胰高血糖素及其受体的核磁共振研究

The glucagon receptor (GCGR) is one of the 15 members of the secretin-like (class B) family of G-protein-coupled receptors (GPCRs) in humans. GCGR is activated by the 29 amino acid hormonal peptide glucagon (GCG), and is a potential drug target for diabetes mellitus. GCGR contains a globular N-terminal extracellular domain (ECD) defined by three conserved disulphide bonds and a seven transmembrane domain (TMD). Although the crystal structure of the ECD and TMD of GCGR has been solved separately, the binding states of full-length receptor and peptide ligand which represented by ensemble of conformations is not known. We propose to use high-resolution Nuclear Magnetic Resonance (NMR) spectroscopy methods, in combination of the state-of-the-art labeling technique, to study the binding interaction and conformational dynamics during the binding of full-length GCGR with GCG peptide ligand. We will use in vitro labeling and unnatural amino acids (UAA) methods to incorporate 19F probe site-specifically to GCGR or GCG, which should give us the ability to observe 19F NMR signal of two major conformational states (inactive and active-like state). Changes in the NMR signals will lead to the information of the impact of ligands and downstream signaling molecules on the equilibrium distribution of the conformational states of the GCGR. We will also use segmental labeling methods, to get the separately labeled full-length GCGR, to study the conformational and dynamics information of the ligand binding. The strategy we proposed will provide direct information about the kinetics of activation of GCGR and this information will be vital for understanding the molecular mechanism of GPCR signaling. The structure-activity relationship studies of GCGR and ligands will demonstrate that NMR is a widely applicable tool for big membrane proteins and the results of the research will serve as a platform for future rational design of specific ligands.

胰高血糖素受体（GCGR）属于人类G蛋白偶联受体（GPCRs）中分泌素家族，被多肽激素肽胰高血糖素（GCG）活化，是用于治疗糖尿病的药物靶标。 GCGR由N-末端胞外结构域（ECD）和跨膜结构域（TMD）组成。GCGR的ECD和TMD的结构已经被分别解析，全长受体和多肽激素配体的结合状态仍是未知的。我们计划使用核磁共振（NMR）波谱学方法研究全长GCGR及GCG多肽配体结合相互作用和构象的动态性质。使用体外标记和非天然氨基酸（UAA）标记的方法将19F探针引入到感兴趣的GCGR或GCG上，结合分段标记的方法，观测各构象状态NMR信号（激活状态和非激活状态），得出配体对GCGR的构象状态的平衡分布影响的动态信息，该研究将对理解GPCR信号传导的分子机制发挥重要作用。GCGR和配体的结构—活性关系（SAR）的研究将扩展核磁共振方法的应用领域，为未来理性设计GPCR特异性配体提供理论基础。

胰高血糖素受体（Glucagon receptor, GCGR）属于人类G蛋白偶联受体（G protein-coupled receptors, GPCRs）中分泌素家族，被胰高血糖素（Glucagon, GCG）活化，是与血糖浓度，糖尿病，肥胖相关的药物靶标之一。本项目主要使用核磁共振（NMR）波谱学方法，结合19F标记与顺磁弛豫增强(PRE)等手段，研究了GCGR与多种正构和别构配体的相互作用。在方法学方面，开发了三甲基硅基探针在GPCR构象表征中的使用；开发了基于配体氟观测的核磁共振方法；进一步优化了分段标记方法，使其能更好的应用于多个结构域蛋白的核磁共振研究。GCGR和配体的结构—活性关系（SAR）的阐明，对理解GPCR信号传导的分子机制发挥重要作用，将扩展核磁共振方法的应用领域，为理性设计GPCR特异性配体提供理论基础。本研究所产生的各种方法学突破也将为此类困难靶标蛋白的液体核磁共振研究提供较好的思路。