Received for publication October 12, 2005.
Revised January 5, 2006.
Accepted for publication January 12, 2006.

Molecular determinants in the second intracellular loop of the 5-hydroxytryptamine(1A) (5-HT1A) receptor for G-protein coupling

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-HT1A receptor function was suggested by the selective loss of G-mediated signaling in the T149A-5-HT1A receptor mutant. Over sixty point mutant 5-HT1A receptors in the alpha-helical Ci2 sequence (¹⁴³DYVNKRTPRR¹⁵²) were generated. Most mutants retained agonist binding and were tested for G signaling to adenylyl cyclase II or phospholipase C and Gi coupling, to detect constitutive and agonist-induced Gi/Go 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; Gi-coupled/weak G-coupled; G-uncoupled; G-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 alpha-helical Ci2 domain that is kinked at Pro150. Polar residues (T149, N146) located in the externally-oriented positively-charged face were required for G but not Gi coupling, suggesting a direct interface with G subunits. The hydrophobic face includes the critical Y144 that directs the specificity of coupling to both G and Gi pathways. The key coupling residues Y144/K147 (Ci2) are predicted to orient internally, forming hydrogen and ionic bonds with D133/R134 (Ni2 DRY motif) and E340 (Ci3) to stabilize the G-protein coupling domain. Thus, the 5-HT1A receptor Ci2 domain determines G specificity and stabilizes Gi-mediated signaling.

Key words: Serotonin, Gi family, cAMP, IP3/DAG, Protein Kinase C, G protein regulation, Desensitization/uncoupling, Structure-activity relationships and modeling

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