Molecular Basis of Glucagon-like Peptide 1 Docking to Its Intact Receptor Studied with Carboxyl-terminal Photolabile Probes

Quan Chen,Delia I Pinon,Laurence J Miller,Maoqing Dong

doi:10.1074/jbc.m109.038109

Abstract

The glucagon-like peptide 1 (GLP1) receptor is a member of Family B G protein-coupled receptors and represents an important drug target for type 2 diabetes. Despite recent solution of the structure of the amino-terminal domain of this receptor and that of several close family members, understanding of the molecular basis of natural ligand GLP1 binding to its intact receptor remains limited. The goal of this study was to explore spatial approximations between specific receptor residues within the carboxyl terminus of GLP1 and its receptor as normally docked. Therefore, we developed and characterized two high affinity, full-agonist photolabile GLP1 probes having sites for covalent attachment in positions 24 and 35. Both probes labeled the receptor specifically and saturably. Subsequent peptide mapping using chemical and proteinase cleavages of purified wild-type and mutant GLP1 receptor identified that the Arg(131)-Lys(136) segment at the juxtamembrane region of the receptor amino terminus contained the site of labeling for the position 24 probe, and the specific receptor residue labeled by this probe was identified as Glu(133) by radiochemical sequencing. Similarly, nearby residue Glu(125) within the same region of the receptor amino-terminal domain was identified as the site of labeling by the position 35 probe. These data represent the first direct demonstration of spatial approximation between GLP1 and its intact receptor as docked, providing two important constraints for the modeling of this interaction. This should expand our understanding of the molecular basis of natural agonist ligand binding to the GLP1 receptor and may be relevant to other family members.

Highlights

Ment of drugs acting at these targets
We performed photoaffinity labeling on the intact glucagon-like peptide 1 (GLP1) receptor with probes incorporating photolabile residues in the carboxyl-terminal region of the GLP1 peptide to test, confirm, and refine the molecular basis of agonist ligand binding to their receptor
Characterization of Photolabile Probes—The Bpa24 and Bpa35 GLP1 probes were synthesized by manual solid phase techniques and purified by reversed-phase HPLC, and their identities were verified by mass spectrometry

Summary

Introduction

Ment of drugs acting at these targets. Currently, the molecular basis of ligand binding and activation of Family B GPCRs is less well understood than that of the larger Family A, where high resolution crystal structures have recently been described for several members [1,2,3,4]. Insights into the structure of the predominant ligand binding domain, the amino terminus of the Family B GPCRs, have substantially advanced with the solution of NMR and crystal structures of the isolated ligand-bound amino terminus of the receptors for corticotrophin-releasing factor [11,12,13], pituitary adenylate cyclase-activating peptide [14], gastric inhibitory polypeptide (GIP) [15], glucagon-like peptide 1 (GLP1) [16], and parathyroid hormone (PTH) [17] Most of these ligands represented amino-terminally truncated hormones or analogues of these hormones. This identified residues Glu133 and Glu125 within the amino-terminal domain of the receptor as the sites of labeling for the position 24 and 35 probes, respectively These represent the first demonstration of direct residue-residue approximations between GLP1 and its receptor and provide two important constraints for future molecular modeling. This should contribute substantially to our understanding of the molecular basis of ligand binding and activation of the clinically important GLP1 receptor

Objectives

Results

Conclusion