Identification of Galpha13 as one of the G-proteins that couple to human platelet thromboxane A2 receptors.
Previous studies have shown that ligand or immunoaffinity chromatography can be used to purify the human platelet thromboxane A2 (TXA2) receptor-Galphaq 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 Galphaq, 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 alpha-subunit, this G-protein was identified as Galpha13. In separate experiments, it was found that the TXA2 receptor agonist U46619 stimulated [35S]guanosine 5'-O-(3-thiotriphosphate) incorporation into G13 alpha-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 Galphaq and Galpha13 association with the receptor protein. These results indicate that both Galphaq and Galpha13 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 Galpha13 is a TXA2 receptor-coupled G-protein, as: 1) its alpha-subunit can be co-purified with the receptor protein using both ligand and immunoaffinity chromatography, 2) TXA2 receptor activation stimulates GTPgammaS binding to Galpha13, and 3) Galpha13 affinity for the TXA2 receptor can be modulated by agonist-receptor activation.
D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid / Q000494:pharmacology; D001792:Blood Platelets / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D051960:GTP-Binding Protein alpha Subunit, Gi2; D044365:GTP-Binding Protein alpha Subunits, G12-G13; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D019204:GTP-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D011518:Proto-Oncogene Proteins / Q000302:isolation & purification / Q000378:metabolism; D017482:Receptors, Thromboxane / Q000302:isolation & purification / Q000378:metabolism*; D014662:Vasoconstrictor Agents / Q000494:pharmacology