Relaxin-like factor (RLF)/insulin-like peptide 3 (INSL3) is secreted from testicular Leydig cells as a monomeric protein comprising three domains B-C-A with full biological activity in boars

Biochem J. 2012 Jan 1;441(1):265-73. doi: 10.1042/BJ20111107.


RLF (relaxin-like factor), also known as INSL3 (insulin-like peptide 3), is a novel member of the relaxin/insulin gene family that is expressed in testicular Leydig cells. Despite the implicated role of RLF/INSL3 in testis development, its native conformation remains unknown. In the present paper we demonstrate for the first time that boar testicular RLF/INSL3 is isolated as a monomeric structure with full biological activity. Using a series of chromatography steps, the native RLF/INSL3 was highly purified as a single peak in reverse-phase HPLC. MS/MS (tandem MS) analysis of the trypsinized sample provided 66% sequence coverage and revealed a distinct monomeric structure consisting of the B-, C- and A-domains deduced previously from the RLF/INSL3 cDNA. Moreover, the N-terminal peptide was four amino acid residues longer than predicted previously. MS analysis of the intact molecule and PMF (peptide mass fingerprinting) analysis at 100% sequence coverage confirmed this structure and indicated the existence of three site-specific disulfide bonds. RLF/INSL3 retained full bioactivity in HEK (human embryonic kidney)-293 cells expressing RXFP2 (relaxin/insulin-like family peptide receptor 2), the receptor for RLF/INSL3. Furthermore, RLF/INSL3 was found to be secreted from Leydig cells into testicular venous blood. Collectively, these results indicate that boar RLF/INSL3 is secreted from testicular Leydig cells as a B-C-A monomeric structure with full biological activity.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cells, Cultured
  • Gene Expression Regulation / physiology*
  • HEK293 Cells
  • Humans
  • Insulin / genetics
  • Insulin / metabolism*
  • Leydig Cells / metabolism*
  • Male
  • Protein Structure, Tertiary
  • Proteins / genetics
  • Proteins / metabolism*
  • Receptors, G-Protein-Coupled / genetics
  • Receptors, G-Protein-Coupled / metabolism
  • Swine / physiology*


  • Insulin
  • Leydig insulin-like protein
  • Proteins
  • RXFP2 protein, human
  • Receptors, G-Protein-Coupled