Molecular Identification of the Human Melanocortin-2 Receptor Responsible for Ligand Binding and Signaling

Abstract

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 MC2R gene have also been identified in humans with familial glucocorticoid deficiency. However, the molecular basis of MC2R responsible for ligand binding and signaling remains unclear. In this study, both truncated ACTH peptides and site-directed mutagenesis studies were utilized to determine the molecular basis of MC2R responsible for ACTH binding and signaling. Our results indicate that ACTH1-16 is the minimal peptide required for MC2R binding and signaling and mutations of the conserved amino acid residues E80 in transmembrane domain 2 (TM2), D107 in TM3, F178 in TM4, F235, H238 in TM6 and F258 in TM7 significantly reduced ACTH binding affinity and signaling, which is similar to other MCRs. 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, TM7 of hMC2R are unique for ACTH binding. Our study suggests that hMC2R may have a broad binding pocket in which both conserved and unique amino acid residues are involved, which may be the reason why α-MSH was not able to bind hMC2R. Keywords stress; POMC; MC2R; agonist; GPCR The melanocortin-2 receptor (MC2R), also known as the adrenocorticotropic hormone (ACTH) receptor, plays an important role in the regulation of adrenal cortisol secretion (9). Mutations of the hMC2R have been identified in a potentially fatal disease called familial glucocorticoid deficiency (FGD) in which affected individuals are deficient in cortisol and likely to succumb to hypoglycemia or overwhelming infection in infancy or childhood if they are not treated (8,10,16,35,39,40,44,49,50,52,(58)(59)(60). Over expression of this receptor is also found in adrenal adenomas or hyperplasia associated with glucocorticoid overproduction (Cushing syndrome) (1,22,37,38).Extensive studies had been performed to determine the molecular basis of ACTH in receptor function in the last two decades but the results are not consistent. In early years, ACTH1-14, ACTH11-19 and ACTH11-24 were reported to increase corticosterone levels in the isolated adrenal cell system and ACTH11-24 plays an important role in adipocyte function (14,24,42,45). However, ACTH1-14 was also reported not able to bind MC2R (48). The explanation for these variations is that different methods of adrenal cell preparation and separate receptors may be involved in these peptide activities (53). Further studies of some ACTH peptide truncations indicated that ACTH1-17 is the minimal peptide required for ligand binding and signaling (14,24,42,45). However, detailed structure-function of ACTH responsible for MC2R binding and signaling is still unclear.Cloning of melanoco...

60 YEARS OF POMC: Melanocortin receptors: evolution of ligand selectivity for melanocortin peptides

“…These observations are consistent with the assumption that MC2R orthologs have a HFRW binding site formed by the close interactions between TM2, TM3, and TM6. Chen et al (2007) also hypothesized that phenylalanine residues may play a role in the interactions between the ligand and the receptor, and a single alanine substitution at F 258 is consistent with TM7 forming part of the HFRW binding site (Fig. 3B).…”

“…Amino acid positions that are also found in the HFRW binding site for hMC4R (Pogozheva et al 2005) are marked with asterisk (*). Amino acid positions where a single alanine substitution decreased the activation of the mutant receptor expressed in OS3 adrenal cells are shaded in blue (Chen et al 2007). Amino acid positions where amino acid substitutions as a result of a spontaneous point mutation that resulted in type 1 FGD and blocked activation of the mutant receptor (Chung et al 2008) Subsequent studies revealed that the protein required for the functional activation of tetrapod and teleost MC2R orthologs is the melanocortin-2 receptor accessory protein (MRAP or MRAP1; Metherell et al 2005).…”

“…Once it was understood that functional expression of mammalian MC2R orthologs required either co-express of the receptor construct with an mrap1 construct in some heterologous mammalian cell line or expression of MC2R in a cell line derived from the adrenal cortex, Chen et al (2007) did a alanine-substitution study on hMC2R, which revealed several domains in the receptor that participate in the activation of hMC2R by ACTH. The results of those experiments are summarized in Fig.…”

Third Transmembrane Domain of the Adrenocorticotropic Receptor Is Critical for Ligand Selectivity and Potency

“…This region was identified as important for ligand binding and activities at hMC1R, hMC2R, hMC3R, hMC4R, and hMC5R (27)(28)(29)(30)(31)(32)(33)(34). Our previous results indicate that [Nle 4 ]ACTH(1-39) (with norleucine substituted for methionine at position 4) is equipotent to ACTH(1-39) in ligand binding and signaling (35). Our previous results also indicate that first 17 amino acid residues in ACTH are crucial for ligand binding and signaling at However, in our present study, D-Nal(2Ј) substitution in ACTH did not switch the ligand from agonist to antagonist at hMC2R, suggesting that an aromatic side chain in position 7 of ␣-MSH is crucial for receptor activation at MC3R and MC4R, but not at MC2R.…”

60 YEARS OF POMC: Adrenal and extra-adrenal functions of ACTH

“…Studies on structure-activity relationships have determined that ACTH(1-16) is the minimal sequence required for ACTH binding to MC2R and downstream signaling (Kapas et al 1996, Chen et al 2007). In addition, some ACTH fragments not only lack activity, but act as competitive antagonists of full-length ACTH, as is the case for ACTH(7-38) (Kapas et al 1996).…”