Distinct activation modes of the Relaxin Family Peptide Receptor 2 in response to insulin-like peptide 3 and relaxin

Shoni Bruell,Qing-Ping Wu,Mohammed Akhter Hossain,Ross A D Bathgate,Ashish Sethi,Emma J Petrie,Zhan-Yun Guo,Paul R Gooley,Daniel J Scott,Nicholas Smith

doi:10.1038/s41598-017-03638-4

Abstract

Relaxin family peptide receptor 2 (RXFP2) is a GPCR known for its role in reproductive function. It is structurally related to the human relaxin receptor RXFP1 and can be activated by human gene-2 (H2) relaxin as well as its cognate ligand insulin-like peptide 3 (INSL3). Both receptors possess an N-terminal low-density lipoprotein type a (LDLa) module that is necessary for activation and is joined to a leucine-rich repeat domain by a linker. This linker has been shown to be important for H2 relaxin binding and activation of RXFP1 and herein we investigate the role of the equivalent region of RXFP2. We demonstrate that the linker’s highly-conserved N-terminal region is essential for activation of RXFP2 in response to both ligands. In contrast, the linker is necessary for H2 relaxin, but not INSL3, binding. Our results highlight the distinct mechanism by which INSL3 activates RXFP2 whereby ligand binding mediates reorientation of the LDLa module by the linker region to activate the RXFP2 transmembrane domains in conjunction with the INSL3 A-chain. In contrast, relaxin activation of RXFP2 involves a more RXFP1-like mechanism involving binding to the LDLa-linker, reorientation of the LDLa module and activation of the transmembrane domains by the LDLa alone.

Highlights

Relaxin and insulin-like peptide 3 (INSL3) are two members of the insulin superfamily of peptides that are best known for their roles in the reproductive tract of females and males respectively[1]
Our previous studies have demonstrated that both receptors require the unique low-density lipoprotein type a (LDLa) module to signal in response to their ligands[23] and suggested that it acts as a tethered ligand[40]
Our more recent work on RXFP1 showed that the linker domain flanking the LDLa module interacts with human gene-2 (H2) relaxin and that this interaction stabilizes a helical structure within the linker allowing the LDLa module and linker residues to interact with the TMD27

Summary

Introduction

Relaxin and insulin-like peptide 3 (INSL3) are two members of the insulin superfamily of peptides that are best known for their roles in the reproductive tract of females and males respectively[1]. RXFP1 and RXFP2 are unique in that they contain a large ectodomain consisting of a series of 10 leucine-rich repeats (LRRs) and an N-terminal low-density lipoprotein type a (LDLa) module (Fig. 1B) This domain structure puts them in the LRR-containing family of GPCRs (LGR) but they make up their own classification within this group, forming class C of the LGRs, as they are the only known mammalian GPCRs to contain an LDLa module. Current knowledge holds that both receptors bind their ligand via a high-affinity interaction between the LRRs and the B-chain of the peptide[21, 22] Both receptors absolutely require the presence of the LDLa module for signalling to occur[23]. Our current model of ligand-mediated activation suggests that binding of H2 relaxin to the linker stabilizes a helix which positions residues of the LDLa module and linker to interact with the TMD to activate the receptor

Methods

Results

Conclusion