The G protein coupling behavior of four human 5-hydroxytryptamine receptor subtypes (5-HT1A, 5-HT1B, 5-HT1D, and 5-HT1E) has been studied in membranes from Sf9 cells expressing the individual receptors. The 5-HT1A and 5-HT1B receptors exhibited both high- and low-affinity states for agonist, with the majority of the receptors in a low-affinity state. Addition of purified G protein subunits to membranes expressing either 5-HT1A or 5-HT1B receptors shifted the majority of the receptors to a high-affinity state in the absence, but not in the presence, of guanine nucleotides. The alphai1, alphai2, alphai3, and alphao subunits were able to shift the receptors to a high-affinity state with either betagammabrain or betagammaretina while alphat subunits were inactive regardless of which betagamma preparation was used. A significantly higher affinity for agonist was observed with both receptors in the presence of alphai3 subunits compared with either alphai2 or alphao subunits, while a significantly lower concentration of alpha subunits was required for a maximal affinity shift of 5-HT1A receptors compared with 5-HT1B receptors (EC50 values of 6.4 and 12. 0 nM, respectively). The 5-HT1D and 5-HT1E receptors exhibited only a single affinity state for agonist. Addition of purified G protein subunits to membranes containing 5-HT1D receptors caused a small increase in affinity for agonist that was only partially reversed by guanine nucleotides while the addition of purified G protein subunits to membranes containing 5-HT1E receptors had no affect on agonist binding. Thus when expressed in an identical membrane environment these four closely related 5-HT1 receptor subtypes exhibit different G protein coupling behaviors.

The molecular basis of selectivity in G-protein receptor coupling has been explored by comparing the abilities of G-protein heterotrimers containing chimeric G␣ subunits, comprised of various regions of G i1 ␣, G t ␣, and G q ␣, to stabilize the high affinity agonist binding state of serotonin, adenosine, and muscarinic receptors. The data indicate that multiple and distinct determinants of selectivity exist for individual receptors. While the A1 adenosine receptor does not distinguish between G i1 ␣ and G t ␣ sequences, the 5-HT 1A and 5-HT 1B serotonin and M2 muscarinic receptors can couple with G i1 but not G t . It is possible to distinguish domains that eliminate coupling and are defined as "critical," from those that impair coupling and are defined as "important." Domains within the N terminus, ␣4-helix, and ␣4-helix-␣4/␤6-loop of G i1 ␣ are involved in 5-HT and M2 receptor interactions. Chimeric G i1 ␣/G q ␣ subunits verify the critical role of the G␣ C terminus in receptor coupling, however, the individual receptors differ in the C-terminal amino acids required for coupling. Furthermore, the EC 50 for interactions with G i1 differ among the individual receptors. These results suggest that coupling selectivity ultimately involves subtle and cooperative interactions among various domains on both the G-protein and the associated receptor as well as the G-protein concentration.A large number of diverse seven transmembrane-spanning cell surface receptors mediate signaling to a variety of intracellular effectors by coupling to the heterotrimeric guanine nucleotide-binding regulatory proteins (G-proteins) 1 (1). The mechanisms responsible for selectivity in G-protein-mediated signaling pathways are not fully understood (2, 3). Although it is known that at the molecular level the selectivity in G-protein receptor coupling is determined by amino acid sequences of both receptor and G-protein, the individual amino acids involved in this selective recognition have not been completely identified. Different receptor systems and different methodologies indicate that the G␣ subunit C terminus and ␣5-helix (4 -7), N terminus, and ␣N-helix (4, 8 -10), ␣4-helix, and ␣4/ ␤6-loop (11-13), ␣2-helix, and ␣2/␤4-loop (14), ␣3/␤5-loop (15), ␣N/␤1-loop (13) and amino acids 110 -119 from the ␣-helical domain (16) are involved in receptor-coupling selectivity. Some of these domains contact the receptor directly, while others regulate receptor-coupling selectivity indirectly by playing a role in nucleotide exchange. Despite the fact that many of the receptor-interacting domains have been identified, the relationship between receptor subtypes and G␣ domains involved in receptor coupling has not been clearly established. Thus, it is difficult to predict which G␣ domains will be utilized by a specific receptor. Here we propose that individual receptors recognize specific patterns formed by amino acids of G␣ thus making G-protein interface look different for different receptors. The C terminus of G␣ is a well accepted receptor recognit...

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...

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