Combined modification of intracellular and extracellular loci on human gonadotropin-releasing hormone receptor provides a mechanism for enhanced expression

The mammalian gonadotropin-releasing hormone (GnRH) receptor (GnRH-R) has b een a therapeutic target for human and animal medicine. This receptor is a unique G-protein-coupled receptor that lacks the intracellular C-terminal d omain commonly associated with this family. Development of highthrough put screens for agents active in humans has been hampered by low expression lev els of the hGnRH-R in cellular models. Two sites have attracted the interes t of laboratories studying regulation of expression, The chimeric addition of the C-terminal tail from catfish GnRH-R (cfGnRH-R) to the rat GnRH-R sig nificantly augmented receptor expression in CH, cells. In addition, rodent GnRH-R contains 327 amino acids, but cow, sheep, and human GnRH-R (hGnRH-R) contain 328 residues, the "additional" residue being a Lys 191. Deletion o f Lys 191 (del 191) from the hGnRH-R resulted in increased receptor express ion levels and decreased internalization rates in both COS-7 and HEK 293 ce lls. In this study, the combined effect of the addition of the C-tail from cfGnRH-R and deletion of the Lys 191 from the hGnRH-R was compared to expre ssion of the wild-type (WT) or either alteration alone in a transient expre ssion system using primate cells. The altered receptor (hGnRH-R[del 191]-C- tail) showed significantly increased receptor expression at the cell surfac e compared with the WT or either modification alone. The inositol phosphate response to stimulation was also significantly elevated in response to GnR H agonist. After treatment with a GnRH agonist, the altered receptors showe d a slower internalization rate. The homologous steady-state regulation of the WT and the altered receptors was similar, although the response of the altered receptors was significantly decreased. These results suggest that t he conformational change in the receptor as a result of the deletion of Lys 191 and the addition of the C-terminus tail substantially increased the st eady-state receptor expression and decreased internalization and homologous regulation. Because the effects on expression are greater than additive, i t appears that these alterations exert their effects by differing means. Th ese techniques for expression of the hGnRH-R in transfected mammalian cells provide the basis for a therapeutic screen for GnRH analogs, agonists, and antagonists of the hGnRH.