B. Byrne et al., Isolation and characterisation of the marmoset gonadotrophin releasing hormone receptor: Ser(140) of the DRS motif is substituted by Phe, J ENDOCR, 163(3), 1999, pp. 447-456
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.