Identification of G alpha(13) as one of the G-proteins that couple to human platelet thromboxane A(2) receptors

Previous studies have shown that ligand or immunoaffinity chromatography ca n be used to purify the human platelet thromboxane A(2) (TXA(2)) receptor-G alpha(q) complex. The same principle of co-elution was used to identify an other G-protein associated with platelet TXA(2) receptors, It was found tha t in addition to G alpha(q), purification of TXA(2) receptors by ligand (SQ 31,491)-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) alpha-subunit, this G-protein was identified as G alpha(13). In separ ate experiments, it was found that the TXA(2) receptor agonist U46619 stimu lated [S-35]guanosine 5'-O-(3-thiotriphosphate) incorporation into G(13) al pha-subunit. Further evidence for functional coupling of G(13) to TXA(2) re ceptors was provided in studies where solubilized platelet membranes were s ubjected to immunoaffinity chromatography using an antibody raised against native TXA(2) receptor protein. It was found that U46619 induced a signific ant decrease in G alpha(q) and G alpha(13) association with the receptor pr otein. These results indicate that both G alpha(q) and G alpha(13) are func tionally 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 d ata provide direct evidence that endogenous G alpha(13) is a TXA(2) recepto r-coupled G-protein, as: 1) its alpha-subunit can be co-purified with the r eceptor protein using both ligand and immunoaffinity chromatography, 2) TXA (2) receptor activation stimulates GTP gamma S binding to G alpha(13), and 3) G alpha(13) affinity for the TXA(2) receptor can be modulated by agonist -receptor activation.