Modulating effects of RAMPs on signaling profiles of the glucagon receptor family

Lijun Shao; Yan Chen; Shikai Zhang; Zhihui Zhang; Yongbing Cao; Dehua Yang; Ming-Wei Wang

doi:10.1016/j.apsb.2021.07.028

Modulating effects of RAMPs on signaling profiles of the glucagon receptor family

Acta Pharm Sin B. 2022 Feb;12(2):637-650. doi: 10.1016/j.apsb.2021.07.028. Epub 2021 Aug 4.

Authors

Lijun Shao^{1

2

3}, Yan Chen⁴, Shikai Zhang⁵, Zhihui Zhang⁵, Yongbing Cao⁵, Dehua Yang^{1

2

3}, Ming-Wei Wang^{1

2

3

4

6}

Affiliations

¹ The National Center for Drug Screening and CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences (CAS), Shanghai 201203, China.
² School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China.
³ University of Chinese Academy of Sciences, Beijing 100049, China.
⁴ School of Pharmacy, Fudan University, Shanghai 201203, China.
⁵ Shanghai TCM-Integrated Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 200082, China.
⁶ Department of Pharmacology, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China.

Abstract

Receptor activity-modulating proteins (RAMPs) are accessory molecules that form complexes with specific G protein-coupled receptors (GPCRs) and modulate their functions. It is established that RAMP interacts with the glucagon receptor family of GPCRs but the underlying mechanism is poorly understood. In this study, we used a bioluminescence resonance energy transfer (BRET) approach to comprehensively investigate such interactions. In conjunction with cAMP accumulation, Gα _q activation and β-arrestin1/2 recruitment assays, we not only verified the GPCR-RAMP pairs previously reported, but also identified new patterns of GPCR-RAMP interaction. While RAMP1 was able to modify the three signaling events elicited by both glucagon receptor (GCGR) and glucagon-like peptide-1 receptor (GLP-1R), and RAMP2 mainly affected β-arrestin1/2 recruitment by GCGR, GLP-1R and glucagon-like peptide-2 receptor, RAMP3 showed a widespread negative impact on all the family members except for growth hormone-releasing hormone receptor covering the three pathways. Our results suggest that RAMP modulates both G protein dependent and independent signal transduction among the glucagon receptor family members in a receptor-specific manner. Mapping such interactions provides new insights into the role of RAMP in ligand recognition and receptor activation.

Keywords: AMY, amylin; Allosteric modulation; BRET, bioluminescence resonance energy transfer; Bmax, maximum measured BRET value; CGRP, calcitonin gene-related peptide; CLR, calcitonin-like receptor; EC50, half maximal effective concentration; ECD, extracellular domain; Emax, maximal response; G protein-coupled receptor; GCGR, glucagon receptor; GHRHR, hormone-releasing hormone receptor; GIPR, gastric inhibitory polypeptide receptor or glucose-dependent insulinotropic polypeptide; GLP-1R, glucagon-like peptide-1 receptor; GLP-2R, glucagon-like peptide-2 receptor; GPCRs, G protein-coupled receptors; GPCR–RAMP interaction; Glucagon receptor family; Ligand selectivity; RAMP, receptor activity-modulating protein; Receptor activity-modulating protein; Receptor pharmacology; Rluc, Renilla luciferase; SBA, suspension bead array; SCTR, secretin receptor; SV, splice variant; Signaling; TMD, transmembrane domain; VPAC2R, vasoactive intestinal polypeptide 2 receptor; cAMP, cyclic adenosine monophosphate; pEC50, negative logarithm of EC50; β2-AR, β2-adrenergic receptor.