Abstract

This study provides the first comprehensive evidence that the second intracellular loop C-terminal domain (Ci2) is critical for receptor-G protein coupling to multiple responses. Although Ci2 is weakly conserved, its role in 5-hydroxytryptamine-1A (5-HT1A) receptor function was suggested by the selective loss of Gβγ-mediated signaling in the T149A-5-HT1A receptor mutant. More than 60 point mutant 5-HT1A receptors in the α-helical Ci2 sequence (¹⁴³DYVNKRTPRR¹⁵²) were generated. Most mutants retained agonist binding and were tested for Gβγ signaling to adenylyl cyclase II or phospholipase C and Gαi coupling to detect constitutive and agonist-induced G_i/G_o coupling. Remarkably, most point mutations markedly attenuated 5-HT1A signaling, indicating that the entire Ci2 domain is critical for receptor G-protein coupling. Six signaling phenotypes were observed: wild-type-like, Gα_i-coupled/weak Gβγ-coupled, Gβγ-uncoupled, Gβγ-selective coupled, uncoupled, and inverse coupling. Our data elucidate specific roles of Ci2 residues consistent with predictions based on rhodopsin crystal structure. The absolute coupling requirement for lysine, arginine, and proline residues is consistent with a predicted amphipathic α-helical Ci2 domain that is kinked at Pro150. Polar residues (Thr149, Asn146) located in the externally oriented positively charged face were required for Gβγ but not Gα_i coupling, suggesting a direct interface with Gβγ subunits. The hydrophobic face includes the critical Tyr144 that directs the specificity of coupling to both Gβγ and Gα_i pathways. The key coupling residues Tyr144/Lys147 (Ci2) are predicted to orient internally, forming hydrogen and ionic bonds with Asp133/Arg134 (Ni2 DRY motif) and Glu340 (Ci3) to stabilize the Gprotein coupling domain. Thus, the 5-HT1A receptor Ci2 domain determines Gβγ specificity and stabilizes Gα_i-mediated signaling.