Signal Transduction


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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. 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.

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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 7 transmembrane spanning domains. These receptors are termed serpentine receptors. 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.

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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 Examples Structural Features of Class
I EGF receptor, NEU/HER2, HER3 cysteine-rich sequences
II insulin receptor, IGF-1 receptor cysteine-rich sequences; characterized by disulfide-linked heterotetramers
III PDGF receptors, c-Kit contain 5 immunoglobulin-like domains; contain the kinase insert
IV FGF receptors contain 3 immunoglobulin-like domains as well as the kinase insert; acidic domain
V vascular endothelial cell growth factor (VEGF) receptor contain 7 immunoglobulin-like domains as well as the kinase insert domain
VI hepatocyte growth factor (HGF) and scatter factor (SF) receptors 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
VII neurotrophin receptor family (TRKA, TRKB, TRKC) and NGF receptor contain no or few cysteine-rich domains; NGFR has leucine rich domain

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). 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.

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 C(γ (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).

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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 identifed. 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 (eg 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 four distinct subunits (alpha: α, beta: β, gamma: γ, and delta: δ). 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.

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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 17 RSTKs have been isolated and can be divided into 2 subfamilies identified as the type I and type II receptors. Ligands first bind to the type II receptors which then leads to interaction with the type I receptors. When the complex between ligand and the 2 receptor subtypes forms, the type II receptor phosphorylates the type I receptor leading to initiation of the signaling cascade. 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.

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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.

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 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.

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 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 which contains an additional 43 residues at the N-terminus. The C1A/C1B motifs function as a DAG-/PMA-binding motif. The regulatory domains of the cPKC isoforms also contain a C2 domain that binds anionic phospholipids in a calcium-dependent manner. 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. 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) domain 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.

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).

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. PKCθ is expressed primarily in skeletal muscle and lymphoid/hematopoietic cells. Expression of PKCγ is primarily detected in neuronal tissues.

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The MAP Kinase (MAPK) Cascades

The mitogen-activated protein (MAP) kinase (MAPK) family constitutes a large family of 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), c-Jun N-terminal kinase (JNK), p38, and 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 response to stress and apoptosis. The JNK and p38 cascades are primarily activated in response to cellular stress, although JNK is known to mediate proliferation under certain conditions. As a result MAPK components of the JNK and p38 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 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.

The ERK Cascade

The ERK cascade is 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 TPL2 (also called MAP3K8 and 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). There are two genes encoding the ERKs designated ERK1 (MAPK3) and ERK2 (MAPK1). 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, GSK3 and serine threonine kinase 11 (STK11; also called LKB1 or Peutz-Jeghers syndrome: PJS), as well as ERK3/4 and ERK7/8, are known substrates for MAPKAPKs, but these latter kinases are not usually considered as integral components of the MAPK cascades.

The p38 Cascade

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 involves 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. The next tier of the p38 MAPK cascade is composed of products of four MAPKs genes, including p38α (SAPK2a) p38β (SAPK2b), p38γ, and p38δ. Due to alternative splicing, these four p38 genes generate at least 10 isoforms. Characterizing these 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 activation of 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

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 ten JNK proteins are translated from three different JNK genes that undergo alternative splicing. All of these proteins are either 46kDa or 52-54kDa where the smaller p46 JNKs are referred to as JNK1α1, JNK1β1, JNK2α1, JNK2β1, and JNK3α1. The p54 JNK proteins are referred to as JNK1α2, JNK1β2, JNK2α2, JNK2β2, and JNK3α2. 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 Cascade

The fourth MAPK cascade is the ERK5 cascade. 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 the MAPK, ERK5. ERK5 can be localized to the cytoplasm and be translocated to the nucleus upon stimulation. However, in some cells MEK5 resides in the nucleus where it is activated by nuclear MEK5. Several transcription factors, such as FOS, MYC, and MEF2 family members, are targets for activated ERK5. Additionally, activated ERK5 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 ERK5 can influence transcription through either direct protein–protein interactions or via its intrinsic transcriptional activity. Thus, ERK5 is a unique dual activity protein that, unlike other MAPKs, catalyzes 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.

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G-Proteins

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 phosphodiesterase (Gαt, Gαgust) leading to increased hydrolysis of cAMP. 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 invovled 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 are 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 reassociation 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, hoewever, 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 (α1–5) 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 βL). 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. 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 was shown to contain a motif consisting of 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 of demonstrated to be regulated by Gβγ was PLCβ. Indeed, the 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 f the larger family of Kir3 channels comprised of four distinct subunits encoded on separate genes identified as Kir3.1 through Kir3.4. All Kir3 channels can all 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 dimers 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 (33 members), Rho (20 members), Rab (70 members), Rap (5 members), Ran, Rheb (2 members), Rad, Rit (2 members), and Arf (30 members). A recent addition to the Ras superfamily is the Miro subfamily of monomeric G-proteins that is composed of two members involved in mitochondrial transport processes.

The Ras subfamily is primarily responsible for regulation of events of cell proliferation. The Rho subfamily is involved in the regulation of cell morphology through control of cytoskeletal dynamics. The Rab subfamily is involved in membrane trafficking events. The Rap subfamily is involved in control of cell adhesion. The Ran subfamily is involved in regulation of nuclear transport. The Rheb subfamily 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 subfamily is involved in intracellular vesicle transport.

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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).

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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. There are at least 791 identified GPCRs in the human genome. Many do not have known ligands and are referred to as orphan GPCRs. The GPCR superfamily consists of three defined families or classes as well as a group termed "others". The latter group consists of at least 92 GPCRs that includes the adhesion, frizzled, and taste type-2 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.


Class A Family: The class A GPCR family is referred to as the rhodopsin family. Class A contains the largest number of members compiled into at least 19 subclasses (subfamilies). Class A GPCRs include opsins, the vast majority of the odorant receptors (at least 290 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 B Family: The class B GPCR family referred to as the secretin-like receptor class. Class B is comprised of 34 subclasses (subfamilies) and members include receptors for peptide hormones, such as parathyroid hormone (PTH), parathyroid hormone-related protein (PTHrP), and calcitonin. The class B family also contains the vast majority of the orphan GPCRs.

Class C Family: The class C family is referred to as the metabotropic receptor or glutamate receptor-like family. Class C is comprised of 8 subclasses (subfamilies) and members all form dimers and include the metabotropic glutamate receptors (mGluR), extracellular Ca2+-sensing receptors, taste (gustatory) receptors, and several odorant receptors, as well as the pheromone receptors.

The vast majority of G-proteins to which GPCRs are coupled are members of the heterotrimeric G-protein family (see above). 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.

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Receptor 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.

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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
ACTH
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

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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. 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).

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 co-activator 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 (also known as nuclear receptor coactivator 1: NCOA1), SRC-2 (also known as NCOA2, glucocorticoid receptor-interacting protein 1: GRIP1; and transcriptional intermediary factor 2: TIF2) and SRC-3 (also known as NCOA3; amplified in breast cancer 1: AIB1; and thyroid hormone receptor activator molecule 1: TRAM-1). 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.

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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 and DAG. Activation of PLC results 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.

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, 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 within 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.

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.

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).

Phosphatidylinositol-3-Kinase (PI3K)

PI3K is tyrosine phosphorylated, and subsequently activated, by various RTKs and receptor-associated PTKs. PI3K is a heterodimeric protein containing an 85 kDa and 110 kDa subunits. The p85 subunit contains SH2 domains that interact with activated receptors or other receptor-associated PTKs and is itself subsequently tyrosine phosphorylated and activated. The 85 kDa subunit is non-catalytic, however, it does contain a domain homologous to GTPase activating (GAP) proteins. It is the 110 kDa subunit that is enzymatically active. PI3K, associates with and is activated by, the PDGF, EGF, insulin, IGF-1, HGF and NGF receptors. PI3K phosphorylates various phosphatidylinositols at the 3 position of the inositol ring. This activity generates additional substrates for PLCγ allowing a cascade of DAG and IP3 to be generated by a single activated RTK or other protein tyrosine kinases.

Lysophospholipids

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, Akt/PKB 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.

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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). One class are transmembrane enzymes which contain the phosphatase activity domain in the intracellular portion of the protein. This class of PTP is commonly called the receptor (R) class of PTP. The other class is intracellularly localized enzymes and are referred to as NT PTPs (for non-transmembrane). Currently over 40 genes have been characterized as encoding one or the other class of PTP. The first transmembrane PTP characterized was the leukocyte common antigen protein, CD45. This protein was shown to have homology to the intracellular PTP, PTP1B. There are at least eight sub-classes of the transmembrane PTPs and ten sub-classes of the NT PTPs.

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).

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 PTP1B in oocytes resulted in a marked retardation in the rate of insulin- and progesterone-induced maturation. These results suggest a role for PTP1B in countering the signals leading to cellular activation.

The above observation as well as several others have demonstrated a link between insulin function and PTP-1B. PTP1B 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. Recently the PTP-B gene was disrupted in mice by targeted deletion. Mice lacking a functional PTP-1B 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 (PTP1C and PTP1D) 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 subunit to associate with a large variety of different regulatory subunits. The representative members of the PPP family include protein phosphatase 1 (PP1), PP2A, PP2B (commonly known as calcineurin), PP4, PP5, PP6, and PP7.

As the name implies the PPM family is represented by protein phosphatases that are dependent upon manganese/magnesium ions (Mn2+/Mg2+), such as PP2C and pyruvate dehydrogenase phosphatase. 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.

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): PP1 is a major protein S/T phosphatase. Expression of PP1 isoforms is found in all eukaryotic cells. There are three PP1 genes in humans encoding the catalytic isoforms designated PP1α (PP1CA gene), PP1γ (PP1CG gene), and PP1β/δ (PP1CB gene, also known as PP1CD). The PP1CG gene is alternatively spliced resulting in PP1γ1 and PP1γ2 isoforms. Expression of the PP1γ2 isoform is enriched in testes but the other forms are ubiquitously expressed. As indicated below, the catalytic subunits of PP1 do not exist as free proteins in cells but are associated with a wide range regulatory proteins that number more than 50.

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.

The phosphatase activity of PP1 is regulated by a number of inhibitory proteins such as inhibitor-1 (I-1; also known as PPI-1), 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 I-1 or I-2. It is this functional difference that was the basis for classification of type 1 (PP1) versus type 2 phosphatases. By analogy with the PP1 inhibitors, the endogenous inhibitors of PP2A were named I1 PP2A and I2 PP2A. These two endogenous PP2A inhibitors are also known as putative HLA class II-associated protein I (PHAP-I) and SET (suppressor of variegation, enhancer of zeste, and Trithorax), respectively.

Protein Phosphatase 2A (PP2A): The protein phosphatases that are involved in the regulation of metabolic processes are of two types: type 1, namely PP1, and type 2, which consisted of three enzymes: PP2A, PP2B, and PP2C. PP2A plays an important role in development, cell proliferation, apoptosis, cell mobility, cytoskeleton dynamics, the control of the cell cycle, and the regulation of numerous signaling pathways. PP2A has also been suggested to be a tumor suppressor. PP2A is one of the most abundant enzymes in cells and can account for up to 1% of total cellular protein in some tissues.

PP2A is highly conserved across a variety of eukaryotic species. The mechanisms of its regulatory action are highly complex. PP2A exists in two basic isoforms: a heterodimeric core enzyme and a heterotrimeric holoenzyme. The PP2A core enzyme consists of a scaffold subunit and a catalytic subunit. The scaffold and the catalytic subunits each exist in two isoforms: α and β. The α 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 regulatory subunits comprise four families: B (also known as B55 or PR55), B′ (B56 or PR61), B′′ (PR48/PR72/PR130), and B′′′ (PR93/PR110). Each family consists of two to five isoforms that are encoded by different gene and some isoforms have multiple splice variants. Except for subunits of the B′′′ family, all members of these regulatory subunit families have been shown to bind directly to the PP2A core enzyme. The level of expression of the various regulatory subunit genes varies greatly in different cell types and tissues. The PP2A scaffold subunit contains 15 tandem HEAT (huntingtin- elongation-A subunit-TOR) repeats. The catalytic subunit of PP2A recognizes 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 subunit. The catalytic subunit of PP2A is a target 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 subunit.

The heterotrimeric PP2A holoenzyme is believed to exhibit exquisite substrate specificity as well as spatially and temporally determined functions. For example, B, but not B′ or B′′, subunit has been shown to be responsible for dephosphorylation of the microtubule-binding protein Tau. Likewise, whereas the PP2A holoenzyme containing the B′ subunit interacts with the catalytic subunit, the B subunit makes few interactions with the catalytic subunit. A shared feature of two PP2A holoenzymes that have been structurally defined is that the potential substrate-binding site is on the top face of the regulatory subunit and close to the active site of the catalytic subunit. This feature supports the notion that a major function of the regulatory subunits is to target substrate phosphoproteins to the phosphatase activity of PP2A.

Reversible methylation of the PP2A core enzyme 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 subunit 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 PP2A 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.

Protein Phosphatase 2B/Calcineurin (PP2B): Protein phosphatase 2B (PP2B, also known as calcineurin) 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. Calcineurin consists of a catalytic subunit (calcineurin A or CNA) and a regulatory subunit (calcineurin B or CNB). CNA contains an N-terminal phosphatase domain, followed by a CNB-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 CNA is structurally similar to the catalytic subunit of PP1 and has the same pattern of metal ion coordination. The two metal ions associated with calcineurin are Zn2+ and Fe3+. CNB consists of a pair of Ca2+-binding domains. All four calcium-binding sites in CNB are bound to Ca2+ and each calcium ion is coordinated by five oxygen atoms. The two Ca2+-binding domains of CNB are organized around the CNB-binding helical domain (BBH). The exposed face of BBH forms a composite binding surface with neighboring residues of CNB. The immunosuppressant complexes, FKBP12-FK506 and cyclophilin A (CyPA)-cyclosporin A (CsA) both associate with an exposed surface through a similar set of interactions. 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 from both CNB and the BBH domains. 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. Structural analysis of calcineurin revealed that the Ca2+-CaM-binding motif forms a contiguous α helix, which organizes two Ca2+-CaM molecules into a head-to-tail dimer indicating that calcineurin may form a dimer upon activation by Ca2+-CaM.

Protein Phosphatase 2C (PP2C): PP2C and pyruvate dehydrogenase (PDH) phosphatases belong to the Mn2+/Mg2+-dependent PPM family. PP2C represents a large family of highly conserved protein phosphatases, with 16 distinct PP2C genes in the human genome that give rise to at least 22 different isoforms. Unlike the PPP family phosphatases, PP2C is insensitive to inhibition by okadaic acid or microcystin. The primary function of PP2C is in the regulation of cellular stress signaling. PP2C also plays a role in metabolism, differentiation, growth, survival, and apoptosis. Several members of the PP2C family are candidate tumor suppressor proteins. These include PP2Cα, PP2Cβ, and plextrin homology (PH) domain leucine-rich repeat protein phosphatase (PHLPP). On the other hand PP2Cδ (also known as Wip1) may contribute to oncogenic transformation.

The conserved catalytic domain of human PP2C 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 PP2C phosphatases is similar to that of the PPP family. The dephosphorylation reaction involves nucleophilic attack of the phosphorous by a metal-activated water nucleophile.

The PP2C family has a large number of isoforms encoded by different genes. The different isoforms have distinct sequences and domain organizations. The different PP2C isoforms also exhibit distinct functions, expression patterns, and subcellular localization. The molecular determinants of substrate specificity for the various PP2C isoforms has yet to be fully elucidated. In addition to the conserved PP2C phosphatase domain, the family member, PHLPP, also contains an N-terminal PH domain and a leucine-rich repeat (LRR) domain. It is believed that PHLPP and PHLPP2 promote apoptosis and act as tumor suppressors via the dephosphorylation of distinct PKB/Akt isoforms.

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.

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Last modified: January 2, 2014