Modulation and molecular mechanisms of GPR56 function

GPR56功能的调节和分子机制

• 批准号: 9300929
• 负责人: Gabriel Salzman
• 金额: $ 4.9万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-01 至 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9300929
• 关键词: ADGR1 gene; Adhesions; Affinity; Antibodies; Bacteriophages; Bilateral; Binding; Binding Proteins; Binding Sites; Biochemical; Biological; Biological Assay; Biological Process; Biology; Brain; Brain Diseases; Categories; Cell Adhesion; Cell Culture Techniques; Cell Line; Cell surface; Cells; Complex; Crystallization; Cytoplasm; Data; Development; Disease; Drug Targeting; Endocytosis; Engineering; Environment; Extracellular Structure; Family; G-Protein-Coupled Receptors; GTP-Binding Proteins; Goals; Imaging Techniques; Insecta; Integral Membrane Protein; Lead; Learning; Libraries; Ligands; Link; Lung diseases; Male Infertility; Malignant Neoplasms; Maps; Measures; Mediating; Melanoma Cell; Microgyria; Molecular; Mutagenesis; Mutation; Oligodendroglia; Pathology; Phage Display; Pharmaceutical Preparations; Pharmacologic Substance; Pharmacology; Play; Protein Engineering; Proteins; Reagent; Research; Resources; Role; Signal Transduction; Signaling Protein; Site; Specificity; Squamous Cell Lung Carcinoma; Structure; Techniques; Technology; Testing; Tissues; Validation; Variant; Work; X-Ray Crystallography; Yeasts; cancer type; combat; deafness; disease-causing mutation; drug development; experience; extracellular; falls; human disease; insight; melanoma; member; nervous system disorder; novel; public health relevance; receptor function; scaffold; small molecule; structural biology; success; targeted treatment; therapy development; tool; transglutaminase 2

 DESCRIPTION (provided by applicant): The overall goal of my project is to engineer proteins that perturb and thereby elucidate the molecular mechanisms by which adhesion G protein-coupled receptors (aGPCRs) function. Members of the superfamily of cell-surface signaling proteins known as G protein-coupled receptors (GPCRs) have been validated as successful drug targets. aGPCRs make up the second largest GPCR family and have recently been implicated in a variety of diseases, including neurological disorders, and many types of cancer. Characterized by their large and diverse extracellular regions (ECRs), aGPCRs play a role in cell adhesion. Due to the importance of aGPCRs in disease, as well as the proven druggability of GPCRs, aGPCRs are an unexplored, yet potentially impactful class of drug targets. To this end, before drug development can begin, it is imperative to systematically define the feasibility of targeting aGPCRs with drugs. One of the best-studied aGPCRs, GPR56, has been linked to the progression of many cancers as well as brain development, including its role in the developmental brain disorder bilateral frontoparietal polymicrogyria. Though two GPR56 ligands have been identified, the molecular mechanisms by which GPR56 functions remain unclear. Key to my project is the synergistic implementation of GPR56 fragment purification, cutting-edge protein engineering, X-ray crystallography, and cell signaling assays. Using these techniques, I will systematically elucidate the molecular mechanisms that govern the biological functions of GPR56 and thereby assess its durggability. My proposal consists of three aims, the first of which is to engineer binding proteins, termed monobodies (Mbs), that specifically bind GPR56 with high affinity. Using purified GPR56 fragments as targets for phage display library selection, I have engineered a Mb that binds the ECR of GPR56 with high affinity and specificity. Using the techniques I have already successfully employed, I will engineer Mbs that bind other sites on GPR56. Second, I will determine the regions of GPR56 that interact with natural and engineered proteins. I have solved the crystal structure of my Mb bound to the ECR of GPR56. This is the first structure of the full ECR of any aGPCR and also the first structure of an aGPCR fragment in complex with a binding partner. I will crystallize soluble fragments of GPR56 in complex with new Mbs in order to precisely map their binding sites. I will also purify tissue transglutaminase 2, a ligand of GPR56, and attempt to solve its structure bound to GPR56. Third, I will characterize the biological roles of distinct regions of GPR56. By establishing GPR56 signaling assays in my lab, I will assess the functional implications of Mb binding and structure-guided GPR56 mutations in parallel. With the ultimate goal of combatting aGPCR-mediated diseases, it is crucial to gain a clear and accurate understanding of the mechanisms that govern GPR56 function such that the feasibility of drugging this important aGPCR can be evaluated.

 描述(由申请人提供)：我的项目的总体目标是设计干扰蛋白质，从而阐明黏附G蛋白偶联受体(AGPCRs)发挥作用的分子机制。被称为G蛋白偶联受体(GPCRs)的细胞表面信号蛋白超家族成员已被证实是成功的药物靶点。AGPCRs是第二大GPCRs家族，最近被发现与多种疾病有关，包括神经系统疾病和许多类型的癌症。AGPCRs以其大而多样的胞外区(ECRs)为特征，在细胞黏附中发挥作用。由于aGPCRs在疾病中的重要性，以及已经证实的GPCRs的可药性，aGPCRs是一类未被探索但具有潜在影响的药物靶点。为此，在开始药物开发之前，必须系统地确定以药物为靶点的aGPCRs的可行性。GPR56是研究最多的aGPCRs之一，它与许多癌症的进展和大脑发育有关，包括它在双侧额顶顶多小脑回发育性大脑障碍中的作用。虽然已经确定了两个GPR56配体，但GPR56发挥作用的分子机制仍不清楚。我的项目的关键是协同实施GPR56片段纯化、尖端蛋白质工程、X射线结晶学和细胞信号分析。利用这些技术，我将系统地阐明支配GPR56生物学功能的分子机制，从而评估其耐受性。我的建议包括三个目标，第一个目标是设计结合蛋白，称为单体(MBS)，它以高亲和力特异性地结合GPR56。利用纯化的GPR56片段作为噬菌体展示文库选择的靶点，设计了一种高亲和力和高特异性结合GPR56的ECR的单链抗体。使用我已经成功运用的技术，我将设计绑定GPR56上其他站点的MBS。其次，我将确定GPR56与天然蛋白和工程蛋白相互作用的区域。我已经解决了我的Mb与GPR56的ECR绑定的晶体结构。这是任何aGPCR的完整ECR的第一个结构，也是与结合伙伴形成的复合体中的aGPCR片段的第一个结构。我会将GPR56的可溶性片段与新的MBS形成络合物结晶，以便准确地定位它们的结合部位。我还将纯化GPR56的配体组织转谷氨酰胺酶2，并试图解决其与GPR56结合的结构。第三，我将描述GPR56不同区域的生物学作用。通过在我的实验室建立GPR56信号分析，我将评估Mb结合和结构引导的GPR56突变并行的功能含义。抗击aGPCR介导的疾病的最终目标是清楚和准确地了解GPR56的功能机制，以便评估给这一重要的aGPCR下药的可行性。

项目成果

Monobodies and other synthetic binding proteins for expanding protein science.

• DOI: 10.1002/pro.3148
• 发表时间: 2017-05
• 期刊: Protein science : a publication of the Protein Society
• 作者: Sha F;Salzman G;Gupta A;Koide S
• 通讯作者: Koide S

A Comprehensive Mutagenesis Screen of the Adhesion GPCR Latrophilin-1/ADGRL1.

• DOI: 10.1016/j.isci.2018.04.019
• 发表时间: 2018-05-25
• 期刊: iScience
• 影响因子: 5.8
• 作者: Nazarko O;Kibrom A;Winkler J;Leon K;Stoveken H;Salzman G;Merdas K;Lu Y;Narkhede P;Tall G;Prömel S;Araç D
• 通讯作者: Araç D