Analogs of insulin-like peptide 3 (INSL3) B-chain are LGR8 antagonists in vitro and in vivo.
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.
Howard Florey Institute, Victoria 3010, Australia; Department of Pharmacology, University of Melbourne, Victoria 3010, Australia.
Mark P
MP
Del Borgo
Department of Pharmacology, University of Melbourne, Victoria 3010, Australia.
Richard A
RA
Hughes
Howard Florey Institute, Victoria 3010, Australia.
Ross A D
RAD
Bathgate
Howard Florey Institute, Victoria 3010, Australia.
Feng
F
Lin
Division of Reproductive Biology, Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, California 94305-5317.
Kazu
K
Kawamura
Howard Florey Institute, Victoria 3010, Australia. Electronic address: j.wade@hfi.unimelb.edu.au.
John D
JD
Wade
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D005786:Gene Expression Regulation; D006801:Humans; D007328:Insulin / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology*; D008297:Male; D008969:Molecular Sequence Data; D011485:Protein Binding; D020033:Protein Isoforms; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D043562:Receptors, G-Protein-Coupled / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D013737:Testis / Q000166:cytology / Q000187:drug effects / Q000378:metabolism
D007328:Insulin; C084797:Leydig insulin-like protein; D020033:Protein Isoforms; D011506:Proteins; C472867:RXFP2 protein, human; D043562:Receptors, G-Protein-Coupled; C511608:Rxfp2 protein, rat