Last Modified: December 4, 2024
Purine and Pyrimidine Nucleotide Signaling
The intracellular signaling pathways that involve nucleotides as effectors are quite broad. The details of the intracellular cyclic nucleotides in signal transduction processes are described in the Signal Transduction Pathways: Cyclic Nucleotides and Kinases page. Extracellularly nucleotides also exert critically important processes of signal transduction. Although numerous extracellular nucleotides are known to exert effects, it is predominantly ATP that is the major initiator.
Receptors that bind purines were initially characterized in the early 1970’s and in 1978 Geoffrey Burnstock, considered the father of purinoreceptor research, proposed a nomenclature to divide the two broad classes of receptors for ATP, adenosine 5′ -diphosphate (ADP), adenosine 5′-monophosphate (AMP) and adenosine. This nomenclature identified the purinoreceptors that were selectively activated by adenosine and AMP, leading to changes in cytoplasmic cAMP levels, and selectively antagonized by methylxanthines, as the P1 receptors. The P2 class of purinoreceptors were defined as those that were selectively stimulated by ATP and ADP, had no effect on cytoplasmic cAMP levels, and were unaffected by methylxanthines. The P1 receptors are now referred to as the adenosine receptors and the P2 receptors have been classified as the P2X and P2Y receptors.
These purinoreceptors are members of either the G-protein coupled receptor (GPCR) family or the ionotropic family of ligand-gated ion channel receptors. The ionotropic purinoreceptors are identified as the P2X receptors and the metabotropic purinoreceptors are identified as the P2Y receptors and the adenosine receptors.
In addition to nucleotide receptor-mediated signal transduction mechanisms occurring directly within target cells, the signaling cascades activated by P2X and P2Y receptors result in intercellular communication through the stimulated release of other extracellular messenger substances. These concerted effects result in the activation of additional signal transduction pathways via the receptors to which the secondary messengers bind.
The signaling substances that are induced to be released following nucleotide receptor activation include neurotransmitters, hormones, growth factors, lipid mediators, nitric oxide (NO), numerous proteins (e.g. enzymes and cytokines), and reactive oxygen species (ROS). Of considerable significance to the overall effects of nucleotide receptor activation is that nucleotides also activate or co-activate numerous growth factor receptors.
P2X Receptors: Ligand-Gated Ion Channels
The P2X family of receptors are ATP-gated non-selective ion channels (ligand-gated ion channels). Each of the P2X receptors are homo- or heterotrimeric channels that primarily transport Na+, K+, or Ca2+ ions in response to the binding of extracellular ATP. There are seven subtypes of P2X receptors in humans identified as P2X1–P2X7. The genes that encode these receptor proteins are identified as P2RX genes (P2RX1–P2RX7). These genes are expressed in a wide array of cell types including both excitatory and non-excitatory cells. The P2RX genes are also identified by the P2X*R (e.g. P2RX1 and P2X1R are synonymous) nomenclature.
All seven P2X receptor proteins are composed of common domains. Each protein possesses two transmembrane (TM) domains (TM1 and TM2) that are separated by a glycosylated, disulfide-rich extracellular loop of about 280 amino acids. The
extracellular loop contains 10 cysteine residues that are bound in five disulfide bridges. Each P2X receptor has its N-terminus and C-terminus intracellularly. The N-terminus is approximately 20 to 30 amino acids in length and is associated with TM1. The C-terminus varies in length from 26 to 239 amino acids and is associated with TM2. The extracellular portion of theses receptors contains a large loop with binding sites for ATP, metal ions, as well as agonist.
Each of the seven receptors are known to form homotrimeric receptor channels. Heterotrimeric P2X receptor channels are predominantly composed of subunits from two different P2X receptor genes and include the P2X1/P2X2, P2X1/P2X4, P2X1/P2X5, P2X2/P2X3, P2X2/P2X6, and P2X4/P2X6 heterotrimers. However, a heterotrimeric P2X receptor composed of P2X2/P2X4/P2X6 has been identified in Leydig cells.
The signal transduction cascades initiated by activation of P2X receptors include neuronal synaptic transmission, nociception (sensation of pain), taste, modulation of inflammatory processes, modulation of cardiovascular processes, and tumorigenesis. As might be expected, the loss of function of several P2X encoding genes has been associated with numerous pathological conditions.
The primary agonists of the P2X receptors are ATP, CTP, and GTP. Other nucleotides including ADP, AMP, adenosine, UTP, UDP, and UMP are weak agonists for some of the P2X receptors, but not all. The initial response to ATP binding to the P2X receptors is channel opening and ion flow. Following prolonged exposure to ATP the receptor becomes desensitized such that ion influx terminates even in the presence of bound ATP. Following ATP dissociation the receptor returns to a sensitized state capable of responding again to ATP binding.
In addition to channel activation by ATP binding, the P2X receptors are also regulated by numerous allosteric effectors such as pH and heavy metals. At pH levels below 7, ATP effects on P2X1, P2X3, and P2X4 are attenuated, whereas, at the P2X1 channel ion currents are inhibited by acidic pH. Zinc ions (Zn2+), which represents the second most abundant trace metal in the body (second to iron) have been shown to have important P2X modulating effects. At low concentration Zn2+ can potentiate the ion current through the P2X2, P2X3, and P2X4 channels following ATP binding, but at higher concentration Zn2+ exerts an inhibitory effect on the channel. Intracellularly phospholipids exert increased ion flux through the channel through interactions with the C-terminus of the protein.
The effects of phospholipids has been shown to be exerted on the P2X1, P2X2, and P2X4 homotrimeric receptors and the P2X1/P2X5 and P2X2/P2X3 heterotrimeric receptors.
P2X1
The P2X1 receptor protein (identified as P2X1R as well as P2RX1) is primarily expressed in smooth muscle, platelets, and the CNS. The primary ion transported through the P2X1R ion channel is Ca2+, however, influx of Na+ ions also occurs in response to P2X1R activation. Within platelets the activation of P2X1R leads to a rapid shape change leading to further platelet activation. Within smooth muscle cells the P2X1R is involved in the vasoconstrictive actions in the arteries and arterioles, as well as in the vas deferens, induced by sympathetic innervation. Function of the P2X1R is essential during the development and functioning of the male reproductive system.
The P2RX1 gene is located on chromosome 17p13.2 and is composed of 13 exons that encode a 399 amino acid protein.
P2X2
The P2X2 receptor protein (identified as P2X2R as well as P2RX2) is expressed in the CNS, autonomic and sensory ganglia, smooth muscle, the pancreas as well as in the pituitary and in urinary bladder cells. P2X2 receptors are important in the transition of neural progenitor cells to functional neurons. The P2X2 receptor is also important to the regulation of hearing function in the cochlea.
The P2RX2 gene is located on chromosome 12q24.33 (near the location of the P2RX4 and P2RX7 genes) and is composed of 10 exons that generate eight alternatively spliced mRNAs encoding eight distinct protein isoforms.
P2X3
The P2X3 receptor protein (identified as P2X3R as well as P2RX3) is expressed the CNS, sympathetic neurons, and in nociceptive (pain sensation) sensory neurons.
The P2RX3 gene is located on chromosome 11q12.1 and is composed of 16 exons that encode a 397 amino acid protein.
P2X4
The P2X4 receptor protein (identified as P2X4R as well as P2RX4) is expressed in the CNS, microglial cells, testis, colon, and endothelial cells.
The P2RX4 gene is located on chromosome 12q24.31 and is composed of 14 exons that generate four alternatively spliced mRNAs each of which encode a distinct protein isoform. Additional non-coding alternative transcripts have been shown to be derived from the P2RX4 gene.
The P2RX4 protein interacts with 2-oxoglutarate (α-ketoglutarate) and in so doing contributes to the inhibitory effect of 2-oxoglutarate on hepatic gluconeogenesis.
P2X5
The P2X5 receptor protein (identified as P2X5R as well as P2RX5) is expressed in the skin, skeletal muscle, and epithelial cells.
The P2RX5 gene is located on chromosome 17p13.2 and is composed of 12 exons that generate four alternatively spliced mRNAs each of which encodes a distinct protein isoform. There is also a read-through transcript that includes the downstream Tax1 binding protein 3 (TAX1BP3) gene.
P2X6
The P2X6 receptor protein (identified as P2X6R as well as P2RX6) is expressed in the CNS and in peripheral nerves.
The P2RX6 gene is located on chromosome 22q11.21and is composed of 14 exons that generate two alternatively spliced mRNAs encoding two distinct protein isoforms.
P2X7
The P2X7 receptor (identified as P2X7R as well as P2RX7) is unique among the seven P2X receptors in that it does not significantly function at normal physiological concentrations of extracellular ATP indicating that it most likely functions in the mediation of potential pathophysiological states such as those associated with inflammation and stress. In addition, activation of P2X7R can trigger signal transduction cascades that involve the activation of caspase 1, phospholipases A2 and D (PLA2 and PLD), the MAP kinases (MAPK), PKC, SRC, and glycogen synthase kinase-3 (GSK-3), as well as several phosphatases.
The ion channel function of P2X7 is modulated by several factors. The ions, Cl–, H+, and Mg2+ inhibit activation of the channel. Protons (H+) exert this effect by ionizing an aspartate residue at position 197 (D197) which resides in the extracellular domain of the receptor. Within the intracellular portion of the channel the C-terminus mediates interactions with other proteins and stimulates channel opening. Within the second transmembrane domain (TM2) there is a critical serine residue (S342) that serves as the gate and the selectivity filter of the channel.
The P2RX7 gene is located on chromosome 12q24.31 and is composed of 14 exons that encode a 595 amino acid protein. Additional P2XR7 transcripts are generated but do not encode functional proteins and are degraded by the nonsense-mediated decay pathway. The P2X7 receptor protein (P2X7R) is expressed in microglial cells, oligodendrocytes, macrophages, mast cells, immune cells, and in the pancreas and skin.
P2Y Receptors: G-Protein Coupled Receptors, GPCR
Extracellular purine and pyrimidine nucleotides bind to and activate a class of metabotropic G-protein coupled receptors (GPCR) termed the P2Y receptor family. Humans express ten P2Y receptor genes encoding the P2Y1, P2Y2, P2Y4, P2Y6, P2Y8, and P2Y10–P2Y14 receptors. The genes that encode the P2Y receptors are identified by P2RY with the corresponding identifying number. The P2RY8 and P2RY10 encoded proteins are defined as being members of the G protein-coupled receptors, class A orphan family.
Several GPCR that are structural members of the P2Y family are activated by ligands that are not nucleotides. This includes the receptor identified as GPR23 which has been shown to be activated by lysophosphatidic acid (LPA) binding, and is also identified as P2Y9 and LPA4. Another LPA receptor, LPA6, is also identified as P2Y5.
Although purine nucleotides, primarily ADP (but in some cases also ATP) are the preferred ligands for the P2Y receptors, the P2Y4 and P2Y6 receptors are only activated by the pyrimidines, UTP and UDP, respectively. The P2Y2 receptor can be activated by both ATP and UTP while the P2Y11 receptor is only activated by ATP.
The natural ligands of the P2Y14 receptor are both UDP and UDP-activated sugars. UDP-glucose was the originally identified activator of P2Y14 and it is now known to be activated by UDP-glucose, UDP-galactose, UDP-glucuronic acid, and UDP-N-acetylglucosamine (UDP-GlcNAc).
In addition to the activation of the associated Gα-subunits, several P2Y receptor-associated G-proteins release functional Gβγ-subunits following Gα-subunit release and activation. The Gβγ subunits of P2Y receptors have been shown to be involved in the activation of numerous signaling cascades that involve phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), inwardly rectifying K+ (GIRK) channels, MAP kinase, monomeric G-proteins of the RHO family, and G-protein receptor kinases (GRK).
The P2RY genes are expressed in numerous tissues with various members having selective significance in specific tissues. For example, the P2Y12 receptor (and likely the P2Y1 receptor) is critical in the involvement of platelet function in blood coagulation.
P2Y1
The P2Y1 receptor activates an associated Gq-type G-protein leading to the production of the second messengers, DAG and IP3.
The P2RY1 gene is an intronless gene located on chromosome 3q25.2 that encodes a 373 amino acid protein.
P2Y2
The P2Y2 receptor activates an associated Gq-type G-protein leading to the production of the second messengers, DAG and IP3. The P2RY2 gene is located on chromosome 11q13.43 is composed of 12 exons that generate three alternatively spliced mRNAs each of which encode the same 377 amino acid protein.
P2Y4
The P2Y4 receptor activates an associated Gq-type G-protein leading to the production of the second messengers, DAG and IP3. The P2RY4 gene is an intronless gene located on the X chromosome (Xq13.1) that encodes a 365 amino acid protein.
P2Y6
The P2Y6 receptor activates an associated Gq-type G-protein leading to the production of the second messengers, DAG and IP3. The P2RY6 gene is located on chromosome 11q13.4 and is composed of 7 exons that generate eight alternatively spliced mRNAs that collectively encode two proteins, isoform 1 is a 328 amino acid protein while isoform 2 is 429 amino acid protein.
P2Y8
The P2RY8 gene is highly expressed in lymphocyte precursor cells. A chromosomal abnormality that involves the P2RY8 gene has been found to be associated 5-7% of pediatric B-cell progenitor acute lymphoblastic leukemia (B-ALL) and also with >50% of Down syndrome-associated acute lymphoblastic leukemia. This chromosomal abnormality results from an interstitial deletion that places the promoter region of the P2RY8 gene upstream of the cytokine receptor-like factor 2 (CRLF2) gene. This chromosomal disruption results in high level expression of full-length function CRLF2 leading to the overgrowth of the B-progenitor cells.
The P2RY8 gene is located on both the X chromosome (Xp22.33) and the Y chromosome (Yp11.2) both of which are composed of 6 exons that encode the same 359 amino acid proteins.
P2Y10
P2Y10 was shown to be a receptor for lysophosphatidylserine (lysoPS) and as such it has been proposed that the nomenclature be changed to LPS2. LysoPS also binds and activates GPR34 (LPS1) and GPR174 (LPS3). P2Y10 has also been shown to be activated by lysophosphatidic acid (lysoPA or LPA).
The P2RY10 gene is located on the X chromosome (Xq21.1) and is composed of 5 exons that generate five alternatively spliced mRNAs encoding three distinct protein isoforms.
P2Y11
The P2Y11 receptor activates both Gq– and Gs-type G-proteins and is considered to be a functional member of the P2Y1 family of receptors despite only ~30% amino acid sequence identity to the other members of this family.
The P2RY11 gene is located on chromosome 19p13.2 and is composed of 2 exons that encode a 374 amino acid protein. A read-through transcription product is generated from the P2RY11 gene and the immediate upstream gene that encodes a human homolog (PPAN) of the Drosophila peter pan gene. This read-through transcript encodes a fusion protein that exhibits functionality of both individual gene products.
P2Y12
The P2Y12 receptor activates an associated Gi-type G-protein resulting in the inhibition of adenylate cyclase and, therefore, reduced levels of cAMP and consequent decreases in PKA activity.
The P2RY12 gene is located on chromosome 3 (3q25.1) near the location of the P2RY1 gene and is composed of 4 exons that generate two alternatively spliced mRNAs that both encode the same 342 amino acid protein. P2Y12 is a significant regulator of platelet function through ADP-promoted inhibition of adenylyl cyclase and ADP-mediated platelet aggregation. In addition, is is expressed in the brain where is mediates cell migration responses of microglia during inflammation.
P2Y13
The P2Y13 receptor activates an associated Gi-type G-protein resulting in the inhibition of adenylate cyclase and, therefore, reduced levels of cAMP and consequent decreases in PKA activity.
The P2RY13 gene is also located in the same region of chromosome 3 (3q25.1) as the P2RY12 gene and is composed of 2 exons that encode a 354 amino acid protein.
P2Y14
The P2Y14 receptor activates an associated Gi-type G-protein resulting in the inhibition of adenylate cyclase and, therefore, reduced levels of cAMP and consequent decreases in PKA activity.
The P2RY14 gene is also located in the same 3q25.1 region as the P2RY12 and P2RY13 genes and is composed of 6 exons that generate two alternatively spliced mRNAs both of which encode the same 338 amino acid protein.