ABSTRACT
A Glu/Asp7.32 residue in the extracellular loop 3 of the mammalian GnRH receptor (GnRHR) is known to interact with Arg8 of mammalian GnRH (mGnRH), which may confer preferential ligand selectivity for mGnRH than for chicken GnRH-II (cGnRH-II). However, some nonmammalian GnRHRs also have the Glu/Asp residue at the same position, yet respond better to cGnRH-II than mGnRH. Amino acids flanking Glu/Asp7.32 are differentially arranged such that mammalian and nonmammalian GnRHRs have an S-E/D-P motif and P-X-S/Y motif, respectively. We presumed the position of Ser7.31 or Pro7.33 of rat GnRHR as a potential determinant for ligand selectivity. Either placing Pro before Glu7.32 or placing Ser after Glu7.32 significantly decreased the sensitivity and/or efficacy for mGnRH, but slightly increased that for cGnRH-II in several mutant receptors. Among them, those with a PEV, PES, or SES motif exhibited a marked decrease in sensitivity for mGnRH such that cGnRH-II had a higher potency than mGnRH, showing a reversed preferential ligand selectivity. Chimeric mGnRHs in which positions 5, 7, and/or 8 were replaced by those of cGnRH-II revealed a greater ability to activate these mutant receptors than mGnRH, whereas they were less potent to activate wild-type rat GnRHR than mGnRH. Interestingly, a mutant bullfrog type I receptor with the SEP motif exhibited an increased sensitivity for mGnRH but a decreased sensitivity for cGnRH-II. These results indicate that the position of Pro and Ser near Glu7.32 in the extracellular loop 3 is critical for the differential ligand selectivity between mammalian and nonmammalian GnRHRs.
Subject(s)
Gonadotropin-Releasing Hormone/analogs & derivatives , Gonadotropin-Releasing Hormone/metabolism , Receptors, LHRH/chemistry , Receptors, LHRH/metabolism , Animals , Chickens , DNA Primers , Glutamic Acid , Kinetics , Ligands , Mammals , Models, Molecular , Molecular Conformation , Mutagenesis, Site-Directed , Polymerase Chain Reaction , Proline , Protein Conformation , Protein Structure, Secondary , Rats , Serine , Species Specificity , Substrate SpecificityABSTRACT
Recently, we have identified three distinct types of bullfrog GnRH receptor (designated bfGnRHR-1, bfGnRHR-2, and bfGnRHR-3). In the present study, we have isolated three GnRHR clones in Rana dybowskii (dyGnRHR-1, dyGnRHR-2, and dyGnRHR-3). Despite high homology of dyGnRHRs with the corresponding bfGnRHRs, dyGnRHRs revealed different signaling pathways and ligand sensitivity compared with the bfGnRHR counterparts. Activation of dyGnRHRs with GnRH stimulated cAMP-mediated gene expression. However, dyGnRHR-3 but not dyGnRHR-1 and -2 induced c-fos promoter-driven gene expression. Consistently, dyGnRHR-1 and dyGnRHR-2 were not able to increase GnRH-induced inositol phosphate accumulation, whereas all bfGnRHRs and dyGnRHR-3 were, indicating that dyGnRHR-1 and dyGnRHR-2 are coupled to solely G(s), whereas all bfGnRHRs and dyGnRHR-3 are coupled to both G(s) and G(q/11). Moreover, dyGnRHR-1 and dyGnRHR-2 showed about 10-fold less sensitivity to each ligand than that of the bfGnRHR counterparts. Using type 1 chimeric and point-mutated receptors, we further elucidated that specific amino acids, Ala/Thr(201) in extracellular loop 2 and Leu/Phe(290) in transmembrane domain 6 of the type 1 receptor, are responsible for ligand sensitivity and signal transduction pathway. Particularly, substitution of Leu(290) to Phe in dyGnRHR-1 increased GnRH-induced inositol phosphate production as well as c-fos promoter-driven gene expression whereas substitution of Phe(290) to Leu in bfGnRHR-1 decreased those activities. Collectively, these results demonstrate the presence of three types of GnRHR in amphibians, and suggest species- and type-specific ligand recognition and different signaling pathways in frog GnRHRs.
