Abstract

We previously reported that residues 299–318 in Gα_i1 participate in the selective interaction between Gα_i1 and the 5-hydroxytryptamine_1B(5-HT_1B) receptor (Bae, H., Anderson, K., Flood, L. A., Skiba, N. P., Hamm, H. E., and Graber, S. G. (1997)J. Biol. Chem. 272, 32071–32077). The present study more precisely defines which residues within this domain are critical for 5-HT_1B receptor-mediated G protein activation. A series of Gα_i1/Gα_t chimeras and point mutations were reconstituted with Gβγ and Sf9 cell membranes containing the 5-HT_1B receptor. Functional coupling to 5-HT_1B receptors was assessed by 1) [³⁵S]GTPγS binding and 2) agonist affinity shift assays. Replacement of the α4 helix of Gα_i1 (residues 299–308) with the corresponding sequence from Gα_tproduced a chimera (Chi22) that only weakly coupled to the 5-HT_1B receptor. In contrast, substitution of residues within the α4-β6 loop region of Gα_i1 (residues 309–318) with the corresponding sequence in Gα_t either permitted full 5-HT_1B receptor coupling to the chimera (Chi24) or only minimally reduced coupling to the chimeric protein (Chi25). Two mutations within the α4 helix of Gα_i1 (Q304K and E308L) reduced agonist-stimulated [³⁵S]GTPγS binding, and the effects of these mutations were additive. The opposite substitutions within Chi22 (K300Q and L304E) restored 5-HT_1B receptor coupling, and again the effects of the two mutations were additive. Mutations of other residues within the α4 helix of Gα_i1 had minimal to no effect on 5-HT_1B coupling behavior. These data provide evidence that α4 helix residues in Gα_i participate in directing specific receptor interactions and suggest that Gln³⁰⁴ and Glu³⁰⁸ of Gα_i1 act in concert to mediate the ability of the 5-HT_1B receptor to couple specifically to inhibitory G proteins.