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

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.