Molecular cloning and expression of the ovine testicular follicle stimulating hormone receptor

Abstract

A sheep testicular cDNA library constructed in pcDNAl vector was screened with a probe generated by polymerase chain reaction (PCR) and corresponding to a 1.6 kb fragment of the rat luteinizing hormone receptor cDNA. Several clones hybridizing to the rat probe at low stringency were sequenced to obtain 95% of the putative full-length ovine follicle-stimulating hormone receptor (oFSH-R) cDNA. The missing 5' region was obtained by PCR amplification of the cDNA library. Sequencing revealed a 2085 nucleotide open reading frame encoding a mature protein of 678 amino acids (74,580 daltons). The oFSH-R is remarkably similar (>; 90%) to the human and rat FSH receptors, has a structural motif like the G protein-coupled family of receptors and contains 3 potential sites for N-linked glycosylation. RNA blot analysis revealed two major transcripts of 2.6 kb and 6.7 kb in size and a smaller transcript of about 1 kb in the sheep testis. A 53 residue segment in the extracellular domain unique to the receptor contains more than 50% of residues bearing functional side chains that could participate in ligand (FSH) interaction and/or signal transduction. Transfection of human fetal kidney cell line (293) with the cloned oFSH receptor cDNA based in pcDNAl/Neo vector revealed functional expression. Labeled oFSH bound to receptor expressed on the membrane with high affinity and specificity. In stably transfected 293 cells, purified oFSH and hFSH but not oLH stimulated cyclic AMP accumulation. Chemically deglycosylated oFSH (DG-oFSH) was inactive in these cells but it effectively blocked the action of native hormone. Thus, the functional characteristics of the cloned receptor are similar to the natural receptor in testis.