Ligand Interaction of Glucagon Receptor by Structural and Functional Analyses

描述(申请人提供)：G蛋白偶联受体(GPCRs)是人类最大的蛋白质家族。它们参与许多生理过程，并与许多人类疾病有关。GPCRs与细胞外配体结合，并将此信号转导到细胞内的G蛋白复合体。A类GPCRs约有700个成员，可识别多种配体。人类A类GPCRs的两个结构：β2-肾上腺素能和A2A腺苷，是在雷·史蒂文斯博士的实验室里解决的。这些结构在杆状病毒蛋白表达、受体稳定性和胆固醇掺杂脂肪立方相结晶方面的技术突破得到了解决。B类GPCRs是明显不同的，因为细胞外的N-末端结构域结合了共同的7-螺旋跨膜结构域。这个亚家族识别在生物学和药物发现中至关重要的激素多肽(例如，血糖调节和骨骼稳态)。鉴于大的胞外N末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。该方案的目的是通过X射线结晶学确定与其配体结合的全长B类人胰高血糖素受体的结构，并利用定点突变技术研究受体中各种氨基酸残基对配体结合和诱导信号转导的影响，从而揭示胰高血糖素受体激活机制的重要细节。为研究胰升糖素受体的结构，已进行了结构设计、表达和纯化的过程。令人鼓舞的初步结果显示，全长胰高血糖素受体高水平表达，纯化的受体与小分子化合物和多肽拮抗剂结合并稳定下来。这些结果表明，确定结构的两个最困难的障碍已经克服。胰升糖素受体-配体复合体的晶体将使用胆固醇掺杂的脂质立方相(LCP)结晶方法获得，并使用位于阿贡国家实验室的先进光子源的X射线微束来解析结构。高血糖素受体跨膜区的突变体将被用来测量配体结合亲和力和信号转导水平。突变研究将揭示参与配体结合和诱导信号相互作用的胰高血糖素受体跨膜区的残基。总之，胰高血糖素受体的结构和突变研究将促进对B类GPCRs的理解，并促进治疗糖尿病和其他B类相关疾病的药物开发。 公共卫生相关性：B类G蛋白偶联受体(GPCRs)识别对糖尿病、骨质疏松症、癌症、肥胖等病理疾病以及包括抑郁和焦虑在内的神经疾病至关重要的激素肽。该方案的目的是通过1)利用X射线结晶学获得配体-受体复合体的高分辨率结构，以及2)通过定点突变研究受体中的各种氨基酸残基在参与配体结合和信号传递中的作用，从而阐明B类胰高血糖素受体的配体结合和诱导信号的细节。该结构和诱变研究将揭示有关高血糖素受体在配体结合和诱导信号传递时的激活机制的重要细节，这将促进对B类GPCRs的理解，最终导致更好地治疗各种流行疾病。

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.