MRAP2作为黑色素皮质激素第四受体MC4R辅助蛋白的进化及调控机制研究

The melanocortin-4 receptor (MC4R) is an unusual G-protein coupled receptor that plays a critical role in energy homeostasis accounting for up to 6% of early on-set obesity syndromes in MC4R haploid insufficient human patients. And it is a well-validated drug target for treatment of obesity and disease cachexia. Melanocortin receptor accessory proteins (MRAPs) modulate signaling of melanocortin receptors in vitro，but its functions in vivo are unknown. We found that MRAP2, a mammalian paralog of MRAP1, highly expressed in the hypothalamus and directly interacts with MC4R. In vitro studies are showing that presence of MRAP2 not only decreases the constitutive activity of MC4R, but also enhances its sensitivity and response to the endogenous ligand alpha-MSH. Whole-body or hypothalamus-specific targeted deletion of MRAP2 develop severe obesity at a young age in mouse model. In a study of humans with severe early-onset obesity, four rare potentially pathogenic genetic variants in MRAP2 were found from European cohorts, suggesting that the gene may also contribute to body weight regulation in humans. Strikingly，MRAP2 proteins own single transmembrane and form anti-parallel dimers on cell surface. More interestingly, two MRAP2 genes were seen in zebrafish due to one more round of genomic duplication than mammals. The larval form MRAP2a inhibits agonist (alpha-MSH) binding and assures the rapid growth of zebrafish embryos. MRAP2b, functionally homologous to the mammalian counterpart, sensitizes the receptor to alpha-MSH and presumably regulates appetite of the adult fish. These findings not only revealed the complexities of MC4R signaling but also provided us another potential in vivo target for drug development to treat MC4R associated disorders. In this project we plan to look into the structural insights of MRAP2 protein with the aim to elucidate the motifs essential for forming functional dual topology and modulating MC4R signaling. Next, we will optimize MRAP2 structure to improve its capability to sensitize MC4R signaling in vitro. In vivo activities will be monitored in hypothalamic specific knock-in mouse model. Out study will elucidate a novel ligand independent mechanism on how to treat GPCR associated disorders in eukaryotic organisms.

黑色素皮质激素第四受体MC4R是最常见的脊椎动物致胖基因之一, 基因突变或缺失能够引起人、小鼠或斑马鱼的过量摄食和早发性肥胖。作为七次跨膜的G蛋白偶联受体，MC4R的正常药理学活性受到内源辅助蛋白MRAP2的调控，相应的MRAP2突变或缺失也能引起类似的肥胖表型。更为重要的是MRAP2是迄今为止发现的唯一以反向同源二聚体行使GPCR调节功能的单次跨膜蛋白，具体的形成原因尚不清楚。此课题中我们将利用分子进化、细胞生物学和药理学等手段重点解析形成MRAP2对称性拓扑结构的分子基础，以及和MC4R活性调控之间的相互关系，进一步简化并优化MRAP2的蛋白结构，建立功能域、拓扑结构和调节活性之间的理论模型。本课题的完成将揭示MRAP2蛋白奇特构象的形成原因，阐明其重要的生物学意义和在生物体内的调控机制和功能，为人体肥胖症的诊断、预防与治疗提供新的思路和视野。

黑色素皮质激素第四受体MC4R及内源其辅助蛋白MRAP2是最常见的脊椎动物致胖基因, 基因突变或缺失能够引起人、小鼠或斑马鱼的过量摄食和早发性肥胖。本课题利用分子进化、细胞生物学和药理学等手段首先解析了MRAP2对称性拓扑结构的分子基础，同时对发现的肥胖患者携带的若干MRAP2氨基酸突变，检测了其对靶体MC4R药理学调节改变。系统进化方面解析了斑马鱼基因组中两个MRAP2蛋白对MC5R的调节机制，罗非鱼中MRAP2对MC4R的调节，以及最原始的脊索动物海七鳃鳗中的MRAP2蛋白对两个MCR的调节机制。最后我们拓展发现了MC4R信号通路在两栖类动物非洲爪蟾中参与机体损伤再生的生理功能，阐明其重要的生物学意义和在生物体内的调控机制和功能，为人体肥胖症的诊断、预防与治疗提供新的思路和视野。执行期内以通讯作者发表标注了该课题的SCI论文10篇，其中IF大于10的两篇，IF大于7的3篇。培养博士后1名，硕士生6名，本科生8名。项目投入经费58万元，累计支出46.47万元，剩余11.53万元，剩余经费计划用于本项目研究的后续支出。