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(t) produced 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.

Indexed keywords

EMTREE drug terms: chimeric protein; serotonin 1b receptor; glutamic acid; glycine; guanine nucleotide binding protein; serotonin receptor

EMTREE medical terms: amino acid sequence; amino acid substitution; article; chimera; gene mutation; nonhuman; priority journal; protein domain; protein structure; genetics; metabolism; mutation; protein secondary structure; protein tertiary structure

MeSH: Glutamic Acid; Glycine; GTP-Binding Proteins; Mutation; Protein Structure, Secondary; Protein Structure, Tertiary; Receptors, Serotonin
Medline is the source for the MeSH terms of this document.

Chemicals and CAS Registry Numbers: Glutamic Acid, 56-86-0; Glycine, 56-40-6; GTP-Binding Proteins, EC 3.6.1.-; Receptors, Serotonin