Nonconventional glucagon and GLP-1 receptor agonist and antagonist interplay at the GLP-1 receptor revealed in high-throughput FRET assays for cAMP

Oleg G Chepurny,Minos-Timotheos Matsoukas,George Liapakis,Colin A Leech,Brandon T Milliken,Robert P Doyle,George G Holz

doi:10.1074/jbc.ra118.005682

Abstract

G protein-coupled receptors (GPCRs) for glucagon (GluR) and glucagon-like peptide-1 (GLP-1R) are normally considered to be highly selective for glucagon and GLP-1, respectively. However, glucagon secreted from pancreatic α-cells may accumulate at high concentrations to exert promiscuous effects at the β-cell GLP-1R, as may occur in the volume-restricted microenvironment of the islets of Langerhans. Furthermore, systemic administration of GluR or GLP-1R agonists and antagonists at high doses may lead to off-target effects at other receptors. Here, we used molecular modeling to evaluate data derived from FRET assays that detect cAMP as a read-out for GluR and GLP-1R activation. This analysis established that glucagon is a nonconventional GLP-1R agonist, an effect inhibited by the GLP-1R orthosteric antagonist exendin(9-39) (Ex(9-39)). The GluR allosteric inhibitors LY2409021 and MK 0893 antagonized glucagon and GLP-1 action at the GLP-1R, whereas des-His1-[Glu9]glucagon antagonized glucagon action at the GluR, while having minimal inhibitory action versus glucagon or GLP-1 at the GLP-1R. When testing Ex(9-39) in combination with des-His1-[Glu9]glucagon in INS-1 832/13 cells, we validated a dual agonist action of glucagon at the GluR and GLP-1R. Hybrid peptide GGP817 containing glucagon fused to a fragment of peptide YY (PYY) acted as a triagonist at the GluR, GLP-1R, and neuropeptide Y2 receptor (NPY2R). Collectively, these findings provide a new triagonist strategy with which to target the GluR, GLP-1R, and NPY2R. They also provide an impetus to reevaluate prior studies in which GluR and GLP-1R agonists and antagonists were assumed not to exert promiscuous actions at other GPCRs.

Highlights

G protein– coupled receptors (GPCRs) for glucagon (GluR) and glucagon-like peptide-1 (GLP-1R) are normally considered to be highly selective for glucagon and GLP-1, respectively
We used HEK293 cells that stably express recombinant GPCRs and that were virally transduced with H188, thereby allowing fluorescence resonance energy transfer (FRET) to be monitored in real time using confluent cell monolayers
Tested were GPCR antagonists previously reported to be selective for the glucagon receptor (GluR) or the GLP-1 receptor (GLP-1R) (exendin(9 –39)) or neuropeptide Y2 receptor (NPY2R) (BIIE0246) (Fig. 1) [29]

Summary

Introduction

G protein– coupled receptors (GPCRs) for glucagon (GluR) and glucagon-like peptide-1 (GLP-1R) are normally considered to be highly selective for glucagon and GLP-1, respectively. The optimization of dual or triagonists requires detailed knowledge of their molecular mechanisms of action In this regard, modeling of GPCR structure/conformation is facilitated by the availability of GPCR agonists and antagonists that aid in the identification of orthosteric and allosteric mechanisms of ligand binding and receptor activation. This assay uses the H188 biosensor designed by Klarenbeek et al [28] in which cAMP binds directly to a modified Epac protein that is flanked by mTurquoise2⌬ FRET donor and tandem cp173 Venus–Venus FRET acceptor chromophores We used this FRET assay to discover nonconventional actions of family B GPCR agonists and antagonists, while investigating the pharmacological properties of a hybrid peptide (GGP817) that incorporates amino acid sequences present within glucagon and PYY. Our analysis reveals unexpected features of GPCR agonist and antagonist action, while establishing GGP817 to be a prototype triagonist at the GluR, GLP-1R, and NPY2R

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