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 A2 (TXA2) receptor- Gα(q) complex. The same principle of co-elution was used to identify another G-protein associated with platelet TXA2 receptors. It was found that in addition to Gα(q), purification of TXA2 receptors by ligand (SQ31,491)- affinity chromatography resulted in the co-purification of a member of the G12 family. Using an antipeptide antibody specific for the human G13 α- subunit, this G-protein was identified as Gα13. In separate experiments, it was found that the TXA2 receptor agonist U46619 stimulated [35S]guanosine 5'-O-(3-thiotriphosphate) incorporation into G13 α- subunit. Further evidence for functional coupling of G13 to TXA2 receptors was provided in studies where solubilized platelet membranes were subjected to immunoaffinity chromatography using an antibody raised against native TXA2 receptor protein. It was found that U46619 induced a significant decrease in Gα(q) and Gα13 association with the receptor protein. These results indicate that both Gα(q) and Gα13 are functionally coupled to TXA2 receptors and dissociate upon agonist activation. Furthermore, this agonist effect was specifically blocked by pretreatment with the TXA2 receptor antagonist, BM13.505. Taken collectively, these data provide direct evidence that endogenous Gα13 is a TXA2 receptor-coupled G-protein, as: 1) its α-subunit can be co-purified with the receptor protein using both ligand and immunoaffinity chromatography, 2) TXA2 receptor activation stimulates GTPγS binding to Gα13, and 3) Gα13 affinity for the TXA2 receptor can be modulated by agonist-receptor activation.