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

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 ␣-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 ␣-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. Insulin-like peptide 3 (INSL3)3 is a member of the insulin superfamily of hormones, which also includes insulin-like growth factors I and II (IGF-I and -II), relaxin-1, -2, and -3, and INSL4, -5, and -6. INSL3 is a 6-kDa peptide consisting of two chains (A and B) linked by two intermolecular disulfide bonds, with a third intermolecular disulfide bond in the A-chain (Fig. 1) (1). It is expressed primarily in the Leydig cells of 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). INSL3 binds with high affinity to LGR8 and has very low affinity for the paralagous receptor LGR7, which is the receptor for relaxin (3). It is now well accepted that INSL3 is a member of the relaxin peptide family (4), and LGR7 and LGR8 have been classified as relaxin family peptide receptors, RXFP1 and RXFP2, respectively (5). Importantly, relaxin can bind to and activate LGR8 in some species, although with lesser affinity and potency than INSL3 (6,7).Although the precise physiological functions of INSL3 have not been fully determined, it has been shown to play a pivotal role in testicular descent and ovarian function. In male mice, deletion of either the ...

The A-chain of Human Relaxin Family Peptides Has Distinct Roles in the Binding and Activation of the Different Relaxin Family Peptide Receptors

“…Therefore, it is likely that there is a different mechanism for secondary binding to the TM exoloops and that this secondary binding site influences overall binding affinity. Further evidence for this concept is the lack of RXFP1 binding activity of B chain-only analogs of H2 relaxin (38), whereas similar B-chain analogs of INSL3 bind to RXFP2 and act as antagonists (21). Interestingly, the behavior of the truncated H2 analogs is similar on RXFP1 and RXFP2, albeit with lower affinity and activity on RXFP2, suggesting that H2 relaxin activates RXFP2 in an "RXFP1-like" manner.…”

“…Similarly, we have recently demonstrated that the INSL3 B-chain interacts with specific residues in the RXFP2 receptor LRRs (19 (20). Importantly, B-chain-only INSL3 peptides can bind to the primary binding sites in the LRRs but do not activate the receptor (21). These peptides are low affinity antagonists and highlight that the A-chain is required for receptor activation.…”

Solution Structure and Characterization of the LGR8 Receptor Binding Surface of Insulin-like Peptide 3

“…Analogs of INSL3 with modifications in the B-chain at positions 25 and 27 have shown that Trp 27 is essential for characteristic INSL3 activity (13). More recently, our demonstration, that B-chain-only INSL3 peptides can bind to the primary ligand binding site in the ectodomain of LGR8 and act as antagonists (12), has highlighted that the B-chain contains the essential residues for primary binding. It has also enabled a detailed structure-activity relationship study of analogs of the linear peptide to be undertaken.…”

“…It has also enabled a detailed structure-activity relationship study of analogs of the linear peptide to be undertaken. The results showed that the minimum length required for binding was residues 11-27 and confirmed the critical importance of the Trp 27 residue (12). On the basis of this information and to more precisely map the primary LGR8 receptor binding domain of human INSL3, we undertook its solution structure determination and correlated this with the LGR8 binding activation of INSL3 analogs with selected alanine substitutions in the B-chain.…”

“…Furthermore, LGR8 antagonists have the potential to be highly specific contraceptives. Indeed, we recently demonstrated that LGR8 antagonists based on cyclic peptide mimetics of the INSL3 B-chain can mimic the effect of gonadotrophinreleasing hormone antagonist treatment in prepubertal rats, suggesting that direct antagonism of LGR8 function may be an effective contraceptive treatment (12).…”