Analogs of Insulin-like Peptide 3 (INSL3) B-chain Are LGR8 Antagonists in Vitro and in Vivo

Ross A.D. Bathgate,Mark P. Del Borgo,Kazu Kawamura,Richard A. Hughes,John D. Wade,Feng Lin

doi:10.1074/jbc.m600472200

Abstract

Insulin-like peptide 3 (INSL3) is a member of the insulin superfamily that plays an important role in mediating testes descent during fetal development. More recently, it has also been demonstrated to initiate oocyte maturation and suppress male germ cell apoptosis. These actions are mediated via a specific G-protein-coupled receptor, LGR8. Little is known regarding the structure and function relationship of INSL3, although it is believed that the principal receptor binding site resides within its B-chain. We subsequently observed that the linear B-chain alone (INSL3B-(1-31)) bound to LGR8 and was able to antagonise INSL3 stimulated cAMP accumulation in HEK-293T cells expressing LGR8. Sequentially N- and C-terminally shortened linear analogs were prepared by solid phase synthesis and subsequent assay showed that the minimum length required for binding was residues 11-27. It was also observed that increased binding affinity correlated with a corresponding increase in alpha-helical content as measured by circular dichroism spectroscopy. Molecular modeling studies suggested that judicious placement of a conformational constraint within this peptide would increase its alpha-helix content and result in increased structural similarity to the B-chain within native INSL3. Consequently, intramolecularly disulfide-linked analogs of the B-chain showed a potentiation of INSL3 antagonistic activity, as well as exhibiting increased proteolytic stability, as assessed in rat serum in vitro. Administration of one of these peptides into the testes of rats resulted in a substantial decrease in testis weight probably due to the inhibition of germ cell survival, suggesting that INSL3 antagonists may have potential as novel contraceptive agents.

Highlights

Fetal and adult testes and in the thecal cells of the ovary [1]
In this study, we synthesized the linear Insulin-like peptide 3 (INSL3) B-chain and examined its ability to bind to the INSL3 receptor LGR8 and its effects on cAMP accumulation in LGR8 expressing cells
We found that the peptide, while devoid of INSL3-like activity, is able to bind to LGR8 and act as an antagonist of INSL3 actions in vitro

Summary

Introduction

Fetal and adult testes and in the thecal cells of the ovary [1]. The receptor for INSL3 has recently been identified and shown to belong to the family of leucine-rich repeat-containing G-protein-coupled receptors (LGRs) [2]. The action of gonadotropin on the maturation of oocytes in the ovary and the suppression of male germ cell apoptosis in the testis has recently been shown to be mediated by INSL3 [14]. It has been demonstrated that LH acts on the ovarian theca and testicular Leydig cells to produce INSL3 [14] This binds to LGR8, which is expressed on germ cells to activate the inhibitory G-protein (Gi), leading to decreased cAMP production. The steric effect of B-chain Pro is likely to be crucial for proper positioning of the indole moiety Further evidence of this is provided by the recent finding that an inactive mutant of human INSL3 identified in cryptorchid patients has a B-chain Pro Ser substitution [18]. Given that the B-chain of INSL3 most likely contains the primary binding site, we undertook to determine whether it alone possesses biological activity, and if so, how much of the B-chain is required and whether conformationally constrained analogs could be designed and prepared which would mimic the action of the native peptide

Methods

Findings

Conclusion