Yingkui Yang

The melanocortin-5 receptor (MC5R) is a subtype receptor of the melanocortin receptor (MCR) family, which is expressed centrally, as well as in a variety of peripheral tissues. MC5R has been implicated in many different physiological fields such as lipid metabolism and exocrine function. However, the specific molecular determinants of MC5R responsible for ligand binding and receptor signaling are currently unknown. The aim of this study is to determine the molecular basis of human MC5R (hMC5R) responsible for ligand binding and receptor signaling. Twenty-four single mutations of hMC5R were created and tested. Our results indicate that (1) substituting charged amino acid residue E92 in transmembrane domain 2 (TM2), aspartic acid 115 (D115) and D119 in TM3, and histidine (H) 257 in TM6 with alanine dramatically reduced NDP-α-MSH binding affinity and receptor signaling and (2) substituting aromatic amino acids phenylalanine (F) 195 in TM5, F254 in TM6, and H276 in TM7 with alanine also significantly decreased NDP-α-MSH binding and receptor activity. Combining pharmacological results and computer modeling, our results suggest that D115 and D119 in TM3, F195 in TM5, and F254 in TM6 may form a binding pocket for NDP-α-MSH binding. Our results provide important information about the structural aspects of hMC5R responsible for ligand binding and receptor signaling.

The melanocortin receptor (MCR) subtype family is a member of the GPCR superfamily, and each of them has a different pharmacological profile with regard to the relative potency of the endogenous and synthetic melanocortin peptides. α-MSH and ACTH are endogenous nonselective agonists for MC1R, MC3R, MC4R, and MC5R. In this study, we examined the role of Phe⁷ in ACTH on human (h) MC1R, MC3R, and MC4R binding and signaling. Our results indicate that substitution of Phe⁷ with d-Nal(2′)⁷ in ACTH1−24 yields a pharmacological profile different from that for substitution of Phe⁷ with d-Nal(2′)⁷ in MSH in hMC1R, hMC3R, and hMC4R. N-d-Nal(2′)⁷-ACTH1−24 is an agonist at hMC3R and hMC4R which did not change the peptide from an agonist to an antagonist at hMC3R and hMC4R. Further experiments indicate that N-d-Nal(2′)⁷-ACTH1−17 is the minimal peptide required for hMC3R and hMC4R activation. Single-amino acid substitution studies of d-Nal(2′)⁷-ACTH1−17 indicate that amino acid residues 15−17 in N-d-Nal(2′)⁷-ACTH1−17 are crucial for hMC3R and hMC4R activation. Substitutions of these amino acid residues reduced or abolished agonist activity at hMC3R and hMC4R. Conformational studies revealed a new β-turn (Arg⁸-Trp⁹-Gly¹⁰-Lys¹¹) in N-d-Nal(2′)⁷-ACTH1−17, compared to the β-turn-like structure at NDP-α-MSH (His⁶-d-Phe⁷-Arg⁸-Trp⁹). Our results suggest that NDP-α-MSH and N-d-Nal(2′)⁷-ACTH1−17 do not share the same binding site; the highly basic C-terminal fragment (Lys¹⁵-Lys¹⁶-Arg¹⁷) of N-d-Nal(2′)⁷-ACTH1−17 induced a new β-turn, and this shift contributed the selective agonist activity at hMC3R and hMC4R.

The melanocortin-2 receptor (MC2R), also known as the adrenocorticotropic hormone (ACTH) receptor, plays an important role in regulating and maintaining adrenocortical function, specifically steroidogenesis. Mutations of the human MC2R (hMC2R) gene have also been identified in humans with familial glucocorticoid deficiency; however, the molecular basis responsible for hMC2R ligand binding and signaling remains unclear. In this study, both truncated ACTH peptides and site-directed mutagenesis studies were used to determine molecular mechanisms of hMC2R binding ACTH and signaling. Our results indicate that ACTH1−16 is the minimal peptide required for hMC2R binding and signaling. Mutations of common melanocortin receptor family amino acid residues E80 in transmembrane domain 2 (TM2), D107 in TM3, F178 in TM4, F235 and H238 in TM6, and F258 in TM7 significantly reduced ACTH-binding affinity and signaling. Furthermore, mutations of unique amino acids D104 and F108 in TM3 and F168 and F178 in TM4 significantly decreased ACTH binding and signaling. In conclusion, our results suggest that the residues in TM2, TM3, and TM6 of hMC2R share similar binding sites with other MCRs but the residues identified in TM4 and TM7 of hMC2R are unique and required for ACTH selectivity. Our study suggests that hMC2R may have a broad binding pocket in which both conserved and unique amino acid residues are required, which may be the reason why α-MSH was not able to bind hMC2R.

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 NH₂terminus, 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.