Molecular cloning, distribution and pharmacological characterization of a novel gonadotropin-releasing hormone ([Trp super(8)] GnRH) in frog brain

To date nine structural variants of GnRH have been identified in vertebrates and two additional forms have been isolated from a tunicate. In amphibians only mammalian GnRH ([Arg super(8)] GnRH) and type II GnRH (chicken GnRH II, [His super(5), Trp super(7), Tyr super(8)] GnRH) have been identified. In the present study, a full-length cDNA encoding a novel type of GnRH was isolated from pituitary of Rana dybowskii. The GnRH gene encodes a GnRH peptide ([Trp super(8)] GnRH) in which tryptophan is substituted for arginine of mammalian GnRH Northern blot analysis revealed the presence of a single 500 bp transcript for the [Trp super(8)] GnRH precursor in forebrain but its absence in testis, ovary, kidney and liver. Restriction digests of genomic DNA demonstrated a single copy of the gene. The [Trp super(8)] GnRH immunoreactive cells were identified in the preoptic area of the frog brain. Synthetic [Trp super(8)] GnRH was tested for its ability to stimulate inositol phosphate production by COS-1 cells transfected with the cloned Xenopus pituitary GnRH receptor and the cloned human GnRH receptor. [Trp super(8)] GnRH had a potency of about 60% compared with mammalian GnRH ([Arg super(8)] GnRH) for the Xenopus receptor, whereas the potency of [Trp super(8)] GnRH was approximately 5% compared with mammalian GnRH for the human receptor. Both mammalian GnRH and [Trp super(8)] GnRH were 1000-fold less potent than type II GnRH for the Xenopus GnRH receptor. The similar potency of [Arg super(8)] GnRH and the novel [Trp super(8)] GnRH for the Xenopus pituitary receptor indicates that, unlike the human receptor, the Xenopus receptor does not discriminate between these amino acids in position eight thereby allowing substitution of the arginine in the mammalian GnRH.