title: Reconstitution of receptors and GTP-binding regulatory proteins (G proteins) in Sf9 cells. A direct evaluation of selectivity in receptor.G protein coupling.
abstract: The selectivity in coupling of various receptors to GTP-binding regulatory proteins (G proteins) was examined directly by a novel assay entailing the use of proteins overexpressed in Spodoptera frugiperda (Sf9) cells. Activation of G proteins was monitored in membranes prepared from Sf9 cells co-expressing selected pairs of receptors and G proteins (i.e. alpha, beta1, and gamma2 subunits). Membranes were incubated with [35S]guanosine 5'-(3-O-thio)triphosphate (GTPgammaS) +/- an agonist, and the amount of radiolabel bound to the alpha subunit was quantitated following immunoprecipitation. When expressed without receptor (but with beta1gamma2), the G protein subunits alphaz, alpha12, and alpha13 did not bind appreciable levels of [35S]GTPgammaS, consistent with a minimal level of GDP/[35S]GTPgammaS exchange. In contrast, the subunits alphas and alphaq bound measurable levels of the nucleotide. Co-expression of the 5-hydroxytryptamine1A (5-HT1A) receptor promoted binding of [35S]GTPgammaS to alphaz but not to alpha12, alpha13, or alphas. Binding to alphaz was enhanced by inclusion of serotonin in the assay. Agonist activation of both thrombin and neurokinin-1 receptors promoted a modest increase in [35S]GTPgammaS binding to alphaz and more robust increases in binding to alphaq, alpha12, and alpha13. Binding of [35S]GTPgammaS to alphas was strongly enhanced only by the activated beta1-adrenergic receptor. Our data identify interactions of receptors and G proteins directly, without resort to measurements of effector activity, confirm the coupling of the 5-HT1A receptor to Gz 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-HT1A receptor, and demonstrate for the first time that the cloned receptors for thrombin and substance P activate G12 and G13.
authors.affiliation: Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.
authors.forename: A J
authors.initials: AJ
authors.lastname: Barr
authors.forename: L F
authors.initials: LF
authors.lastname: Brass
authors.forename: D R
authors.initials: DR
authors.lastname: Manning
mesh_terms: D000818:Animals; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D018342:Receptors, Adrenergic, beta-1 / Q000378:metabolism; D018040:Receptors, Neurokinin-1 / Q000378:metabolism; D011985:Receptors, Serotonin / Q000378:metabolism; D044263:Receptors, Serotonin, 5-HT1; D018179:Receptors, Thrombin / Q000378:metabolism; D018411:Spodoptera
chemical_list: D018342:Receptors, Adrenergic, beta-1; D018040:Receptors, Neurokinin-1; D011985:Receptors, Serotonin; D044263:Receptors, Serotonin, 5-HT1; D018179:Receptors, Thrombin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019204:GTP-Binding Proteins