MOLECULAR-CLONING, STRUCTURE, AND EXPRESSION OF A TESTICULAR FOLLITROPIN RECEPTOR WITH SELECTIVE ALTERATION IN THE CARBOXY-TERMINUS THAT AFFECTS SIGNALING FUNCTION

During the molecular cloning of the ovine testicular follicle-stimulat ing (FSH) receptor that couples to the Gs;type effector systems, we di scovered novel cDNA clones that were highly homologous. Some of these clones contained an insert of 1,584 bp, which consisted of a divergent 3' region spliced with a 5' region that was identical to nucleotides 724-1,924, forming part of the 9th and 10th exons, of the coding regio n of the ovine FSH receptor gene. The prominence of alternately splice d clone, which suggested important functional implications, prompted t his detailed investigation. Screening of the library by polymerase cha in reaction and Northern analysis of testicular messenger RNA with a s pecific ribo-probe directed to the divergent 3' region of this transcr ipt suggested existence of a full-length transcript of roughly 2.4 kb size, The cDNA was assembled and characterized for its structure, The predicted full-length sequence consisting of nucleotides -121-1,924 of the ovine FSH receptor and the novel 3' region (nucleotides 1,925-2,3 07) encoded a protein of 670 amino acids containing the entire extrace llular and transmembrane domains of the ovine FSH receptor. However, a frame-shift in the coding sequence at the point of divergence resulte d in a shorter (40 residues vs. 65 for ovine FSH receptor) C-terminus with three cysteine residues and a reduced number of potential phospho rylation sites. Two of the cysteine residues were adjacent and are app arently potential double palmitoylation sites compared to the single s ite present in the Gs coupled ovine FSH receptor. Stable expression of this novel transcript in human embryonic kidney (HEK 293) cells revea led the complete absence of cyclic AMP inducible functions, but presen ce of specific hormone binding activity on plasma membranes and promin ent cell surface immunostaining by antireceptor antiserum. There was n o alteration in hormone binding specificity because the structurally a nalogous luteinizing hormone (LH) did not bind to the receptor. The lo ss of cyclic AMP stimulation in the transfected cells was completely o pposite to the properties of the cells expressing the active wild-type receptor. When cells expressing active receptors were cotransfected w ith the altered FSH receptor cDNA, hormone action was inhibited, sugge sting that it could be functioning as a dominant negative receptor, Th e existence of this ovine FSH receptor with an altered carboxyl termin us predicts the utilization of an alternative splicing mechanism for r egulation of receptor expression, signalling and gonadal function. Our study reveals that the modular structure of the FSH receptor gene gen erates motifs that allows coupling to different effecters. This could become a common feature for all glycoprotein hormone receptors. (C) 19 97 Wiley-Liss, Inc.