP1C
PTPN7 protein tyrosine phosphatase, nonreceptor type 7 HEPTP
PTPN9 protein tyrosine phosphatase, nonreceptor type 9 MEG2
PTPN11 protein tyrosine phosphatase, nonreceptor type 11 PTP2C, SHP-2, SH-PTP2
PTPN12 protein tyrosine phosphatase, nonreceptor type 12 PTPG1
PTPN13 protein tyrosine phosphatase, nonreceptor type 13 [APO-1/CD95(Fas)-associated phosphatase] PTP1E, PTPL1
PTPN14 protein tyrosine phosphatase, nonreceptor type 14 PEZ
PTPN18 protein tyrosine phosphatase, nonreceptor type 18 (brain-derived) BDP1
PTPN20 protein tyrosine phosphatase, nonreceptor type 20 PTPN20A, PTPN20B
PTPN21 protein tyrosine phosphatase, nonreceptor type 21 PTPD1, PTPRL10
PTPN22 protein tyrosine phosphatase, nonreceptor type 22 (lymphoid) PTPN8, Lyp1, Lyp2
PTPN23 protein tyrosine phosphatase, nonreceptor type 23 HD-PTP

The above observation as well as several others have demonstrated a link between insulin function and PTPN1. PTPN1 directly interacts with the insulin receptor and removes the tyrosine phosphates incorporated by autophosphorylation in response to insulin binding, thereby, negatively affecting the activity of the insulin receptor. Mice lacking a functional PTPN1 gene exhibit increased insulin sensitivity as well as resistance to obesity induced by a high fat diet.

As with the transmembrane PTPs little is known about the regulation of the activity of the intracellular PTPs. Two intracellular PTPs (PTPN6 and PTPN21) have been shown to contain SH2 domains. These SH2 domains allow these PTPs to directly interact with tyrosine phosphorylated RTKs and PTKs, thereby, dephosphorylating tyrosines in these proteins. Following receptor stimulation of signal transduction events, the SH2 containing PTPs are directed to several of the RTKs and/or PTKs with the net effect being a termination of the signaling events by tyrosine dephosphorylation.

Protein Serine/Threonine Phosphatases

Other phosphatases that recognize serine and/or threonine phosphorylated proteins also exist in cells. These are referred to as protein serine phosphatases (PSPs). The PSPs are grouped into three major families: phosphoprotein phosphatases (PPPs), metal-dependent protein phosphatases (PPMs), and the aspartate-based phosphatases represented by FCP/SCP. This latter family name is derived from transcription factor IIF (TFIIF)-associating component of RNA polymerase II C-terminal domain (CTD) phosphatase/small CTD phosphatase.

The broad spectrum of activity associated with the members of the PPP family stems from the ability of the catalytic subunits to associate with a large variety of different regulatory/inhibitory subunits. The regulatory/inhibitory subunits are grouped into five subfamilies termed the protein phosphatase (1, 2, 3, 4, and 6) regulatory subunit families. The designations for these regulatory gene familes is PPP1R, PPP2R, PPP3R, PPP4R, and PPP6R. The number of PPP regulatory subunit genes is vast with the PPP1R family being composed of 181 genes in humans, PPP2R composed of 15 genes, PPP3R composed of 2 genes, PPP4R composed of 3 genes, and PPP6R composed of 6 genes. Although there are several PPP regulatory subunits genes involved in the regulation of metabolism, the proteins encoded by the PPP1R3A and PPP1R3B genes are critical to the regulation of glycogen homeostasis.

Human Serine/Threonine Phosphatases, Catalytic Subunits (PPPs)

Symbol Enzyme Name Comments; Common Abbreviations
PPP1CA protein phosphatase 1, catalytic subunit, alpha (α) isozyme originally identified as PP1; PP1A, PPP1A, PP1alpha (PP1α)
PPP1CB protein phosphatase 1, catalytic subunit, beta (β) isozyme originally identified as PP1; PP1B, PP1beta (PP1β); also referred to as PP1delta (PP1δ)
PPP1CC protein phosphatase 1, catalytic subunit, gamma (γ) isozyme originally identified as PP1; PP1C, PP1gamma; two major isoforms identified as PP1Cγ1 and PP1Cγ2
PPP2CA protein phosphatase 2, catalytic subunit, alpha (α) isozyme originally identified as PP2A; PP2Calpha (PP2Cα)
PPP2CB protein phosphatase 2, catalytic subunit, beta (β) isozyme originally identified as PP2A; PP2Abeta (PP2Aβ)
PPP3CA protein phosphatase 3, catalytic subunit, alpha (α) isozyme originally identified as PP2B or calcineurin A; PPP2B, CALN, CALNA
PPP3CB protein phosphatase 3, catalytic subunit, beta (β) isozyme originally identified as PP2B or calcineurin B; PP2Bbeta, CALNA2, CALNB
PPP3CC protein phosphatase 3, catalytic subunit, gamma (γ) isozyme originally identified as PP2B or calcineurin; PP2Bgamma, CALNA3
PPP4C protein phosphatase 4, catalytic subunit PP4, PPX
PPP5C protein phosphatase 5, catalytic subunit PP5
PPP6C protein phosphatase 6, catalytic subunit PP6
PPEF1 protein phosphatase, EF-hand calcium binding domain 1 PPP7CA
PPEF22 protein phosphatase, EF-hand calcium binding domain 2 PPP7CB

As the name implies the PPM family is represented by protein phosphatases that are dependent upon manganese/magnesium ions (Mn2+/Mg2+), such as PPM1A (originally identified as PP2C) and the two pyruvate dehydrogenase phosphatases (PDP1 and PDP2). Unlike the members of the PPP family, phosphatases of the PPM family do not have regulatory subunits but they do contain additional domains and conserved sequence motifs that are involved in the determination of substrate specificity. For both the PPP and PPM families, metal ions play a catalytic and central role through the activation of a water molecule for the dephosphorylation reaction. The PPM family includes 17 genes expressed in humans.

In contrast to the mechanism of action of the PPP and PPM phosphatases, the FCP/SCP phosphatases use an aspartate-based catalytic mechanism. There is currently only one known substrate for FCP/SCP and as the name implies it is the the C-terminal domain (CTD) of RNA polymerase II. The CTD of RNA polymerase II contains tandem repeats of a serine-rich heptapeptide and the state of phosphorylation of several serines in this repeat is critical for the regulation of polymerase activity (see the RNA Synthesis page for more details). The conserved structural core of FCP/SCP is the FCP homology (FCPH) domain. FCPs, but not SCPs, also contain a BRCA1 C-terminal domain-like (BRCT) domain that is C-terminal to the FCPH domain.

Protein Phosphatase 1 (PP1)

Protein Phosphatase 1 (PP1) represents a major subfamily of protein serine/threonine phosphatases (see Table above). Expression of various PP1 catalytic subunit isoforms is found in all eukaryotic cells. There are three PP1 catalytic subunit genes in humans encoding the isoforms designated PP1α (PPP1CA gene), PP1β (PPP1CB gene), and PP1γ (PPP1CC gene). All three PP1 catalytic subunit genes are subject to alternative splicing with the result that there are three isoforms derived from the PPP1CA gene, one from the PPP1CB gene (the two mRNAs from this gene encode the same protein), and two from the PPP1CC gene. The catalytic subunits of PP1 do not exist as free proteins in cells but are associated with a wide range regulatory/inhibitory proteins that number more than 200 (see above and below). The original regulatory/inhibitory protein shown to regulate the activity of PP1 was identifed as inhibitor of protein phosphatase 1 (IPP-1, also identified as PPI-1). The current designation for PPI-1 is protein phosphatase 1, regulatory (inhibitory) subunit 1A, encoded by the PPP1R1A gene.

PP1 plays an important role in a wide range of cellular processes, including protein synthesis, metabolism, regulation of membrane receptors and channels, cell division, apoptosis, and reorganization of the cytoskeletal architecture. Although the many isoforms of PP1 collectively exhibit broad substrate specificity, when assembled each PP1 enzyme is believed to display highly specific substrate specificity and thus, will elicit a specific biological responses. Each functional PP1 enzyme is composed of a catalytic subunit and a regulatory subunit. The catalytic subunit of PP1 is highly conserved among all eukaryotes. At least 100 putative PP1-binding regulatory subunits have been identified. The regulatory subunits are involved in targeting the PP1 catalytic subunits to specific subcellular compartments, act to modulate substrate specificity, and also can serve as substrates themselves. The interactions between a given PP1 catalytic subunit and a specific regulatory subunit are central to the functions of each PP1. The catalytic subunit of PP1 contains a domain that interacts with two metal ions. This metal-binding domain is highly conserved in all members of the PPP family. The two metal ions play a role in the activation of a water molecule, which initiates a nucleophilic attack on the phosphorous atom.

In addition to the above mentioned PPI-1, the phsopahtase activity of PP1 is regulated by a number of other inhibitory proteins such as inhibitor-2 (I-2), CPI-17 (a 17 kDa PKA activated PP1 inhibitor also known as PP1 regulatory subunit 14A, PPP1R14A, found in smooth muscle and inhibits myosin light chain phosphatase, MLCP), and DARPP-32 (dopamine- and cAMP-regulated phosphoprotein 32 kDa; also known as PP1 regulatory subunit 1B, PPP1R1B). Despite the fact that there is sequence conservation between PP1 and PP2A and PP2B, the latter two phosphatases are not sensitive to inhibition by PPI-1 or I-2. It is this functional difference that was the basis for classification of type 1 (PP1) versus type 2 phosphatases.

Protein Phosphatase 2A (PP2A)

The protein phosphatases that are involved in the regulation of metabolic processes are of two types: type 1, namely the PP1 isoforms, and the type 2 isoforms, which consist of three catalytic enzymes: PP2A, PP2B, and PP2C. The phosphatase originally designated as PP2C is a member of the metal-dependent protein phosphatase (PPM) family.

PP2A exists in two distinct catalytic isoforms encoded by two genes identified as PPP2Ca and PPP2CB. These PP2A isoforms play important roles in development, cell proliferation, apoptosis, cell mobility, cytoskeleton dynamics, the control of the cell cycle, and the regulation of numerous signaling pathways. PP2A isoforms represent some of the most abundant enzymes in cells and can account for up to 1% of total cellular protein in some tissues. PP2A has also been suggested to be a tumor suppressor. PP2A is highly conserved across a variety of eukaryotic species. The mechanisms of its regulatory action are highly complex.

As indicated, PP2A exists in two basic isoforms: a heterodimeric core enzyme and a heterotrimeric holoenzyme. The PP2A core enzyme consists of a scaffold subunit (originally termed the A subunit) and a catalytic subunit (the C subunits). The scaffold and the catalytic subunits each exist in two isoforms: alpha (α) and beta (β). The catalytic α subunit is encoded by the PPP2CA gene and the catalytic β subunit is encoded by the PPP2CB gene. The scaffold α subunit is encoded by the PPP2R1A gene and the β subunit by the PPP2R1B gene. The α catalytic isoform is approximately 10 times more abundant than the β isoform. The PP2A core enzyme interacts with a variable regulatory subunit to assemble into a holoenzyme. The heterotrimeric PP2A holoenzyme is believed to exhibit exquisite substrate specificity as well as spatially and temporally determined functions.

The PP2A regulatory subunits comprise four families originally classified as the B subunits (B, B', B'', and B'''). Each family consists of multiple isoforms that are encoded by different genes. There are currently 11 different PP2A Bsubunit genes expressed in humans. Several of these genes generate multiple isoforms as a result of alternative splicing. Except for subunits of the B''' family, all members of these regulatory subunit families have been shown to bind directly to the PP2A catalytic subunits. The level of expression of the various regulatory subunit genes varies greatly in different cell types and tissues. The PP2A scaffold subunits contains 15 tandem HEAT (huntingtin- elongation-A subunit-TOR) repeats. The catalytic subunits of PP2A recognize a conserved domain of HEAT repeats 1. Although other PPP family members share extensive sequence similarity with the catalytic subunit of PP2A, they do not associate with the PP2A scaffold subunits. The catalytic subunits of PP2A are targets of a number of potent tumor-inducing toxins, such as okadaic acid (OA) and microcystin-LR (MCLR). Both OA and MCLR interact with a similar set of amino acids surrounding the active site of the catalytic subunits. The major function of the PP2A regulatory subunits is to target phosphorylated substrate proteins to the phosphatase activity of the PP2A catalytic subunits.

Reversible methylation of the PP2A catalytic subunits is a conserved mechanism for the regulation of PP2A function. Methylation of leucine 309 (L309) in the C-terminus, within a conserved motif of the catalytic α subunit (the PPP2CA encoded protein), has been shown to enhance the affinity of the PP2A core enzyme for some, but not all, regulatory subunits. This implies that changes in PP2A methylation may modulate the specificity and activity of PP2A in cells. The reversible methylation of the PP2A catalytic α subunit is catalyzed by two conserved and PP2A-specific enzymes, leucine carboxyl methyltransferase (LCMT1) and PP2A methylesterase (PME-1). PME-1 catalyzes the removal of the methyl group, thus reversing the activity of LCMT1. The methylated C-terminus of the catalytic subunit may allow it to be targeted to specific cellular location for holoenzyme assembly. In addition, the methylated C-terminus may recruit other proteins that facilitate the assembly of the PP2A holoenzymes within the cell.

By analogy with the PP1 inhibitors, the endogenous inhibitors of PP2A were named I1 PP2A and I2 PP2A. The I1 PP2A inhibitor was subsequently shown to be encoded by the acidic nuclear phosphoprotein 32 family member A (ANP32A) gene which was also known as putative HLA class II-associated protein I (PHAP-I). The I2 PP2A inhibitor was subsequently shown to be encoded by the SET proto-oncogene. The name SET relates to the fact that the protein encoded by this gene was shown to have homology to three related Drosophila genes called Suppressor of variegation variant 3-9 [Su(var)3-9], Enhancer of zeste, and Trithorax. There are numerous proteins in humans that are now known to contain a similar domain now called the SET domain. Several of the SET domain-containing proteins are involved in histone methylation which alters chromatin structure and thus, gene expression.

Protein Phosphatase 2B/Calcineurin (PP2B)

Protein phosphatase 2B (PP2B, also known as calcineurin) is more correctly identified as protein phosphatase 3 (PP3). Calcineurin (PP3) plays an important role in numerous calcium-dependent biological processes that include signal transduction, immune responses, muscle development, neural development and memory, and cardiac hypertrophy. Like the other members of the PPP superfamily of serine/threonine phosphatases, calcineurin (PP3) is encoded by several catalytic subunit genes and the activity of the catalytic subunits regulated by several regulatory/inhibitory subunits. Humans express three calcineurin catalytic subunit genes: PPP3CA (encoding the α isoform), PPP3CB (encoding the β isoform), and PPP3CC (encoding the γ isoform). There are two calcineurin (PP3) regulatory subunit genes expressed in humans identified as PPP3R1 (encoding regulatory subunit B, alpha) and PPP3R2 (encoding regulatory subunit B, beta). The catalytic subunits of calcineurin contain an N-terminal phosphatase domain, followed by a regulatory subunit-binding helical domain, a calcium (Ca2+)-calmodulin-binding motif, and an autoinhibitory element. Calcineurin is inactive alone and is active only upon association with Ca2+-calmodulin (Ca2+-CaM). The autoinhibitory element of calcineurin forms an α helix and blocks access to the catalytic site. Given this structural orientation of the catalytic site and autoinhibitory domain indicates that displacement of the autoinhibitory domain may be required for the activation of the protein.

The phosphatase domain of the catalytic subunits of calcineurin is structurally similar to the phosphatase domain of the catalytic subunits of PP1 and has the same pattern of metal ion coordination. The two metal ions associated with calcineurin are Zn2+ and Fe3+. The regulatory subunits of calcineurin contain a pair of Ca2+-binding domains forming four calcium-binding sites. All four calcium-binding sites in the regulatory subunits are bound to Ca2+ and each calcium ion is coordinated by five oxygen atoms.

The immunosuppressant complexes, FKBP12-FK506 and cyclophilin A (CyPA)-cyclosporin A (CsA) both associate with an exposed surface of the regulatory subunits of calcineurin. Binding by these immunosuppressant complexes is thought to inhibit calcineurin-mediated dephosphorylation of the transcription factor, nuclear factor of activated T cells 1 (NFAT1), ultimately resulting in the suppression of T cell activation. In both cases, the immunosuppressants make direct contacts with residues contained in the calcium-binding domains of the regulatory subunits. This observation explains why interactions between calcineurin and immunophilins strictly depend on the presence of the immunosuppressants.

Extensive studies on the interactions of calcineurin with its substrates, especially using NFAT1, have revealed a consensus recognition motif of PxIxIT (where x represents any amino acid). Variations in substrate binding affinities can be attributed to sequence variations within the PxIxIT motif in different substrates. Although the presence of the PxIxIT motif is necessary for substrate recognition, additional binding elements from the substrate are likely to be required for the specific activity of calcineurin.

Protein Phosphatase 2C Family (PP2C: PPM)

Protein phosphatase 2C actually represents a subfamily (PPM1) of the Mn2+/Mg2+-dependent PPM family of protein phosphatases. The PPM family represents a large family of highly conserved protein phosphatases, with 17 distinct PPM encoding genes in the human genome that give rise to at least 22 different isoforms. The PPM1 (PP2C) subfamily consists of 12 genes. Two additional, highly important metabolic regulating enzymes of the PPM family are the pyruvate dehydrogenase phosphatases. The different PPM isoforms exhibit distinct functions, expression patterns, and subcellular localization.

Unlike the PPP family phosphatases, PPM family phosphatases are insensitive to inhibition by okadaic acid or microcystin. The primary function of PPM1 subfamily phosphatase is in the regulation of cellular stress signaling, and they also play roles in the regulation of metabolism, differentiation, growth, survival, and apoptosis. Several members of the PPM family are candidate tumor suppressor proteins. These include PPM1A (originally called PP2Cα), PPM1B (originally called PP2Cβ), and the plextrin homology (PH) domain leucine-rich repeat protein phosphatases (PHLPP1 and PHLPP2). On the other hand PPM1D (originally identified as PP2Cδ and Wip1) may contribute to oncogenic transformation.

The conserved catalytic domain of human PPM enzymes contains a central β-sandwich, with each β-sheet flanked by a pair of α-helices, the orientation of which generates a cleft between the two β-sheets. The two metal ions are found at the base of the cleft with each metal ion hexacoordinated by amino acids and water molecules. The catalytic activity of PPM phosphatases is similar to that of the PPP family. The dephosphorylation reaction involves nucleophilic attack of the phosphorous by a metal-activated water nucleophile.

Human PPM Family Phosphatases

Symbol Enzyme Name Other Common Abbreviations
PPM1A protein phosphatase, Mg2+/Mn2+ dependent, 1A PP2CA, PP2Calpha
PPM1B protein phosphatase, Mg2+/Mn2+ dependent, 1B PP2CB, PP2Cbeta
PPM1D protein phosphatase, Mg2+/Mn2+ dependent, 1D PP2Cdelta, Wip1
PPM1E protein phosphatase, Mg2+/Mn2+ dependent, 1E PP2CH, CaMKP-N, POPX1
PPM1F protein phosphatase, Mg2+/Mn2+ dependent, 1F CaMKPase, CAMKP, POPX2
PPM1G protein phosphatase, Mg2+/Mn2+ dependent, 1G PP2CG, PP2Cgamma
PPM1H protein phosphatase, Mg2+/Mn2+ dependent, 1H ARHCL1
PPM1J protein phosphatase, Mg2+/Mn2+ dependent, 1J PP2CZ, PP2Czeta
PPM1K protein phosphatase, Mg2+/Mn2+ dependent, 1K PP2Ckappa, PP2Cm
PPM1L protein phosphatase, Mg2+/Mn2+ dependent, 1L PP2CE
PPM1M protein phosphatase, Mg2+/Mn2+ dependent, 1M P2Ceta6
PPM1N protein phosphatase, Mg2+/Mn2+ dependent, 1N  
PDP1 pyruvate dehydrogense phosphatase catalytic subunit 1 PPM2C, PDP
PDP2 pyruvate dehydrogense phosphatase catalytic subunit 2 PPM2C2
PHLPP1 PH domain and leucine rich repeat protein phosphatase 1 PHLPP, SCOP
PHLPP2 PH domain and leucine rich repeat protein phosphatase 1 PHLPPL
ILKAP integrin-linked kinase-associated serine/threonine phosphatase  

The Aspartate-Based Phosphatases: FCP/SCP

The phosphatases that are members of the FCP/SCP family utilize the aspartic acids of the sequence motif DxDxT/V for phosphatase activity. Unlike the other S/T phosphatases described thus far, the FCP/SCP family has only one primary substrate, that being the CTD of RNA polymerase II. The CTD of RNA polymerase II contains tandem repeats of the sequence YSPTSPS. There are eight putative CTD phosphatases in the human genome. The core structure of the FCP/SCP phosphatases resembles phosphoserine phosphatases from several different bacteria, the hexose phosphate phosphatase from Bacteroides (a common human intestinal bacterium), and the haloacid dehalogenase (HAD) from Xanthobacter autotrophicus.

The level, as well as the pattern of phosphorylation in the CTD repeat changes throughout the cycles of transcription, with hypophosphorylation in the preinitiation complex and hyperphosphorylation during transcription elongation. Phosphorylated serine 5 (pSer5), the serine at the fifth position in the tandem repeat, is enriched at transcription initiation and early transcription elongation, whereas phosphorylation of the serine at the second position in the tandem repeat (pSer2) is favored during transcription elongation and through the end of transcription (see the RNA Synthesis page for additional information). Fcp1 is the main serine phosphatase for the CTD. This phosphatase can dephosphorylate both pSer2 and pSer5. By comparison, Scp1 exhibits little activity for pSer2 and prefers pSer5 by a factor of 70-fold.

The catalytic mechanism of Fcp1/Scp1 likely involves two sequential steps. First, an oxygen atom from the N-terminal aspartate in the DxDxT motif initiates a nucleophilic attack on the phosphorous atom of a pSer. Second, a water nucleophile, likely activated by the second aspartate in the DxDxT motif, attacks the phosphorous atom releasing an inorganic phosphate. The Mg2+ ion likely facilitates both of these steps in the reaction by neutralizing the negative charges of the phosphate group. Of note is the fact that the role of the Mg2+ ion in Fcp1/Scp1 is different from that in the PPP or PPM family, where the metal ions are directly involved in catalysis through the activation of a water nucleophile.

Chronophin, a member of the HAD family, is also an aspartate-based PSP. Like FCP/SCP, it contains the signature sequence motif DxDxT and has a similar active site. Also, like FCP/SCP, chronophin has only one known substrate protein. Chronophin dephosphorylates pSer3 of cofilin, which is an important regulator of actin dynamics, leading to its activation.

back to the top
Return to The Medical Biochemistry Page
Michael W King, PhD | © 1996–2016, LLC | info @

Last modified: January 10, 2017