title: Third transmembrane domain of the adrenocorticotropic receptor is critical for ligand selectivity and potency.
abstract: The ACTH receptor, known as the melanocortin-2 receptor (MC2R), plays an important role in regulating and maintaining adrenocortical function. MC2R is a subtype of the melanocortin receptor (MCR) family and has unique characteristics among MCRs. Endogenous ACTH is the only endogenous agonist for MC2R, whereas the melanocortin peptides α-, β-, and γ-melanocyte-stimulating hormone and ACTH are full agonists for all other MCRs. In this study, we examined the molecular basis of MC2R responsible for ligand selectivity using ACTH analogs and MC2R mutagenesis. Our results indicate that substitution of Phe(7) with D-Phe or D-naphthylalanine (D-Nal(2')) in ACTH(1-24) caused a significant decrease in ligand binding affinity and potency. Substitution of Phe(7) with D-Nal(2') in ACTH(1-24) did not switch the ligand from agonist to antagonist at MC2R, which was observed in MC3R and MC4R. Substitution of Phe(7) with D-Phe(7) in ACTH(1-17) resulted in the loss of ligand binding and activity. Molecular analysis of MC2R indicated that only mutation of the third transmembrane domain of MC2R resulted in a decrease in D-Phe ACTH binding affinity and potency. Our results suggest that Phe(7) in ACTH plays an important role in ligand selectivity and that the third transmembrane domain of MC2R is crucial for ACTH selectivity and potency.
authors.affiliation: From the Department of Surgery, State University of New York at Buffalo, Buffalo, New York 14203 and yingkuiy@buffalo.edu.
authors.forename: Yingkui
authors.initials: Y
authors.lastname: Yang
authors.affiliation: the Departments of Medicine.
authors.forename: Vinod
authors.initials: V
authors.lastname: Mishra
authors.affiliation: Genetics, and.
authors.forename: Chiquito J
authors.initials: CJ
authors.lastname: Crasto
authors.affiliation: From the Department of Surgery, State University of New York at Buffalo, Buffalo, New York 14203 and.
authors.forename: Min
authors.initials: M
authors.lastname: Chen
authors.affiliation: Pediatrics, University of Alabama at Birmingham, Birmingham, Alabama 35233.
authors.forename: Reed
authors.initials: R
authors.lastname: Dimmitt
authors.affiliation: From the Department of Surgery, State University of New York at Buffalo, Buffalo, New York 14203 and.
authors.forename: Carroll M
authors.initials: CM
authors.lastname: Harmon
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D005434:Flow Cytometry; D057809:HEK293 Cells; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D018043:Receptors, Corticotropin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D008024:Ligands; D018043:Receptors, Corticotropin
keywords: 7-Helix Receptor; Cyclic AMP (cAMP); G Protein-coupled Receptor (GPCR); Membrane Protein; Peptide Hormone