Two amino acids within the α4 helix of Gαi1 mediate coupling with 5-hydroxytryptamine1B receptors

We previously reported that residues 299-318 in Ga-i1 participate in the selective interaction between Ga-i1 and the 5-hydroxytryptamine(1B) (5-HT1B) 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-HT1B receptor-mediated G protein activation. A series of Ga-i1/G alpha(t) chimeras and point mutations were reconstituted with G beta gamma and Sf9 cell membranes containing the 5-HT1B receptor. Functional coupling to 5-HT1B receptors was assessed by 1) [S-35]GTP gamma S binding and 2) agonist affinity shift assays. Replacement of the alpha 4 helix of Ga-i1 (residues 299-308) with the corresponding sequence from G alpha(t) produced a chimera (Chi22) that only weakly coupled to the 5-HT1B receptor. In contrast, substitution of residues within the alpha 4-beta 6 loop region of Ga-i1 (residues 309-318) with the corresponding sequence in G alpha(t) either permitted full 5-HT1B receptor coupling to the chimera (Chi24) or only minimally reduced coupling to the chimeric protein (Chi25), Two mutations within the alpha 4 helix of Ga-i1 (Q304K and E308L) reduced agonist-stimulated [S-35]GTP gamma S binding, and the effects of these mutations were additive. The opposite substitutions within Chi22 (K300Q and L304E) restored Ga-i1 receptor coupling, and again the effects of the two mutations were additive. Mutations of other residues within the alpha 4 helix of Ga-i1 had minimal to no effect on 5-HT1B coupling behavior. These data provide evidence that alpha 4 helix residues in G alpha(i) participate in directing specific receptor interactions and suggest that Gln(304) and Glu(308) of Ga-i1 act in concert to mediate the ability of the 5-HT1B receptor to couple specifically to inhibitory G proteins.