The Structural Determinants of Insulin-Like Peptide 3 Activity

Ross A D Bathgate,Richard A Hughes,K Johan Rosengren,Soude Zhang,John D Wade

doi:10.3389/fendo.2012.00011

Ross A D Bathgate, Richard A Hughes + Show 3 more

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https://doi.org/10.3389/fendo.2012.00011

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Abstract

Insulin-like peptide 3 (INSL3) is a hormone and/or paracrine factor which is a member of the relaxin peptide family. It has key roles as a fertility regulator in both males and females. The receptor for INSL3 is the leucine rich repeat (LRR) containing G-protein coupled receptor 8 (LGR8) which is now known as relaxin family peptide receptor 2 (RXFP2). Receptor activation by INSL3 involves binding to the LRRs in the large ectodomain of RXFP2 by residues within the B-chain of INSL3 as well as an interaction with the transmembrane exoloops of the receptor. Although the binding to the LRRs is well characterized the features of the peptide and receptor involved in the exoloop interaction are currently unknown. This study was designed to determine the key INSL3 determinants for RXFP2 activation. A chimeric peptide approach was first utilized to demonstrate that the A-chain is critical for receptor activation. Replacement of the INSL3 A-chain with that from the related peptides INSL5 and INSL6 resulted in complete loss of activity despite only minor changes in binding affinity. Subsequent replacement of specific A-chain residues with those from the INSL5 peptide highlighted that the N-terminus of the A-chain of INSL3 is critical for its activity. Remarkably, replacement of the entire N-terminus with four or five alanine residues resulted in peptides with near native activity suggesting that specific residues are not necessary for activity. Additionally removal of two amino acids at the C-terminus of the A-chain and mutation of Lys-8 in the B-chain also resulted in minor decreases in peptide activity. Therefore we have demonstrated that the activity of the INSL3 peptide is driven predominantly by residues 5–9 in the A-chain, with minor additional contributions from the two C-terminal A-chain residues and Lys-8 in the B-chain. Using this new knowledge, we were able to produce a truncated INSL3 peptide structure which retained native activity, despite having 14 fewer residues than the parent peptide.

Highlights

Insulin-like peptide 3 (INSL3) was originally identified as a novel gene highly expressed in the Leydig cells of the testis, and was initially named Leydig insulin-like peptide (Ley-I-L; Adham et al, 1993)
CHIMERIC PEPTIDES The INSL3 B-chain was combined with the A chains of other relaxin family peptides to produce the chimeric peptides H2A/INSL3-B, INSL6-A/INSL3-B, and INSL5-A/INSL3-B (Table 1)
While human gene-2 (H2)-A/INSL3-B was able to bind to RXFP2 with high affinity, and it showed a similar pEC50 to both INSL3 and H2 relaxin it was only able to stimulate to 45% of the maximal INSL3 effect demonstrating properties of a partial agonist (Table 1, Figure 2B)

Summary

Introduction

Insulin-like peptide 3 (INSL3) was originally identified as a novel gene highly expressed in the Leydig cells of the testis, and was initially named Leydig insulin-like peptide (Ley-I-L; Adham et al, 1993). INSL3 is its common name and it is known to be a member of the relaxin peptide family. The relaxin peptides are all characterized by a common structure comprising two chains (A and B) that are cross-linked by disulfide bonds (Figure 1). This arrangement results in a general structure in which three helical segments (two in the A-chain and one in the B-chain) enclose a hydrophobic core (Rosengren et al, 2009). INSL3 binds with high affinity to the leucine-rich repeat-containing, G protein coupled receptor 8 (LRG8; Kumagai et al, 2002), known as the relaxin family peptide receptor 2 (RXFP2; Bathgate et al, 2006a)

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