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

Abstract

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...

Receptor-Mediated Activation of Heterotrimeric G-Proteins: Current Structural Insights

“…D2N, in common with several other receptor loop peptides, exhibits modest GEF activity on G␣ subunits in vitro with a selectivity profile analogous to the cognate full-length receptor (Nanoff et al, 2006). We found that D2N binds to the ␣4/␤6 loop region of G␣, previously identified as a critical receptor contact site important for G␣-coupling selectivity (Hamm et al, 1988;Onrust et al, 1997;Slessareva et al, 2003; (Figs. 4 and 6).…”

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

“…Notably, the ␣4 helix residues Q304 and E308 contacted directly by D2N basic residues (Fig. 2B) have been implicated in the G protein coupling specificity of the 5-HT 1A serotonin receptor (21). Mutation of these two ␣4 helix residues within G␣ i1 to the corresponding residues in G␣ s (Q304R/ E208L; Fig.…”

“…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.…”

Estudos conformacionais de seqüências hidrofóbicas de domínios de receptores acoplados a proteínas G (GPCR) e da proteína G. Um estudo de CD e espectroscopia de fluorescência

“…Além de Wess et aI. (240), Graber et alo (242,243) também mostraram através de mutações que os últimos cinco resíduos do C-terminal de Ga são importantes para o acoplamento da proteína G com o receptor muscarínico. Essa região forma uma dobra (3, conforme discutido acima (seção 5.2.1).…”

“…Pin et aI. (243,244) também mostraram que a posição -3 e --4 (-1 corresponde ao último resíduo) em G u é importante para o acoplamento com o receptor de glutamato.…”