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

Abstract

In order to facilitate the understanding of gonadotrophinreleasing hormone (GnRH) agonist and antagonist action in the primate animal model, the marmoset GnRH receptor (GnRH-R) was cloned and characterised. It was shown to have 95% and 85% sequence identity with the human and rat GnRH-Rs, respectively, and, when transiently expressed in COS-7 cells, it exhibited high-affinity des-Gly 10 , ]-GnRH binding, with a K d value similar to those of both the rat and human forms, but with a greatly reduced B max value. The ED 50 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 the 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 interesting feature of the marmoset GnRH-R was the substitution of this motif with DRF. In order to investigate the DRS to DRF substitution, a Ser 140 Phe rat GnRH-R mutant was generated. The mutant had a K d value similar to that of the wild-type rat receptor, although the B max value was slightly lower, indicating that expression of functional mutant receptor at the cell surface was reduced. The ED 50 value for IP production was also similar to that of the wild-type receptor, with a reduction in maximal response. The level of internalisation for the rat wild-type and mutant GnRH-R constructs was also assessed and the Ser 140 Phe mutant was shown to have an increased rate of receptor internalisation, suggesting a role for this residue in regulating internalisation. These results show that the marmoset GnRH-R exhibits a substitution in the DRS motif and that this substitution may play a part in desensitisation and internalisation events.

Alternative splicing of a single precursor mRNA generates two subtypes of Gonadotropin-Releasing Hormone receptor orthologues and their variants in the bivalve mollusc Crassostrea gigas

“…EC: Extracellular loop, IC: Intracellular loop. Accession numbers in EMBL/Genbank: Cg-GnRH-R: (oyster) (AJ890150); Cg-GnRH-RII-S (AM904566); Cg-GnRH-RII-L (AM904565); Cg-GnRH-R-TF (AM904567); Cj-GnRH-RII: (marmoset) Callithrix jacchus (AF368286); Clg-GnRH-RI: (catfish) Clarias gariepinus (X97497); Dm-AKH-R: Drosophila melanogaster (AF077299); Gg-GnRH-RI: (chicken) Gallus gallus (AJ304414); Xl-GnRH-RI: Xenopus laevis (AF172330) and Cj-GnRH-RI is from (Byrne et al, 1999). Fig.…”

Review on testicular development, structure, function, and regulation in common marmoset

“…The Tyr/Ser residue is believed to be a potential phosphorylation site, acting as a substrate for a G‐protein receptor kinase and thus is important for receptor desensitization and internalization (Arora et al, 1997; Millar et al, 2004). Byrne et al (1999) found that the GnRH‐R in male marmosets had 95% and 85% sequence identity with that in humans and rats, respectively. However, the highly conserved DRY motif in the GnRH‐R in marmoset is substituted with DRF, i.e., Ser is substituted with phenylalanine (Phe), instead of DRS as in all other mammals that have been studied so far.…”

A role of Histidine151 in the lamprey gonadotropin-releasing hormone receptor-1 (lGnRHR-1): Functional insight of diverse amino acid residues in the position of Tyr of the DRY motif in GnRHR from an ancestral type II receptor

“…In the present study, the lamprey GnRHR and three of the four mutants of lamprey GnRHR all induced a decrease in receptor expression except the mutant DRY 151 which induced an increase in receptor expression. Other studies on the DRS motif in mammalian type I GnRHRs revealed that Ser could be involved in ligand binding and receptor internalization events (Arora et al, 1997; Arora et al, 1995; Byrne et al, 1999). Substituting Ser with Tyr increased both the ligand binding affinity and internalization (Arora et al, 1995) but substituting with Ala had no effect (Arora et al, 1997).…”

“…This suggests that the type II GnRH receptor mutants may not be capable of augmenting internationalization. In addition, Byrne et al (1999) suggested that the third amino acid position in DRY and the phosphorylation in the site are not essential for internalization. This would support the result of our study that the rate of internalization of the DRE 151 , DRS 151 and DRY 151 mutants was similar to that of wild-type because the c-terminal tail was more essential for internalization, although DRX 151 , point deletion of His 151 deletion in DRH, decreased the rate of internalization compared to other mutants and the wild-type receptors.…”

“…1). To date, there are few reports about the functional significance of the Ser in DRS of type I receptors (Arora et al, 1997; Arora et al, 1995; Byrne et al, 1999). Thus, the functional significance of this variation of the DRY motif, particularly the type II GnRHR, is not established.…”