N. Kawate et al., ROLE OF PALMITOYLATION OF CONSERVED CYSTEINE RESIDUES OF LUTEINIZING-HORMONE HUMAN CHORIOGONADOTROPIN RECEPTORS IN RECEPTOR DOWN-REGULATION, Molecular and cellular endocrinology, 127(2), 1997, pp. 211-219
The conserved cysteine residues 621 and 622 of luteinizing hormone/hum
an chorionic gonadotropin receptors were converted to serine (C621S, C
622S, C621/622S) and glycine residues (C621/C622G) by site directed mu
tagenesis. The wild type and mutant receptor cDNAs were cloned into th
e mammalian expression vector (PCMV4) and human embryonic kidney cells
(293 cells) were transiently transfected with these constructs. Equil
ibrium binding studies with [I-125]hCG (human chorionic gonadotropin)
showed that the mutant and wild type receptors expressed on the cell s
urface exhibited similar K-d. The effect of mutation of the conserved
cysteine residues on the ability of the receptors to undergo ligand-in
duced down-regulation was then tested. In vitro exposure of cells expr
essing the wild type receptor to a saturating concentration of human c
horionic gonadotropin (100 ng/ml) for 24 h resulted in modest down-reg
ulation of receptors. The palmitoylation deficient mutants, C621S, C62
2S, C621/622S and C621/622G, showed increased down-regulation compared
with the wild type receptor. The extent of down-regulation of the mut
ant receptors correlated with increased internalization of the recepto
r. Additionally, the G protein coupling efficiency of the palmitoylati
on deficient mutants was not different from the wild type since the EC
(50)s for cyclic AMP (cAMP) production were identical in both groups.
These studies demonstrate that palmitoylation deficient mutants are mo
re prone to ligand-induced receptor down-regulation. Furthermore, abro
gation of palmitoylation by mutagenesis showed no effect on the effici
ency of the palmitoylation deficient mutants to couple to Gs protein.
(C) 1997 Elsevier Science Ireland Ltd.