IDENTIFICATION OF G(Q) AS ONE OF THE G-PROTEINS WHICH COPURIFY WITH HUMAN PLATELET THROMBOXANE-A(2) PROSTAGLANDIN-H(2) RECEPTORS

The present study employed ligand affinity and immunoaffinity chromato graphy to isolate human platelet thromboxane A2/prostaglandin H-2 (TXA 2/PGH2) receptor-coupled G-proteins. Purification of TXA2/PGH2 recepto rs by ligand (SQ31,491)-affinity chromatography resulted in the elutio n of receptor binding and GTPase activity in the same fraction. GTPase activity of this fraction was enriched (6-fold) relative to solubiliz ed platelet membranes, was stimulated (65%) by 500 nM U46619, and was blocked (74%) by 250 nM SQ29,548. Furthermore, GTP (100 muM) increased [H-3]SQ29,548 receptor binding by 48%. Immunoblotting of this fractio n against QL antiserum identified a 42-kDa protein as a member of the G(q) family. In separate experiments, TXA2/PGH2 receptors were purifie d by immunoaffinity chromatography using P1Ab, P2Ab, and TxAb affinity columns. QL-immunoreactive proteins at 42 kDa were found in all three column eluates. Studies using G(alpha,common) antiserum (GA/1) demons trated immunoblotting of two proteins of approximately 42 and 85 kDa i n both the ligand and P2Ab affinity column fractions. On the other han d, the P1Ab and TxAb affinity column eluates contained GA/1 immunoreac tivity only in the 42 kDa region. Collectively, these data identify G( q) as a TXA2/PGH2 receptor-coupled G-protein and suggest the associati on of this receptor with additional G(alpha) subunits.