Molecular Mechanism of the Constitutive Activation of the L250Q Human Melanocortin‐4 Receptor Polymorphism‡

Department of Medicinal Chemistry, University of Florida, PO Box 100485, Gainesville, FL 32610-0485, USA.
Chemical Biology & Drug Design (Impact Factor: 2.49). 04/2006; 67(3):215-29. DOI: 10.1111/j.1747-0285.2006.00362.x
Source: PubMed


The Melanocortin-4 Receptor is a G-protein coupled receptor that has been physiologically linked to participate in the regulation of energy homeostasis. The Melanocortin-4 Receptor is stimulated by endogenous melanocortin agonists derived from the pro-opiomelanocortin gene transcript and antagonized by the endogenous antagonist agouti-related protein. Central administration of melanocortin agonists has been demonstrated to decrease food intake and conversely, treatment with antagonists resulted in increased food intake. Deletion of the Melanocortin-4 Receptor gene from the mouse genome results in an obese and hyperphagic phenotype. Polymorphisms of the human Melanocortin-4-Receptor have been found in severely obese individuals, suggesting that Melanocortin-4 Receptor malfunction might be involved in human obesity and obesity-associated diabetes. Herein, we have performed experiments to understand the molecular mechanisms associated with the L250Q human Melanocortin-4-Receptor polymorphism discovered in an extremely obese woman. This L250Q human Melanocortin-4-Receptor has been pharmacologically characterized to result in a constitutively active receptor. The fact that a constitutively active human Melanocortin-4-Receptor mutation was found in an obese person is a physiologic contradiction, as chronic activation of the human Melanocortin-4-Receptor and subsequently high cyclic adenosine monophosphate levels should theoretically result in a normal or lean phenotype. In this study, we demonstrated that agouti-related protein acts as an inverse agonist at this constitutively active receptor, and we propose a mechanism by which agouti-related protein might contribute to the obese phenotype in the L250Q patient. In addition, using receptor mutagenesis, pharmacology, and computer modeling approaches, we investigated the molecular mechanism by which modification of the L250 residue results in constitutive activation of the human Melanocortin-4-Receptor.

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Available from: Irina D Pogozheva
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    • "Using this new prototype structure (the PDB entry 3DQB) as a template for the activated state of MC4R, recent modeling suggested that the cytoplasmic part of TM6 moves away from TM7 and toward TM5 upon transition from the presumed ground state (template provided by the X-ray structure of b2AR; Cherezov et al., 2007) to the presumed activated state of MC4R (Tao et al., 2010). Also, in the activated state, the side chain of D146 3.49 is involved in a strong salt bridge with the side chain of R165 4.42 , and the R147 3.50 side chain changes orientation possibly contacting L250 6.40 (similar to the model above; Proneth et al., 2006). These modeling results explained the constitutive activity of mutants D146 3.49 N and L250 6.40 Q, which are naturally occurring mutations identified in severe childhood obesity. "
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    ABSTRACT: In the past decade, an increasing number of studies using computational modeling procedures have focused on the structural aspects of constitutive activity in G protein-coupled receptors (GPCRs). This chapter reviews various conceptual approaches in computational modeling of constitutively active mutants (CAMs) including analyzing three-dimensional models of the ground states of GPCRs based on structural homology with the known X-ray templates; molecular dynamics simulations starting from the ground states; and modeling of CAMs based on the experimentally suggested templates of the possible activated states. The developed buildup procedure of rotational sampling of the TM regions of GPCRs is highlighted in more detail. Experimental data on CAMs of the complement factor 5a receptor (C5aR) are used to validate the rotational sampling results.
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    • "Fluorescence-activated cell sorting (FACS) analysis of N-terminally Flag-tagged WT and mutant hMC4R was performed as described previously [19]. For cell surface and intracellular detection of the Flag-hMC4Rs an allophycocyanin (APC)-conjugated anti-Flag monoclonal antibody (Prozyme, San Leandro, CA, USA) was used. "
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    ABSTRACT: Melanocortin-4-receptor (MC4R) mutations represent the most frequent genetic cause of non-syndromic early onset obesity. Children carrying MC4R mutations seem to show a particular phenotype characterized by early onset, severe obesity and high stature. To verify whether MC4R mutations are associated with this particular phenotype in the Italian pediatric population, we decided to screen the MC4R gene in a group of obese children selected on the basis of their phenotype. To perform this study, a multicentric approach was designed. Particularly, to be enrolled in the study subjects needed to meet the following criteria: Body mass index > or = 3 deviation scores according to age and sex, familiar history of obesity (at least one parent obese), obesity onset before the 10 years old, height > or = 2 deviation scores. The coding region of MC4R gene was screened in 240 obese children (mean age 8.3 +/- 3.1, mean BMI 30.8 +/- 5.4) and in 200 controls (mean age 8.1 +/- 2.8; mean BMI 14.2 +/- 2.5). Three mutations have been found in five obese children. The S127L (C380T), found in three unrelated children, had been described and functionally characterized previously. The Q307X (C919T) and the Y332H (T994C) mutations were found in two patients. Functional studies showed that only Q307X impaired protein function. The low prevalence of MC4R mutations (1.6%) in this group of obese children selected according to the obesity degree, the tall stature and the family history of obesity was similar to the prevalence observed in previous screenings performed in obese adults and in not phenotypically selected obese children.
    Full-text · Article · Feb 2009 · BMC Medical Genetics
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    ABSTRACT: The melanocortin pathway, specifically the melanocortin-4 receptor and the cognate endogenous agonist and antagonist ligands, have been strongly implicated in the regulation of energy homeostasis and satiety. Genetic studies of morbidly obese human patients and normal weight control patients have resulted in the discovery of over 70 human melanocortin-4 receptor (MC4R) polymorphisms observed as both heterozygous and homozygous forms. A number of laboratories have been studying these hMC4R polymorphisms attempting to understand the molecular mechanism(s) that might explain the obese human phenotype. Herein, we have studied 13 polymorphic hMC4Rs that have been identified to possess statistically significant decreased endogenous agonist potency with synthetic peptides and small molecules attempting to identify ligands that can pharmacologically rescue the hMC4R polymorphic agonist response. The ligands examined in this study include NDP-MSH, MTII, Ac-His-DPhe-Arg-Trp-NH2 (JRH887-9), Ac-Anc-DPhe-Arg-Trp-NH2 (amino-2-naphtylcarboxylic acid, Anc, JRH420-12), Ac-His-(pI)DPhe-Arg-Trp-NH2 (JRH322-18), chimeric AGRP-melanocortin based ligands (Tyr-c[Cys-His-DPhe-Arg-Trp-Asn-Ala-Phe-Cys]-Tyr-NH2, AMW3-130 and Ac-mini-(His-DPhe-Arg-Trp)-hAGRP-NH2, AMW3-106), and the small molecules JB25 and THIQ. The hMC4R polymorphisms included in this study are S58C, N97D, I102S, L106P, S127L, T150I, R165Q, R165W, L250Q, G252S, C271Y, Y287Stop, and I301T. These studies resulted in the NDP-MSH, MTII, AMW3-130, THIQ, and AMW3-106 ligands possessing nanomolar to subnanomolar agonist potency at the hMC4R polymorphisms examined in this study. Thus, these ligands could generically rescue the potency and stimulatory response of the abnormally functioning hMC4Rs studied and may provide tools to further clarify the molecular mechanism(s) involving these receptor modifications.
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