ROLE OF PALMITOYLATION OF CONSERVED CYSTEINE RESIDUES OF LUTEINIZING-HORMONE HUMAN CHORIOGONADOTROPIN RECEPTORS IN RECEPTOR DOWN-REGULATION

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.