Department of Pharmacodynamics, University of Florida, Gainesville, Florida 32610, USA.
Biochemistry. 2010 Jun 8;49(22):4583-600. doi: 10.1021/bi100068u.
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.
黑素皮质素 4 受体（MC4R）是一种 G 蛋白偶联受体（GPCR），在中枢神经系统中表达，在调节摄食行为、肥胖、能量平衡、男性勃起反应和血压方面发挥作用。自 1997 年报告 MC4R 敲除小鼠以来，该领域一直在寻找这种遗传生物标志物与人类肥胖和 2 型糖尿病之间的联系。已经从肥胖和非肥胖对照患者中鉴定出超过 80 个单核苷酸多态性（SNP）。许多重要的研究已经进行了，检查这些 hMC4R SNP 的药理学特征，试图确定分子缺陷/见解，可能将遗传因素与肥胖表型联系起来，这些突变存在于患者中。我们实验室之前已经报告了 40 种这些多态 hMC4 受体与多种内源性和合成配体的药理学特征。目前研究的目标是使用多种内源性激动剂 [α-、β-和γ（2）-黑色素细胞刺激激素（MSH）和促肾上腺皮质激素（ACTH）]、拮抗剂 agouti 相关蛋白 hAGRP（87-132）和合成激动剂 [NDP-MSH、MTII 和四肽 Ac-His-dPhe-Arg-Trp-NH2（JRH887-9）]，对另外 30 种具有单核苷酸多态性的人 hMC4R 进行类似的全面并排特征描述。这些体外数据在某些情况下，为功能失调的 hMC4R 和人类肥胖之间提供了一个假定的分子联系。这 30 种 hMC4R SNP 包括 R7H、R18H、R18L、S36Y、P48S、V50M、F51L、E61K、I69T、D90N、S94R、G98R、I121T、A154D、Y157S、W174C、G181D、F202L、A219V、I226T、G231S、G238D、N240S、C271R、S295P、P299L、E308K、I317V、L325F 和 750DelGA。除了 N240S hMC4R 之外，所有这些都在肥胖患者中被鉴定出来。此外，我们还对一个双 I102T/V103I hMC4R 进行了特征描述。除了药理学特征外，还通过流式细胞术评估了 hMC4R 变体的表面表达。F51L、I69T 和 A219V hMC4R 具有完全激动剂活性，并显著降低了内源性激动剂配体的效力。在 E61K、D90N、Y157S 和 C271R hMC4R 中，所有检查的激动剂配体仅在产生最大信号反应（部分激动剂）方面具有部分效力，并显著降低了内源性激动剂配体的效力。只有 A219V、G238D 和 S295P hMC4R 具有显著降低的 AGRP（87-132）拮抗剂效力。这些数据为 GPCR 计算开发提供了新的信息，并深入了解 MC4R 的结构和功能。
