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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.

endocrinology

Ala/Thr201 in Extracellular Loop 2 and Leu/Phe290 in Transmembrane Domain 6 of Type 1 Frog Gonadotropin-Releasing Hormone Receptor Confer Differential Ligand Sensitivity and Signal Transduction