Pharmacological characterization of 30 human melanocortin-4 receptor polymorphisms with the endogenous proopiomelanocortin-derived agonists, synthetic agonists, and the endogenous agouti-related protein antagonist

Abstract

The melanocortin-4 receptor (MC4R) is a G-protein-coupled receptor (GPCR) that is expressed in the central nervous system and has a role in regulating feeding behavior, obesity, energy homeostasis, male erectile response, and blood pressure. Since the report of the MC4R knockout mouse in 1997, the field has been searching for links between this genetic biomarker and human obesity and type 2 diabetes. More then 80 single nucleotide polymorphisms (SNPs) have been identified from human patients, both obese and nonobese controls. Many significant studies have been performed examining the pharmacological characteristics of these hMC4R SNPs in attempts to identify a molecular defects/insights that might link a genetic factor to the obese phenotype observed in patients possessing these mutations. Our laboratory has previously reported the pharmacological characterization of 40 of these polymorphic hMC4 receptors with multiple endogenous and synthetic ligands. The goal of the current study is to perform a similar comprehensive side-by-side characterization of 30 additional human hMC4R with single nucleotide polymorphisms using multiple endogenous agonists [alpha-, beta-, and gamma(2)-melanocyte stimulating hormones (MSH) and adrenocorticotropin (ACTH)], the antagonist agouti-related protein hAGRP(87-132), and synthetic agonists [NDP-MSH, MTII, and the tetrapeptide Ac-His-dPhe-Arg-Trp-NH(2) (JRH887-9)]. These in vitro data, in some cases, provide a putative molecular link between dysfunctional hMC4R's and human obesity. These 30 hMC4R SNPs include R7H, R18H, R18L, S36Y, P48S, V50M, F51L, E61K, I69T, D90N, S94R, G98R, I121T, A154D, Y157S, W174C, G181D, F202L, A219 V, I226T, G231S, G238D, N240S, C271R, S295P, P299L, E308K, I317V, L325F, and 750DelGA. All but the N240S hMC4R were identified in obese patients. Additionally, we have characterized a double I102T/V103I hMC4R. In addition to the pharmacological characterization, the hMC4R variants were evaluated for cell surface expression by flow cytometry. The F51L, I69T, and A219V hMC4Rs possessed full agonist activity and significantly decreased endogenous agonist ligand potency. At the E61K, D90N, Y157S, and C271R hMC4Rs, all agonist ligands examined were only partially efficacious in generating a maximal signaling response (partial agonists) and possessed significantly decreased endogenous agonist ligand potency. Only the A219V, G238D, and S295P hMC4Rs possessed significantly decreased AGRP(87-132) antagonist potency. These data provide new information for use in GPCR computational development as well as insights into MC4R structure ad function.

Figure 1

Small molecule and amino acid sequences of the endogenous and synthetic melanocortin ligands examined in this study.

Figure 2

Fluorescence cell activated sorting analysis (FACS) of the hMC4R polymorphisms in stably expressed in HEK-293. The total cell receptor expression levels were determined using permeabilized cells measuring both cell surface and intracellular protein expression. The cell surface expression levels were determined using non-permeabilized cells. Cell expression levels are presented relative to the wild type hMC4R control.

Figure 3

Summarizes the total specific binding counts (cpms) of ¹²⁵I-NDP-MSH binding to the polymorphic hMC4R stable HEK-293 cell lines generated in this study. These data indicate that receptor protein expressed at the cell surface is properly folded to allow for ligand binding.

Figure 4

Illustration of the wild type hMC4R full agonist and partial agonist activity of the endogenous melanocortin agonists at the E61K, D90N, Y157S, and C271R hMC4Rs. The ligand efficacy is relative to non-receptor dependent maximal stimulation by the forskolin control compound which is defined as 1.0 on the Y-axis.

Figure 5

Illustration of the hAGRP(87-132) antagonist pharmacology at the wild type (WT), E61K, D90N, Y157S, and C271R hMC4Rs. The MTII ligand is a full agonist at the WT hMC4R and a partial agonist at the E61K, D90N, Y157S, and C271R hMC4Rs relative to non-receptor dependent maximal stimulation by the forskolin control compound which is defined as 1.0 on the Y-axis.

Figure 6

Summary of the synthetic agonist peptide and small molecule agonist ligand pharmacology (Figure 2 and Table 4) at the wild type (WT) hMC4R and the F51L, E61K, I69T, D90N, Y157S, A219V, and C271R hMC4Rs. The ligands were full agonists at the wild type hMC4R and displayed different combinations of a) no stimulatory activity (up to 100 μM concentrations), b) partial agonist efficacy as normalized to the forskolin control (defined as 1.0 on the Y-axis), and c) to full agonist activity. Notable results include the THIQ small molecule to reach a full agonist response at the Y157S.

Figure 7

Summary of the 30 hMC4R polymorphisms examined in this study. The dashed line indicates the N-terminal FLAG sequence used for immunohistochemical cell expression studies. Changes in key receptor pharmacological profiles are indicated by either a change in font color and/or a colored callout circle, as indicated in the figure. The lavender color indicates hMC4R residues postulated to be involved in the putative ligand binding pocket.