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/human chorionic gonadotropin receptors were converted to serine (C621S, C622S, C621/622S) and glycine residues (C621/C622G) by site directed mutagenesis. The wild type and mutant receptor cDNAs were cloned into the mammalian expression vector (PCMV4) and human embryonic kidney cells (293 cells) were transiently transfected with these constructs. Equilibrium binding studies with [(125)I]hCG (human chorionic gonadotropin) showed that the mutant and wild type receptors expressed on the cell surface exhibited similar K(d). The effect of mutation of the conserved cysteine residues on the ability of the receptors to undergo ligand-induced down-regulation was then tested. In vitro exposure of cells expressing the wild type receptor to a saturating concentration of human chorionic gonadotropin (100 ng/ml) for 24 h resulted in modest down-regulation of receptors. The palmitoylation deficient mutants, C621S, C622S, C621/622S and C621/622G, showed increased down-regulation compared with the wild type receptor. The extent of down-regulation of the mutant receptors correlated with increased internalization of the receptor. Additionally, the G protein coupling efficiency of the palmitoylation 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 more prone to ligand-induced receptor down-regulation. Furthermore, abrogation of palmitoylation by mutagenesis showed no effect on the efficiency of the palmitoylation deficient mutants to couple to Gs protein.
Kawate
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Department of Obstetrics/Gynecology, University of Michigan Medical School, Ann Arbor 48109, USA.
Peegel
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Menon
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KM
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D006063:Chorionic Gonadotropin / Q000378:metabolism / Q000494:pharmacology; D017124:Conserved Sequence; D000242:Cyclic AMP / Q000032:analysis; D003545:Cysteine / Q000378:metabolism*; D018076:DNA, Complementary; D015536:Down-Regulation / Q000235:genetics*; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000196:embryology; D007700:Kinetics; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D010168:Palmitates / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D051381:Rats; D011974:Receptors, LH / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D006063:Chorionic Gonadotropin; D018076:DNA, Complementary; D008024:Ligands; D010168:Palmitates; D011974:Receptors, LH; D000242:Cyclic AMP; D019205:GTP-Binding Protein alpha Subunits, Gs; D003545:Cysteine