Ligand Interaction of Glucagon Receptor by Structural and Functional Analyses

描述（由申请人提供）：G蛋白偶联受体（gpcr）是最大的人类蛋白家族。它们参与许多生理过程，并与许多人类疾病有关。gpcr结合细胞外配体并将信号转导到细胞内G蛋白复合物。a类gpcr包含约700个成员，可识别多种配体。两种人类a类gpcr的结构：β 2-肾上腺素能和A2A腺苷，在Ray Stevens博士的实验室中得到了解决。这些结构在杆状病毒蛋白表达、受体稳定和胆固醇掺杂脂质立方相结晶方面的技术突破得到了解决。b类gpcr具有较大的胞外N端结构域与常见的7-螺旋跨膜结构域相结合的特点。该亚家族识别在生物学和药物发现（如血糖调节和骨稳态）中至关重要的激素肽。鉴于大细胞外n端结构域的关键作用，b类受体的配体结合和信号转导的激活机制尚不清楚。本研究的目的是通过x射线晶体学确定与配体结合的全长b类人胰高血糖素受体的结构，并利用位点定向诱变研究受体中各种氨基酸残基对配体结合和诱导信号传导的影响，揭示胰高血糖素受体激活机制的重要细节。进行了胰高血糖素受体结构研究的构建体设计、表达和纯化过程。令人鼓舞的初步结果显示全长胰高血糖素受体的高水平表达，纯化的受体与小化合物和肽拮抗剂结合并稳定。这些结果表明，两个最困难的结构确定障碍已经克服。胰高血糖素受体-配体复合物的晶体将使用胆固醇掺杂脂质立方相（LCP）结晶方法获得，并使用阿贡国家实验室先进光子源的x射线微型光束解决结构。胰高血糖素受体跨膜结构域的突变体将用于测量配体结合亲和力和信号转导水平。诱变研究将揭示胰高血糖素受体跨膜区域的残基参与配体结合和诱导信号的相互作用。总之，胰高血糖素受体的结构和突变研究将促进对b类gpcr的认识，并促进糖尿病和其他b类相关疾病的药物开发。

DESCRIPTION (provided by applicant): G protein-coupled-receptors (GPCRs) are the largest family of human proteins. They are involved in many physiological processes and implicated in numerous human diseases. GPCRs bind to extracellular ligands and transduce this signal to the intracellular G protein complex. Class-A GPCRs contain about 700 members and recognize a variety of ligands. The structures of two human class-A GPCRs: beta2-adrenergic and A2A adenosine, were solved in Dr. Ray Stevens' laboratory. These structures were solved with technological breakthroughs in baculovirus protein expression, receptor stabilization, and crystallization in cholesterol doped lipid cubic phase. Class-B GPCRs are distinctly different given the large extracellular N- terminal domain combined with the common 7-helix transmembrane domain. This subfamily recognizes hormone peptides that are of critical importance in biology and drug discovery (e.g. blood glucose regulation and bone homeostasis). The activation mechanisms of ligand binding and signal transduction for the class-B receptors are unclear given the critical role of the large extracellular N-terminal domain. The goal of this proposal is to reveal important details regarding the activation mechanisms of the glucagon receptor by determining the structure of the full-length class-B human glucagon receptor bound to its ligand using X-ray crystallography, and studying the effects of various amino acid residues in the receptor on ligand binding and induced signaling using site-directed mutagenesis. The processes of construct design, expression, and purification for the structural study of the glucagon receptor have been conducted. Encouraging preliminary results show high-level expression of the full-length glucagon receptor, and the purified receptor is bound and stabilized with small compound and peptide antagonists. These results demonstrate two of the most difficult structure determination hurdles have been overcome. Crystals of the glucagon receptor-ligand complex will be obtained using a cholesterol doped lipid cubic phase (LCP) crystallization method, and the structure solved using an X-ray mini-beam at the Advanced Photon Source at Argonne National Laboratory. Mutants of the transmembrane domain of the glucagon receptor will be used to measure ligand binding affinities and signal transduction levels. The mutagenesis study will reveal residues in the transmembrane domain of the glucagon receptor involved in the interaction of ligand binding and induced signaling. Together, the structure and mutagenesis study of the glucagon receptor will advance the understanding of class-B GPCRs, and facilitate drug development for diabetes and other class-B related diseases. PUBLIC HEALTH RELEVANCE: Class-B G protein-coupled receptors (GPCRs) recognize hormone peptides that are critical to pathological conditions such as diabetes, osteoporosis, cancer, obesity, and neurological disorders, including depression and anxiety. The goal of this proposal is to elucidate details of ligand binding and induced signaling of the class-B glucagon receptor by 1) obtaining a high-resolution structure of the ligand-receptor complex using X-ray crystallography, and 2) studying the role of various amino acid residues in the receptor involved in ligand binding and signaling by site-directed mutagenesis. The structure and the mutagenesis study will reveal important details regarding the activation mechanisms upon ligand binding and induced signaling of the glucagon receptor that will advance the understanding of class-B GPCRs and ultimately, leading to better treatment of various prevalent diseases.