Pharmacological characterization of canine melancortin-4 receptor and its natural variant V213F

Abstract

Dogs have become one of the most important companion animals in modern society. However, it is estimated that 20% to 40% of owned dogs are obese, suggesting that obesity has become one of the most important canine health problem. In addition, obesity in dogs also leads to type II diabetes. Because the melanocortin-4 receptor (MC4R) has been shown to be essential in maintaining energy homeostasis in several different species, including rodents and humans, we initiated studies toward elucidating the roles of MC4R in obesity pathogenesis in dogs. Canine MC4R has been cloned, and a missense variant V213F was identified. We designed primers and successfully cloned canine MC4R and generated the variant V213F by site-directed mutagenesis. The objective of this study was to investigate the pharmacological properties of canine MC4R and its natural variant V213F. We measured ligand binding and signaling properties with the use of both natural and synthetic ligands. Human MC4R was also included in the experiments for comparison. Both wild-type canine MC4R and its natural variant V213F functioned normally in terms of binding and signaling. Of the ligands we used, [Nle⁴, D-Phe⁷]-α-melanocyte-stimulating hormone is the most potent ligand. We conclude that the cloned canine MC4R is a functional receptor, and the natural variant V213F does not have any functional defect and therefore is not likely to cause obesity in dogs.

Introduction

The melanocortin system is essential for regulation of energy homeostasis and body weight [1]. This system includes neurons that express pro-opiomelanocortin (POMC) and agouti-related peptide (AgRP) as well as melanocortin receptors [2]. Tissue-specific posttranslational processing of POMC generates various peptides, including α, β, and γ melanocyte-stimulating hormone (MSH) produced in the hypothalamus and the neurointermediate lobe of pituitary as well as other peripheral tissues [3]. There are five subtypes of melanocortin receptors [2]. The melancortin-4 receptor (MC4R) is stimulated by its endogenous agonists α- and β-MSHs and inhibited by the endogenous antagonist AgRP [4].

The MC4R is a member of G protein–coupled receptor superfamily [5]. It consists of seven transmembrane domains connected by alternating extracellular and intracellular loops, with an intracellular COOH terminus and an extracellular NH₂ terminus. On agonist stimulation, MC4R couples to the stimulatory heterotrimeric G protein and activates adenylyl cyclase to promote the intracellular production of the second messenger cAMP [4].

Studies in the last decade have shown MC4R plays a central role in regulating energy homeostasis in rodents [1]. Both Pomc- and Mc4r-null mice developed a marked obesity syndrome associated with hyperphagia, hyperinsulinemia, hyperglycemia, and an increase in linear growth [6], [7]. In addition, mice that overexpress AgRP show a hyperphagic, obese, and hyperinsulinemic phenotype [8], [9], whereas mice that were administrated α-MSH or its analog melanotan II have decreased food intake [10], [11]. Therefore, either genetic inactivation of MC4R or pharmacological inhibition through overexpression of its endogenous antagonist can increase food intake and develop obesity.

Human genetic studies also unraveled the important role of MC4R in regulation of food intake and body weight. Since 1998, when the first frameshift mutation was identified in the MC4R gene associated with dominant-inherited morbid obesity in humans [12], [13], >150 distinct MC4R mutations have been identified in obese patients from various ethnic populations [4]. These mutations include frameshift, in-frame deletion, nonsense mutations, and missense mutations, scattered throughout the coding region of the MC4R gene. Functional analysis of human MC4R showed that MC4R mutations could lead to receptor defects, including receptor biosynthesis, cell surface expression, ligand binding, or signaling [14], [15]. In one large study, 5.8% of subjects with severe early onset obesity were identified to have mutations in MC4R [16]. Therefore, MC4R deficiency represents the most common form of monogenic obesity.

In 2007, canine MC4R (cMC4R) was cloned, and three single nucleotide polymorphisms were identified, one of which (G637T) is a missense variant, valine to phenylalanine at position 213 (V213F) [17]. Of the 31 dogs genotyped in this study, 84% had 637G. It was suggested that the variant might be functioning normally because both valine and phenylalanine are hydrophobic [17]. This variant was identified in another more recent study [18]. In preliminary analysis of 187 Golden Retrievers, no association between the polymorphisms and morphologic measures were found. However, those investigators suggested that the variant probably affects receptor function, because this missense variant changed an amino acid residue that is highly conserved and under strong purifying selection. Furthermore, PolyPen program predicted that this polymorphism is probably damaging, although the investigators conceded that this prediction needed to be experimentally verified by in vitro assays [18].

We hypothesize that the central melanocortin system is also critical for regulating energy homeostasis in dogs. Therefore, as the initial step for our long-term goal of understanding the melanocortin regulation of energy balance in dogs, we studied the pharmacology of cMC4R and its natural variant V213F. Herein, we performed functional studies on cMC4R and its natural variant V213F with the use of two endogenous ligands, α-MSH and β-MSH, and the superpotent analog [Nle⁴, D-Phe⁷]-α-MSH (NDP-MSH). Ligand binding and signaling properties of these receptors were determined.