title: Structural insights into the role of the ACTH receptor cysteine residues on receptor function.
abstract: The ACTH receptor, also known as the melanocortin-2 receptor (MC2R), is critical for ACTH-mediated adrenal glucocorticoid release. Human MC2R (hMC2R) has 10 cysteine residues, which are located in extracellular loops (ELs), transmembrane domains (TMs), and intracellular loops (ILs). In this study, we examined the importance of these cysteine residues in receptor function and determined their involvement in disulfide bond formation. We replaced these cysteines with serine and expressed the mutated receptors in adrenal OS3 cells, which lack endogenous MC2R. Our results indicate that four mutations, C21S in NH(2) terminus, C245S, C251S, and C253S in EL3, resulted in significant decrease both in receptor expression and receptor function. Mutation of cysteine 231 in TM6 significantly decreased ACTH binding affinity and potency. In contrast, the five other mutated receptors (C64S, C158S, C191S, C267S, and C293S) did not significantly alter ACTH binding affinity and potency. These results suggest that extracellular cysteine residue 21, 245, 251, and 253, as well as transmembrane cysteine residue 231 are crucial for ACTH binding and signaling. Further experiments suggest that a disulfide bond exists between the residue C245 and C251 in EL3. These findings provide important insights into the importance of cysteine residues of hMC2R for receptor function.
authors.affiliation: Division of Pediatric Surgery, University of Alabama at Birmingham, 1600 7th Avenue South, Birmingham, AL 35233, USA. ying-kui.yang@ccc.uab.edu
authors.forename: Yingkui
authors.initials: Y
authors.lastname: Yang
authors.forename: Min
authors.initials: M
authors.lastname: Chen
authors.forename: Robert Allen
authors.initials: RA
authors.lastname: Kesterson
authors.forename: Carroll M
authors.initials: CM
authors.lastname: Harmon
mesh_terms: D000324:Adrenocorticotropic Hormone / Q000378:metabolism; D000595:Amino Acid Sequence; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry*; D004220:Disulfides / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D044103:Receptor, Melanocortin, Type 2 / Q000737:chemistry* / Q000502:physiology*; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014162:Transfection
chemical_list: D004220:Disulfides; D044103:Receptor, Melanocortin, Type 2; D013439:Sulfhydryl Reagents; D000324:Adrenocorticotropic Hormone; D000242:Cyclic AMP; D003545:Cysteine; D004229:Dithiothreitol