Role of the Transmembrane Domain 4/Extracellular Loop 2 Junction of the Human Gonadotropin-releasing Hormone Receptor in Ligand Binding and Receptor Conformational Selection

Zhi-Liang Lu,Rachel Forfar

doi:10.1074/jbc.m111.240341

Abstract

Recent crystal structures of G protein-coupled receptors (GPCRs) show the remarkable structural diversity of extracellular loop 2 (ECL2), implying its potential role in ligand binding and ligand-induced receptor conformational selectivity. Here we have applied molecular modeling and mutagenesis studies to the TM4/ECL2 junction (residues Pro(174(4.59))-Met(180(4.66))) of the human gonadotropin-releasing hormone (GnRH) receptor, which uniquely has one functional type of receptor but two endogenous ligands in humans. We suggest that the above residues assume an α-helical extension of TM4 in which the side chains of Gln(174(4.60)) and Phe(178(4.64)) face toward the central ligand binding pocket to make H-bond and aromatic contacts with pGlu(1) and Trp(3) of both GnRH I and GnRH II, respectively. The interaction between the side chains of Phe(178(4.64)) of the receptor and Trp(3) of the GnRHs was supported by reciprocal mutations of the interacting residues. Interestingly, alanine mutations of Leu(175(4.61)), Ile(177(4.63)), and Met(180(4.66)) decreased mutant receptor affinity for GnRH I but, in contrast, increased affinity for GnRH II. This suggests that these residues make intramolecular or intermolecular contacts with residues of transmembrane (TM) domain 3, TM5, or the phospholipid bilayer, which couple the ligand structure to specific receptor conformational switches. The marked decrease in signaling efficacy of I177A and F178A also indicates that IIe(177(4.63)) and Phe(178(4.64)) are important in stabilizing receptor-active conformations. These findings suggest that the TM4/ECL2 junction is crucial for peptide ligand binding and, consequently, for ligand-induced receptor conformational selection.

Highlights

The crystal structures of bovine rhodopsin, the ␤-ARs, and A2A adenosine receptor reveal the conformational diversity of extracellular loops (ECLs), especially extracellular loop 2 (ECL2) [35], but all of the above G protein-coupled receptors (GPCRs) structures predict ECL2 as part of the ligand binding pocket [36, 37]
In our current gonadotropin-releasing hormone (GnRH) receptor models, the 7-TM domains were constructed based on ␤2-AR and opsin, whereas the ECLs and intracellular loops were modeled ab initio by means of a molecular mechanics-based algorithm implemented in LOOPER [28], which often finds near native loop conformations
Our molecular modeling shows that the residues at the N terminus of ECL2 (Gly172(4.58)–Met180(4.66)) of the human GnRH receptor form a helical extension from TM4 (Fig. 1B), which leaves ECL2 with a trajectory similar to that of ␤-ARs

Summary

Introduction

Binding of GnRH I and II to the GnRH receptor may cause different intramolecular interactions to be broken, allowing the receptor to adopt varied conformations and enabling divergent signaling pathways. We have termed this ligand-induced selective signaling [3, 8, 9]. Alanine-scanning mutagenesis of residues Pro173(4.59)–Met180(4.66) (receptor residues are identified by the amino acid sequence number followed in parentheses by Ballesteros and Weinstein numbering, where the position of the most conserved amino acid in the TM domain, X, is designated X.50) was employed to better understand the role of this TM4/ECL2 junction of the GnRH receptor in terms of structure, ligand binding, and function

Objectives

Results

Conclusion