Two Amino Acids within the α4 Helix of Gαi1Mediate Coupling with 5-Hydroxytryptamine1BReceptors

Abstract

We previously reported that residues 299 -318 in G␣ i1 participate in the selective interaction between G␣ 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 receptormediated G protein activation. A series of G␣ i1 /G␣ t chimeras and point mutations were reconstituted with G␤␥ and Sf9 cell membranes containing the 5-HT 1B receptor. Functional coupling to 5-HT 1B receptors was assessed by 1) [35 S]GTP␥S binding and 2) agonist affinity shift assays. Replacement of the ␣4 helix of G␣ i1 (residues 299 -308) with the corresponding sequence from G␣ t produced a chimera (Chi22) that only weakly coupled to the 5-HT 1B receptor. In contrast, substitution of residues within the ␣4-␤6 loop region of G␣ i1 (residues 309 -318) with the corresponding sequence in G␣ 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 ␣4 helix of G␣ i1 (Q304K and E308L) reduced agonist-stimulated [ 35 S]GTP␥S 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 ␣4 helix of G␣ i1 had minimal to no effect on 5-HT 1B coupling behavior. These data provide evidence that ␣4 helix residues in G␣ i participate in directing specific receptor interactions and suggest that Gln 304 and Glu 308 of G␣ i1 act in concert to mediate the ability of the 5-HT 1B receptor to couple specifically to inhibitory G proteins.The interaction of heptahelical receptors with their cognate heterotrimeric guanine nucleotide-binding proteins (G proteins) represents an initial step in the transmission of extracellular signals across the plasma membrane (2-4). The receptor-G protein interaction modulates specific second messenger systems that result in a unique physiologic response to the extracellular signal. The particular downstream effect of G protein activation is not the result of an explicit interaction between each heptahelical receptor and a unique heterotrimeric G protein. On the contrary, G protein-coupled receptors have repeatedly been demonstrated to couple to several related members within the same family of G protein ␣ subunits, albeit with differing levels of efficiency (5-11). Clawges et al. (12) demonstrated that the serotonin (5-HT) 1 1B receptor couples to heterotrimers containing either G␣ i1 , G␣ i2 , G␣ i3 , or G␣ o . Nevertheless, this receptor does not couple to heterotrimers containing another member of this same family, the G␣ t subunit (12). Therefore, the 5-HT 1B receptor represents one receptor system that can be exploited to investigate the precise molecular determinants governing selective receptor-G protein interactio...

RETRACTED: Structural basis for nucleotide exchange on Gα i subunits and receptor coupling specificity

“…Furthermore, these results support a role for charge-charge complementation in receptor/G␣ coupling ( Fig. 3 A and B) as postulated for other GPCRs, including rhodopsin and the 5-HT 1A serotonin receptor (20).…”

“…D2N engages a triad of residues on the ␣4 helix and ␤6 strand (Q304/E308 and T321, respectively; Fig. 2B) that were implicated in studies using G␣ chimera (19)(20)(21) in the coupling specificity and GEF activity of the 5-HT 1A serotonin receptor. Binding of D2N results in displacement of the G␣ i1 ␤6 strand with respect to the unbound state ( Fig.…”

Rhodopsin Recognition by Mutant Gsα Containing C-terminal Residues of Transducin

“…Conservation of the Cterminal residues within one family requires participation of additional regions of G␣ to achieve fine receptor tuning. For example, Gln 304 and Glu 308 in the ␣ 4 helix of G i ␣ 1 are important to the selectivity of the 5-hydroxytryptamine receptor for G i ␣ 1 over G t ␣ (20). Chimeric G t ␣/G i ␣ proteins have been very instrumental in gaining insight into the G i ␣ 1 /5-hydroxytryptamine receptor selectivity (20), but would not be helpful in elucidating the C-terminal determinants of the G t ␣/R* interaction since the C termini of G t ␣ and G i ␣ are almost identical, and R* potently stimulates G i ␣ in vitro (13).…”

Closely Related G-protein-coupled Receptors Use Multiple and Distinct Domains on G-protein α-Subunits for Selective Coupling

“…The ␣4-helix-␣4/␤6-loop domain, first described as an effector domain, has been shown to be important for 5-HT 1B receptor coupling to G i1 (11). Later it was demonstrated that Gln-304 and Glu-308 in the ␣4-helix of G i1 ␣ are important for 5-HT 1B receptor coupling (18). However the generality of the role for the ␣4-helix-␣4/␤6-loop domain in receptor coupling selectivity has not been determined.…”

“…The digested PCR fragment was inserted into the BglII and HindIII sites of the Chi13 plasmid (11). Functional characterization of all bacterial subunits included GTP␥S binding, AlF 4 Ϫ -dependent conformational change (measured as an increase in intrinsic tryptophan fluorescence) or binding to the cGMP phosphodiesterase ␥-subunit (11,18,19).…”