Mechanism of Activation of a G Protein-coupled Receptor, the Human Cholecystokinin-2 Receptor

Ingrid Langer,Daniel Fourmy,Chantal Escrieut,Esther Marco,Irina G. Tikhonova,Magali Foucaud

doi:10.1074/jbc.m700349200

Abstract

G protein-coupled receptors (GPCRs) represent a major focus in functional genomics programs and drug development research, but their important potential as drug targets contrasts with the still limited data available concerning their activation mechanism. Here, we investigated the activation mechanism of the cholecystokinin-2 receptor (CCK2R). The three-dimensional structure of inactive CCK2R was homology-modeled on the basis of crystal coordinates of inactive rhodopsin. Starting from the inactive CCK2R modeled structure, active CCK2R (namely cholecystokinin-occupied CCK2R) was modeled by means of steered molecular dynamics in a lipid bilayer and by using available data from other GPCRs, including rhodopsin. By comparing the modeled structures of the inactive and active CCK2R, we identified changes in the relative position of helices and networks of interacting residues, which were expected to stabilize either the active or inactive states of CCK2R. Using targeted molecular dynamics simulations capable of converting CCK2R from the inactive to the active state, we delineated structural changes at the atomic level. The activation mechanism involved significant movements of helices VI and V, a slight movement of helices IV and VII, and changes in the position of critical residues within or near the binding site. The mutation of key amino acids yielded inactive or constitutively active CCK2R mutants, supporting this proposed mechanism. Such progress in the refinement of the CCK2R binding site structure and in knowledge of CCK2R activation mechanisms will enable target-based optimization of nonpeptide ligands.

Highlights

Ever, these drugs address only a small fraction of the G protein-coupled receptors (GPCRs) repertoire
Modeled Structure of Active cholecystokinin-2 receptor (CCK2R) (CCK2R*)—Because CCK2R is a member of the family I of rhodopsin-like GPCRs, the three-dimensional model of the inactive state of the CCK2R (CCK2R°) was constructed by homology modeling using coordinates from the inactive rhodopsin crystal isolated in the dark (19)
The procedure of modeling, which is described in detail under “Experimental Procedures,” included the following main steps: rotation of conserved Trp-6.48 side chain (22); docking of CCK4 followed by steered molecular dynamics simulation in vacuum; steered molecular dynamics simulation based on constrained distances indicated in Table 1 and derived from published data on GPCR activation; and refinement in a lipid bilayer

Summary

Introduction

The activation of GPCRs is generally explained by the extended ternary complex model whereby the population of receptors exists as an equilibrium between inactive and active states (1). According to this model, the active conformation of the receptor may be stabilized or induced by agonists. Converging studies have documented that activation of wild-type CCK2R and/or expression of a constitutively active variant may contribute to human diseases (8, 9) These new findings have generated considerable interest in the identification of antagonists of CCK2R. The aim of the present study was to increase our knowledge of the molecular mechanisms whereby CCK2R is activated For this purpose, three-dimensional models of inactive and active CCK2R were constructed on the basis of available structural data. The activation mechanism of the receptor was further investigated using targeted molecular dynamics simulations and site-directed mutagenesis

Objectives

Results

Conclusion