PALMITOYLATION OF LUTEINIZING-HORMONE HUMAN CHORIOGONADOTROPIN RECEPTORS IN TRANSFECTED CELLS - ABOLITION OF PALMITOYLATION BY MUTATION OF CYS-621 AND CYS-622 RESIDUES IN THE CYTOPLASMIC TAIL INCREASES LIGAND-INDUCED INTERNALIZATION OF THE RECEPTOR
N. Kawate et Kmj. Menon, PALMITOYLATION OF LUTEINIZING-HORMONE HUMAN CHORIOGONADOTROPIN RECEPTORS IN TRANSFECTED CELLS - ABOLITION OF PALMITOYLATION BY MUTATION OF CYS-621 AND CYS-622 RESIDUES IN THE CYTOPLASMIC TAIL INCREASES LIGAND-INDUCED INTERNALIZATION OF THE RECEPTOR, The Journal of biological chemistry, 269(48), 1994, pp. 30651-30658
We have examined whether the two cysteine residues (621 and 622) of th
e carboxyl-terminal cytoplasmic domain of the rat luteinizing hormone
(LH/hCG) receptor are potential sites for palmitoylation. The full-len
gth LH/hCG receptor cDNA was cloned into an expression vector (hCGR-pC
MV4). A human embryonic kidney cell line expressing large T antigen (2
93T cells) was transiently transfected with hCGR-pCMV4 by the calcium
phosphate precipitation technique. The functional expression of the re
ceptor was confirmed by S-35-labeled cysteine incorporation into the r
eceptor as well as by I-125-human chorionic gonadotropin (hCG) binding
. The transfected cells were then labeled with [H-3]palmitic acid, and
the labeled receptors purified on hCG-Affi-Gel matrix and subjected t
o SDS polyacrylamide gel electrophoresis and autoradiography. The hCGR
-pCMV4 trans fected cells incorporated [H-3]palmitic acid into a 92-kD
a band corresponding to the mature form of the LH/hCG receptor; this b
and was absent in cells transfected with vector alone. Site directed m
utagenesis of either cysteine 621 or 622 to serine residue was partial
ly inhibitory, whereas mutation of both cysteine residues (621 and 622
) completely abolished palmitoylation. Scatchard analyses revealed tha
t the mutant and wild type receptors have similar affinities for I-125
-hCG. The biological function of palmitoylation was then examined in t
he transfected cells. The results showed that although the intracellul
ar trafficking of the receptor and the ability to stimulate cyclic AMP
production were unaffected, the rate of ligand-induced internalizatio
n of the receptor was higher in palmitoylation-deficient mutants compa
red to the wild type receptors. The first order rate constants of inte
rnalization of the mutant receptors were over 2-fold higher than the w
ild type. Intracellular degradation of the receptor-bound ligand was a
lso higher in the mutants. These studies suggest that the native LH/hC
G receptor is palmitoylated at cysteine residues 621 and 622, creating
a membrane-anchoring site at the putative cytoplasmic domain. This pa
lmitic acid-mediated anchoring decreases the ligand-induced receptor i
nternalization thereby prolonging the retention of the ligand-bound re
ceptor on the cell surface.