Inhibition of human type i gonadotropin-releasing hormone receptor (GnRHR) function by expression of a human type II GnRHR gene fragment.

Humans possess only one functional GnRH receptor, the type I GnRH receptor (GnRHR-I). A type II GnRH receptor (GnRHR-II) gene homolog exists, but it is disrupted by a frame shift and premature stop codon, suggesting that a conventional receptor is not translated from this gene. However, the gene remains transcriptionally active and displays alternative splicing. We identified a putative translational start site 117 bp downstream of the premature stop codon. Use of this start codon encodes a protein (designated as the GnRHR-II-reliquum) corresponding to the domains from the cytoplasmic end of transmembrane domain-5 to the carboxyl terminus of the putative full-length receptor. Immunocytochemistry revealed that GnRHR-II-reliquum expression appeared to be localized throughout the cytoplasm. Transient cotransfection of GnRHR-I and GnRHR-II-reliquum constructs into COS-7 cells resulted in reduced expression of the GnRHR-I at the cell surface and impaired signaling via the GnRHR-I as revealed by reduction of GnRH-induced inositol phosphate accumulation. This inhibitory effect was specific and dependent on the degree of GnRHR-II-reliquum coexpressed. Immunoblot analysis revealed that the total cell GnRHR-I complement, i.e. both cell-surface and nascent intracellular receptors, was markedly reduced by coexpression of the GnRHR-II-reliquum. Treatments with cell-permeable agents that blocked either de novo protein synthesis (cycloheximide) or proteinase-mediated degradation (leupeptin and phenylmethylsulfonyl fluoride) failed to alter the inhibitory effect of GnRHR-II-reliquum coexpression, suggesting that the inhibitory effect is exerted at the nucleus/endoplasmic reticulum or Golgi apparatus level, possibly by perturbing normal processing of GnRHR-I from these sites. We suggest that the GnRHR-II-reliquum plays a modulatory role in GnRHR-I expression.

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