13 Ann. N.Y. Acad. Sci. 1041: 13–16 (2005). © 2005 New York Academy of Sciences. doi: 10.1196/annals.1282.003 Characterization of the Rat INSL3 Receptor D.J. SCOTT, a P. FU, a P.-J. SHEN, a A. GUNDLACH, a S. LAYFIELD, a A. RIESEWIJK, b H. TOMIYAMA, c J.M. HUTSON, c G.W. TREGEAR, a AND R.A.D. BATHGATE a a Howard Florey Institute, University of Melbourne, Victoria, Australia 3010 b Department of Pharmacology, NV Organon, Oss, The Netherlands c F. Douglas Stephens Surgical Research Laboratory, Murdoch Children’s Research Institute, Victoria, Australia ABSTRACT: Human LGR8, initially discovered as a low-affinity relaxin recep- tor, has now been characterized as the INSL3 receptor. To investigate LGR8 function in the rat, an LGR8 ortholog was identified in the rat genome, and the full-length sequence was cloned and expressed. Rat LGR8 bound INSL3 with high affinity, clearly demonstrating that it is the rat INSL3 receptor. Interest- ingly, native rat relaxin did not activate rat LGR8, indicating that relaxin is not an endogenous ligand for rat LGR8. LGR8 mRNA expression was demonstrat- ed in the gubernaculum at the time of testis descent and in the testis associated with germ cells. KEYWORDS: INSL3; receptor; LGR8; relaxin IDENTIFICATION AND EXPRESSION OF RAT LGR8 The leucine-rich repeat containing G-protein receptor 8 (LGR8), initially described as a relaxin receptor, has clearly been demonstrated as the receptor for insulin-like peptide 3 (INSL3). 1–3 To further study the function of LGR8, we cloned the rat LGR8 ortholog and studied its ligand binding profile and expression pattern. Human LGR8 was used with the BLASTn to identify 1,612 bp of rat sequence homologous to human LGR8 in the NCBI HTGS database (accession number AC098990). A putative rat LGR8 sequence was assembled from the fragmented database hits, confirmed, and completed (2214-bp coding region) by sequencing multiple, overlapping RT-PCR products (Genebank accession number AY906861). At the protein level, rat LGR8 shares 82.6% and 95.4% protein identity with human and mouse LGR8, respectively. This high level of sequence identity is indicative of strict evolutionary conservation. With the use of RT-PCR, rat LGR8 mRNA expression was previously reported in the gubernaculum of fetal male rats, where it plays a pivotal role in INSL3-mediated differentiation and development of the gubernaculum. 4 The expression of LGR8 mRNA in the gubernaculum was further investigated in the rat fetus using in situ hy- bridization. As demonstrated in FIGURE 1, LGR8 mRNA is expressed in the guber- Address for correspondence: Dr. R. Bathgate, Howard Florey Institute, University of Mel- bourne, Victoria 3010, Australia. Voice: +61 3 8344 5648; fax: +61 3 9348 1707. r.bathgate@hfi.unimelb.edu.au