Mutational and Computational Analysis of the α1b-Adrenergic Receptor: INVOLVEMENT OF BASIC AND HYDROPHOBIC RESIDUES IN RECEPTOR ACTIVATION AND G PROTEIN COUPLING

Peter J Greasley,Francesca Fanelli,Alexander Scheer,Liliane Abuin,Monique Nenniger-Tosato,Pier G Debenedetti,Susanna Cotecchia

doi:10.1074/jbc.m105791200

Abstract

To investigate their role in receptor coupling to G(q), we mutated all basic amino acids and some conserved hydrophobic residues of the cytosolic surface of the alpha(1b)-adrenergic receptor (AR). The wild type and mutated receptors were expressed in COS-7 cells and characterized for their ligand binding properties and ability to increase inositol phosphate accumulation. The experimental results have been interpreted in the context of both an ab initio model of the alpha(1b)-AR and of a new homology model built on the recently solved crystal structure of rhodopsin. Among the twenty-three basic amino acids mutated only mutations of three, Arg(254) and Lys(258) in the third intracellular loop and Lys(291) at the cytosolic extension of helix 6, markedly impaired the receptor-mediated inositol phosphate production. Additionally, mutations of two conserved hydrophobic residues, Val(147) and Leu(151) in the second intracellular loop had significant effects on receptor function. The functional analysis of the receptor mutants in conjunction with the predictions of molecular modeling supports the hypothesis that Arg(254), Lys(258), as well as Leu(151) are directly involved in receptor-G protein interaction and/or receptor-mediated activation of the G protein. In contrast, the residues belonging to the cytosolic extensions of helices 3 and 6 play a predominant role in the activation process of the alpha(1b)-AR. These findings contribute to the delineation of the molecular determinants of the alpha(1b)-AR/G(q) interface.

Highlights

To investigate their role in receptor coupling to Gq, we mutated all basic amino acids and some conserved hydrophobic residues of the cytosolic surface of the ␣1badrenergic receptor (AR)
The functional analysis of the receptor mutants in conjunction with the predictions of molecular modeling supports the hypothesis that Arg254, Lys258, as well as Leu151 are directly involved in receptor-G protein interaction and/or receptor-mediated activation of the G protein
Evidence suggests that a conformational adjustment within the helical bundle of the receptor underlies the process of agonist-induced activation of G protein-coupled receptors (GPCRs)

Summary

Introduction

To investigate their role in receptor coupling to Gq, we mutated all basic amino acids and some conserved hydrophobic residues of the cytosolic surface of the ␣1badrenergic receptor (AR). Residues located in the helical bundle and at the boundary between the membrane and the cytosol may play a role in the “conformational switch” underlying receptor activation, amino acids in the intracellular loops are believed to be more directly involved in receptor-G protein interaction and/or receptor-induced G protein activation. The combination of these two latter events, which cannot be unequivocally separated experimentally, is generally indicated with the term of receptor-G protein coupling. Following a combination of experimental and computer-simulated mutagenesis of the ␣1b-AR, we have hypothesized that protonation of the aspartate (Asp142) and a shift of the arginine (Arg143) out of a conserved “polar pocket” are crucial steps in the transition of the receptor from the inactive (R) to active (R*) state (4 – 6)

Results

Discussion

Conclusion