Identification of Gα13 as One of the G-proteins That Couple to Human Platelet Thromboxane A2 Receptors

Abstract

Previous studies have shown that ligand or immunoaffinity chromatography can be used to purify the human platelet thromboxane A 2 (TXA 2 ) receptor-G␣ q complex. The same principle of co-elution was used to identify another G-protein associated with platelet TXA 2 receptors. It was found that in addition to G␣ q , purification of TXA 2 receptors by ligand (SQ31,491)-affinity chromatography resulted in the co-purification of a member of the G 12 family. Using an antipeptide antibody specific for the human G 13 ␣-subunit, this G-protein was identified as G␣ 13 . In separate experiments, it was found that the TXA 2 receptor agonist U46619 stimulated [35 S]guanosine 5-O-(3-thiotriphosphate) incorporation into G 13 ␣-subunit. Further evidence for functional coupling of G 13 to TXA 2 receptors was provided in studies where solubilized platelet membranes were subjected to immunoaffinity chromatography using an antibody raised against native TXA 2 receptor protein. It was found that U46619 induced a significant decrease in G␣ q and G␣ 13 association with the receptor protein.These results indicate that both G␣ q and G␣ 13 are functionally coupled to TXA 2 receptors and dissociate upon agonist activation. Furthermore, this agonist effect was specifically blocked by pretreatment with the TXA 2 receptor antagonist, BM13.505. Taken collectively, these data provide direct evidence that endogenous G␣ 13 is a TXA 2 receptor-coupled G-protein, as: 1) its ␣-subunit can be co-purified with the receptor protein using both ligand and immunoaffinity chromatography, 2) TXA 2 receptor activation stimulates GTP␥S binding to G␣ 13 , and 3) G␣ 13 affinity for the TXA 2 receptor can be modulated by agonist-receptor activation.

Purification, Cloning, Expression, and Mechanism of Action of a Novel Platelet Aggregation Inhibitor from the Salivary Gland of the Blood-sucking Bug, Rhodnius prolixus

“…To investigate such a mechanism, platelet aggregation was studied using an aggregometer. Platelet response to U46619 is mediated by two different receptors or by the same receptor with two different affinities as follows: one binding site that binds U46619 with high affinity that mediates platelet shape change, Ca 2ϩ increase, and cortactin tyrosine phosphorylation (15)(16)(17), and a second binding site that binds U46619 with low affinity and mediates platelet aggregation, inositol-phosphate production, and protein kinase C activation (18 -20). Fig.…”

“…A number of studies have shown that either TXA 2 has two distinct TP receptors on platelets or, alternatively, two different affinity states of the same receptor may exist as follows: one of high affinity that mediates platelet shape change, Ca 2ϩ mobilization, and cortactin tyrosyl phosphorylation, and a second of low affinity that mediates PKC activation and platelet aggregation (15)(16)(17)(18)(19)72). Because U46619-induced platelet shape change and calcium increase was preserved by RPAI-1, it was clear that the high affinity TP receptor on platelet membrane was not the target of RPAI-1.…”

“…Activation of both P2Y 1 and P2Y AC receptors is necessary for full platelet activation by ADP (12)(13)(14). Finally, a number of papers (15)(16)(17) have identified, through binding experiments, two binding sites for thromboxane A 2 (TXA 2 ) in platelets. One site binds TXA 2 mimetics with high affinity and mediates shape change, calcium increase, and cortactin phosphorylation by a G␣ 12/13 -coupled receptor.…”

Thromboxane A2 Signalling in Humans: a ‘Tail’ of Two Receptors

“…Whereas many of the latter reported studies have implicated various G protein α subunits in mediating TP activation [45][46][47][48][49][50][51][52][53][54][55] and the latter studies [33,60] mobilization by TPα and TPβ indicating that the TP isoforms may mediate opening of L-type channels in a Gα 12 dependent signalling mechanism [61]. Taken together, these studies investigating the signalling by the TP isoforms indicate that both TPα and TPβ couple to both the G q and G 12 family with no apparent isoform specific differences in their signalling behaviour.…”

Protein kinase C- and calcium-regulated pathways independently synergize with Gi pathways in agonist-induced fibrinogen receptor activation

“…Two GPCRs for ADP, the P2Y1 and P2Y12 receptors, stimulate the G q and G i signalling pathways, respectively, and their co-stimulation is needed for ADP-induced primary aggregation [9][10][11][12]. The thromboxane A # receptor also couples to G q , as well as G "# and G "$ [13], but requires agonists secreted from platelets for activation of G i pathways and aggregation [7]. Thrombin activates a family of G-protein-coupled protease-activated receptors (PARs).…”