RECONSTITUTION OF RECEPTORS AND GTP-BINDING REGULATORY PROTEINS (G-PROTEINS) IN SF9 CELLS - A DIRECT EVALUATION OF SELECTIVITY IN RECEPTOR-CENTER-DOT-G PROTEIN COUPLING

The selectivity in coupling of various receptors to GTP-binding regula tory proteins (G proteins) was examined directly by a novel assay enta iling the use of proteins overexpressed in Spodoptera frugiperda (Sf9) cells, Activation of G proteins was monitored in membranes prepared f rom Sf9 cells co-expressing selected pairs of receptors and G proteins (i.e. alpha, beta(1), and gamma(2) subunits), Membranes were incubate d with [S-35]guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) +/- an agonist, and the amount of radiolabel bound to the (alpha subunit was quantitated following immunoprecipitation. When expressed without rece ptor (but with beta(1) gamma(2)), the G protein subunits alpha(z), alp ha(12), and alpha(13) did not bind appreciable levels of [S-35]GTP gam ma S consistent with a minimal level of GDP/[S-35]GTP gamma S exchange . In contrast, the subunits alpha(s) and alpha(q) bound measurable lev els of the nucleotide, Co-expression of the 5-hydroxytryptamine(1A) (5 -HT1A) receptor promoted binding of [S-35]GTP gamma S to alpha(z) but not to alpha(12), alpha(13), or alpha(s). Binding to alpha(z) was enha nced by inclusion of serotonin in the assay, Agonist activation of bot h thrombin and neurokinin-l receptors promoted a modest increase in [S -35]GTP gamma S binding to alpha(z) and more robust increases in bindi ng to alpha(q), alpha(12), and alpha(13). Binding of [S-35]GTP gamma S to alpha(s) was strongly enhanced only by the activated beta(1)-adren ergic receptor, Our data identify interactions of receptors and G prot eins directly, without resort to measurements of effector activity, co nfirm the coupling of the 5-HT1A receptor to G(z) and extend the list of receptors that interact with this unique G protein to the receptors for thrombin and substance P, imply constitutive activity for the 5-H T1A receptor, and demonstrate for the first time that the cloned recep tors for thrombin and substance P activate G(12) and G(13).