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.

Keywords: 7-Helix Receptor; Cyclic AMP (cAMP); G Protein-coupled Receptor (GPCR); Membrane Protein; Peptide Hormone.

FIGURE 1.

ACTH analog sequences. The core amino acid residue substituted is shown in boldface. AA, amino acids.

FIGURE 2.

Schematic representation of the chimeric hMCRs used in these studies. A schematically depicts the seven-transmembrane structures of WT MC4R (thin lines) and MC2R (thick lines). B depicts the structures of chimeric MC4R.

FIGURE 3.

Binding affinities and potencies of ACTH(1–24) analogs at WT hMC2R. Cells transfected with hMC2R were incubated with ¹²⁵I-ACTH(1–24) at 37 °C for 1 h in the presence of the indicated amounts of unlabeled ligands. Total ¹²⁵I-ACTH(1–24) binding was then determined on duplicate wells as described under “Experimental Procedures.” Data points represent the mean ± S.E. of at least three independent experiments. A depicts the binding affinities of ACTH(1–24) analogs at WT hMC2R. B demonstrates the abilities of ACTH(1–24) analogs to stimulate the production of intracellular cAMP at WT hMC2R.

FIGURE 4.

Binding affinities and potencies of ACTH(1–17) analogs at WT hMC2R. Cells transfected with hMC2R were incubated with ¹²⁵I-ACTH(1–24) in the presence of the indicated amounts of unlabeled ligands. Total ¹²⁵I-ACTH(1–24) binding was then determined on duplicate wells as described under “Experimental Procedures.” Data points represent the mean ± S.E. of at least three independent experiments. A depicts the binding affinities of ACTH(1–17) analogs at WT hMC2R. B demonstrates the abilities of ACTH(1–17) analogs to stimulate the production of intracellular cAMP at WT hMC2R.

FIGURE 5.

Binding affinity and potency of ACTH(1–24) at the chimeric receptors. HEK cells transfected with hMC4R chimeras were incubated with ¹²⁵I-ACTH at 37 °C for 1 h in the presence of the indicated amounts of unlabeled ligands, and total ¹²⁵I-ACTH(1–24) binding was determined. For the cAMP assay, the cells transfected with hMC4R chimeras were incubated with the indicated amounts of [Nle⁴,d-Phe⁷-ACTH(1–24) for 30 min, and total cAMP accumulation was determined on duplicate wells. Data points represent the mean ± S.E. of at least three independent experiments. A depicts the binding affinity of ACTH at the chimeric receptors. B demonstrates the ability of ACTH to stimulate the production of intracellular cAMP at the chimeric receptors.

FIGURE 6.

Binding affinity and potency of ACTH at the chimeric receptors. HEK cells transfected hMC4R chimeras were incubated with ¹²⁵I-ACTH at 37 °C for 1 h in the presence of the indicated amounts of unlabeled ligands, and total ¹²⁵I-ACTH binding was determined on duplicate wells as described under “Experimental Procedures.” For the cAMP assay, the cells transfected with hMC4R chimeras were incubated with the indicated amounts of [d-Phe⁷]ACTH(1–24) for 30 min, and total cAMP accumulation was determined on duplicate wells. Data points represent the mean ± S.E. of at least three independent experiments. A depicts the binding affinity of [d-Phe⁷]ACTH(1–24) at the chimeric receptors. B demonstrates the ability of [d-Phe⁷]ACTH(1–24) to stimulate the production of intracellular cAMP at the chimeric receptors.

FIGURE 7.

Effects of different ligands on cAMP production at the chimeric receptor. HEK cells transfected with hMC4R/TM3 MC2R were incubated with different ligands for 30 min, and total cAMP accumulation was determined on duplicate wells. Data points represent the mean ± S.E. of at least three independent experiments.