Isolation and characterisation of the marmoset gonadotrophin releasing hormone receptor: Ser(140) of the DRS motif is substituted by Phe

In order to facilitate the understanding of gonadotrophin-releasing hormone (GnRH) agonist and antagonist action in the primate animal model, the marm oset GnRH receptor (GnRH-R) was cloned and characterised. It was shown to h ave 95% and 85% sequence identity with the human and rat GnRH-Rs, respectiv ely, and, when transiently expressed in COS-7 cells, it exhibited high-affi nity des-Gly(10),[D-Trp(6)]-GnRH binding, with a K-d value similar to those of both the rat and human form, but with a greatly reduced B-max value. Th e ED50 for production of GnRH-induced total inositol phosphate (IP) for the marmoset GnRH-R was also similar to those of the rat and the human, but th e maximal response compared with the rat receptor was markedly reduced. In all mammalian forms of the GnRH-R cloned to date, the conserved DRY region of G-protein-coupled receptors is substituted with DRS. The most interestin g feature of the marmoset GnRH-R was the substitution of this motif with DR F. In order to investigate the DRS to DRF substitution, a Ser(140)Phe rat G nRH-R mutant was generated. The mutant had a K-d value similar to that of t he wild-type rat receptor, although the B-max value was slightly lower, ind icating that expression of functional mutant receptor at the cell surface w as reduced. The ED50 value for IP production was also similar to that of th e wild-type receptor, with a reduction in maximal response. The level of in ternalisation for the rat wild-type and mutant GnRH-R constructs was also a ssessed and the Ser(140)Phe mutant was shown to have an increased rate of r eceptor internalisation, suggesting a role for this residue in regulating i nternalisation. These results show that the marmoset GnRH-R exhibits a subs titution in the DRS motif and that this substitution may play a part in des ensitisation and internalisation events.