The mammalian GnRH receptor is an atypical G protein-coupled receptor which lacks the C-terminal cytoplasmic tail that is present in all other seven-transmembrane domain receptors. The mouse and rat GnRH receptors contain 327 amino acids, whereas human, sheep, and bovine receptors have an additional residue in the second extracellular loop at position 191. Another notable species difference is that human receptors undergo agonist-induced internalization much more rapidly than the mouse receptor. In this report, the role of the additional amino acid (Lys191) in GnRH receptor function was studied in transiently expressed mutant and wild-type human and mouse GnRH receptors. Deletion of Lys191 from the human GnRH receptor caused a 4-fold increase in receptor expression in COS-1 and HEK 293 cells and a modest increase in binding affinity. The magnitude of the agonist-induced inositol phosphate response mediated by the deltaK191 human receptor was similar to that of the wild-type receptor, but the EC50 was decreased by about 5-fold. In addition, the rate of internalization of the deltaK191 human receptor was significantly reduced and was similar to that of the mouse receptor. In contrast to these effects of deletion of Lys191, its replacement by Arg, Glu, Gln, or Ala caused no significant change in receptor expression or function. These findings demonstrate that a specific residue in the extracellular region of the human GnRH receptor is a significant determinant of receptor expression, agonist-induced activation, and internalization.

The coupling of agonist-activated heptahelical receptors to their cognate G proteins is often dependent on the amino-terminal region of the third intracellular loop. Like many G protein-coupled receptors, the gonadotropin-releasing hormone (GnRH) receptor contains an apolar amino acid in this region at a constant distance from conserved Pro and Tyr/Asn residues in the fifth transmembrane domain (TM V). The hypothalamic decapeptide, gonadotropin-releasing hormone (GnRH), 1 controls the activity of the reproductive system by regulating the synthesis and release of luteinizing hormone and follicle-stimulating hormone from the anterior pituitary gland (1, 2). In pituitary gonadotrophs, the actions of GnRH are mediated by specific high affinity receptors that promote G protein-dependent stimulation of phosphoinositide turnover and calcium mobilization (3). The cloned GnRH receptors of mouse, rat, human, sheep, cow, and pig exhibit more than 85% amino acid identity among species (4). The hydropathy analysis of the GnRH receptor coding region is consistent with the seven transmembrane domain structure that is characteristic of the G protein-coupled receptor (GPCR) superfamily. Mammalian GnRH receptors exhibit several unique structural features, including the absence of an intracellular carboxyl-terminal tail, reciprocal exchange of the conserved Asp and Asn residues in transmembrane domains (TM) II and VII, and replacement of Tyr with Ser in the Asp-Arg-Tyr motif located at the junction of TM III and the second intracellular loop (IL2) (3, 4).In several GPCRs, mutational analysis has shown that regions of the third intracellular loop (IL3), in particular its amino-and carboxyl-terminal portions, and sometimes the cytoplasmic tail of the receptor, are important determinants of receptor-G protein coupling (5-10). In some GPCRs, the first and second intracellular loops have also been shown to be important in interaction with G proteins and activation of signal transduction (10). For example, we have recently demonstrated the dependence of cAMP-induced signaling from the GnRH receptor on specific residues in IL1 that are not essential for activation of the phosphoinositide signaling pathway (11). The role of IL3 in GnRH receptor function has not been examined in detail, but recent evidence has indicated its ability to couple the receptor to G s -and G q/11 -mediated signal transduction pathways. However, this study did not identify specific amino acid residues within the loop that are functionally important for receptor-G protein coupling and selectivity (12). Although the GnRH receptor has the unusual structural features mentioned above, it also contains several conserved residues and sequences in its TM helices and loops that are typical of other members of the GPCR superfamily. One of these is a hydrophobic amino acid (Leu 237 ) located at the NH 2 -terminal region of IL3. This residue is positioned at a constant distance from the conserved Pro and Tyr residues in TM V (Tyr is replaced by Asn in the GnRH receptor), and ...

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