INHIBITION OF GONADOTROPIN-RELEASING-HORMONE RECEPTOR SIGNALING BY EXPRESSION OF A SPLICE VARIANT OF THE HUMAN RECEPTOR

GnRH binds to a specific G protein-coupled receptor in the pituitary t o regulate synthesis and secretion of gonadotropins. Using RT-PCR and human pituitary poly(A)(+) RNA as a template, the full-length GnRH rec eptor (wild type) and a second truncated cDNA characterized by a 128-b p deletion between nucleotide positions 522 and 651 were cloned. The d eletion causes a frame shift in the open reading frame, thus generatin g new coding sequence for further 75 amino acids. The truncated cDNA a rises from alternative splicing by accepting a cryptic splicing accept or site in exon 2. Distinct translation products of approximately 45-5 0 and 42 kDa were immunoprecipitated from COS-7 cells transfected with cDNA coding for wild type GnRH receptor and the truncated splice vari ant, respectively. Immuno-cytochemical and enzyme-linked immunosorbent assay studies revealed a membranous expression pattern for both recep tor isoforms. Expression of the splice variant, however, occurred at a significantly lower cell surface receptor density. In terms of ligand binding and phospholipase C activation, the wild type receptor showed characteristics of a typical GnRH receptor, whereas the splice varian t was incapable of ligand binding and signal transduction. Coexpressio n of wild type and truncated proteins in transiently or stably transfe cted cells, however, resulted in impaired signaling via the wild type receptor by reducing maximal agonist-induced inositol phosphate accumu lation. The inhibitory effect depended on the amount of splice variant cDNA cotransfected and was specific for the GnRH receptor because sig naling via other G(q/11)-coupled receptors, such as the thromboxane A( 2), M-5 muscarinic, and V-1 vasopressin receptors, was not affected. I mmunological studies revealed that coexpression of the wild type recep tor and the truncated splice Variant resulted in impaired insertion of the wild type receptor into the plasma membrane. Thus, expression of truncated receptor proteins may highlight a novel principle of specifi c functional inhibition of G protein-coupled receptors.