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 the carboxyl-terminal cytoplasmic domain of the rat luteinizing hormone (LH/hCG) receptor are potential sites for palmitoylation. The full-length LH/hCG receptor cDNA was cloned into an expression vector (hCGR-pCMV4). A human embryonic kidney cell line expressing large T antigen (293T cells) was transiently transfected with hCGR-pCMV4 by the calcium phosphate precipitation technique. The functional expression of the receptor was confirmed by 35S-labeled cysteine incorporation into the receptor as well as by 125I-human chorionic gonadotropin (hCG) binding. The transfected cells were then labeled with [3H]palmitic acid, and the labeled receptors purified on hCG-Affi-Gel matrix and subjected to SDS-polyacrylamide gel electrophoresis and autoradiography. The hCGR-pCMV4-transfected cells incorporated [3H]palmitic acid into a 92-kDa band corresponding to the mature form of the LH/hCG receptor; this band was absent in cells transfected with vector alone. Site-directed mutagenesis of either cysteine 621 or 622 to serine residue was partially inhibitory, whereas mutation of both cysteine residues (621 and 622) completely abolished palmitoylation. Scatchard analyses revealed that the mutant and wild type receptors have similar affinities for 125I-hCG. The biological function of palmitoylation was then examined in the transfected cells. The results showed that although the intracellular trafficking of the receptor and the ability to stimulate cyclic AMP production were unaffected, the rate of ligand-induced internalization of the receptor was higher in palmitoylation-deficient mutants compared to the wild type receptors. The first order rate constants of internalization of the mutant receptors were over 2-fold higher than the wild type. Intracellular degradation of the receptor-bound ligand was also higher in the mutants. These studies suggest that the native LH/hCG receptor is palmitoylated at cysteine residues 621 and 622, creating a membrane-anchoring site at the putative cytoplasmic domain. This palmitic acid-mediated anchoring decreases the ligand-induced receptor internalization thereby prolonging the retention of the ligand-bound receptor on the cell surface.