Discovery of antibodies that bind G protein-coupled receptors

发现结合 G 蛋白偶联受体的抗体

• 批准号: 8091868
• 负责人: MATTHEW P DELISA
• 金额: $ 23.85万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-15 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8091868
• 关键词: Address; Adenosine A2A Receptor; Adenylate Cyclase; Antibodies; Antibody Affinity; Bacteria; Binding; Biological Assay; Biology; Cannabinoids; Carrier Proteins; Cell membrane; Cells; Cellular biology; Cessation of life; Code; Communication; Communities; Crystallization; Cytosine deaminase; Detection; Development; Diabetes Mellitus; Diagnosis; Dimerization; Disease; Dopamine; Dopamine D2 Receptor; Drug Delivery Systems; Drug abuse; Engineering; Environment; Enzymes; Epitopes; Escherichia coli; Family; G-Protein-Coupled Receptors; Genetic; Goals; HIV Infections; Heart; Homo; Human Genome; Human body; Immunoglobulin Fragments; Integral Membrane Protein; Ion Channel; Lactamase; Libraries; Life; Link; Malignant Neoplasms; Mammalian Cell; Mammals; Mediating; Medical; Membrane; Membrane Proteins; Methods; Molecular Conformation; Neurotensin Receptors; Parkinson Disease; Physiological; Play; Process; Protein Engineering; Protein Fragment; Proteins; RNA Splicing; Reagent; Recombinant Antibody; Reporter; Reporting; Rhodopsin; Role; Signal Transduction; Source; Structure; System; Testing; Therapeutic Studies; Transport Process; United States National Institutes of Health; Variant; Yeasts; base; combinatorial; cost effective; extracellular; genetic selection; human disease; innovation; interest; novel; novel strategies; periplasm; protein expression; protein protein interaction; protein structure; receptor; reconstitution; tool

DESCRIPTION (provided by applicant): G protein-coupled receptors (GPCRs) are integral membrane proteins that transduce extracellular signals across the cell membrane and have been directly linked to several diseases including cancer, diabetes and HIV infection. GPCRs comprise the largest class of membrane proteins in mammalian cells - the human genome encodes more than 800 - and recent estimates indicate that GPCRs represent 60-70% of all drug targets. Yet despite their widespread occurrence and potential medical relevance, the structure, function and biology of most GPCRs remains elusive due to difficulties in protein expression, purification and crystallization, as well as the general lack of reagents for their manipulation in native environments. As a result, only four unique GPCR structures have been reported. In several of these cases, GPCR structure determination was greatly aided by specifically binding antibodies and antibody fragments (e.g., Fab or Fv). Unfortunately, antibodies that bind specifically and tightly to integral membrane proteins have been challenging to generate using conventional means. Hence, the goal of this project is to develop new genetic and protein engineering tools that allow fast access to high-affinity antibodies that bind GPCRs. Specifically, two novel protein-fragment complementation assays (PCAs) will be engineered in E. coli for detecting protein-protein interactions involving GPCRs. These PCA strategies are based on the principle of split protein complementation, in which two inactive fragments derived from an enzyme are fused to a pair of interacting partners. When the two partners interact, the two inactive fragments are brought into proximity and an intact enzyme is reconstituted. The first strategy employs TEM-1 2-lactamase PCA to detect interactions between periplasmically expressed antibody fragments and native epitopes of GPCRs expressed in the cytoplasmic membrane (specific aim 1). The second strategy uses yeast cytosine deaminase PCA to report antibody fragment-mediated blocking of GPCR dimerization (specific aim 2). In each of these selection strategies, the coding sequence of the GPCR of interest is fused to a protein fragment derived from a selectable marker enzyme. Consequently, the reassembly of the marker and survival on selective media is dependent upon an interaction between the GPCR and a partner protein fused to the other half of the selectable marker. The advantage of this approach is that molecular interactions involving GPCRs can be directly and rapidly detected in the context of living E. coli cells using simple clonal selection. Studies here will focus on the following mammalian GPCRs from the rhodopsin family of receptors: adenosine A2A, cannabinoid CB1 and CB2, the long splice variant of dopamine D2 and neurotensin receptor-1. The development of convenient genetic tools for rapidly isolating GPCR- specific antibodies in a high-throughput and cost-effective way would address a major technological gap that has hampered the GPCR field and the NIH scientific community as a whole. PUBLIC HEALTH RELEVANCE: Antibodies have been used extensively for studying and manipulating pharmacologic protein targets that are soluble, but also have the potential to overcome many of the bottlenecks associated with the functional and structural characterization of membrane proteins. Unfortunately, high-quality antibodies against membrane proteins such as G protein-coupled receptors (GPCRs) have been challenging to generate using conventional means. Therefore, in this project innovative new approaches for isolating recombinant antibodies against GPCRs will be developed that will provide a rich source of potent antibody-based reagents for diagnosis, therapy, and research related to GPCRs and numerous other membrane protein systems.

描述（由申请人提供）：G蛋白偶联受体（GPCR）是跨细胞膜传递细胞外信号的整合膜蛋白，与包括癌症、糖尿病和HIV感染在内的多种疾病直接相关。GPCR包括哺乳动物细胞中最大的一类膜蛋白-人类基因组编码超过800种-最近的估计表明GPCR占所有药物靶标的60-70%。然而，尽管它们广泛存在并具有潜在的医学相关性，但由于蛋白质表达、纯化和结晶的困难，以及在天然环境中普遍缺乏用于其操作的试剂，大多数GPCR的结构、功能和生物学仍然难以捉摸。因此，只报告了四种独特的气相化学还原反应结构。在这些情况中的几种情况下，GPCR结构测定极大地通过特异性结合抗体和抗体片段（例如，Fab或Fv）。不幸的是，特异性地和紧密地结合到整合膜蛋白的抗体一直具有挑战性，使用常规手段产生。因此，该项目的目标是开发新的遗传和蛋白质工程工具，允许快速获得结合GPCR的高亲和力抗体。具体来说，将在大肠杆菌中设计两种新型蛋白片段互补测定（PCA）。大肠杆菌，用于检测涉及GPCR的蛋白质-蛋白质相互作用。这些PCA策略基于分裂蛋白互补的原理，其中来自酶的两个无活性片段融合到一对相互作用的伴侣。当两个伴侣相互作用时，两个无活性片段被带到附近，并重建完整的酶。第一种策略采用TEM-1 2-内酰胺酶PCA来检测周质表达的抗体片段与细胞质膜中表达的GPCR的天然表位之间的相互作用（具体目的1）。第二种策略使用酵母胞嘧啶脱氨酶PCA来报告抗体片段介导的GPCR二聚化的阻断（具体目的2）。在这些选择策略中的每一种中，感兴趣的GPCR的编码序列与来源于选择性标记酶的蛋白质片段融合。因此，标记物的重新组装和在选择性培养基上的存活取决于GPCR和与选择性标记物的另一半融合的伴侣蛋白之间的相互作用。这种方法的优点是，涉及GPCR的分子相互作用可以在活的E. coli细胞中进行克隆选择。这里的研究将集中在以下哺乳动物GPCR从视紫红质家族的受体：腺苷A2 A，大麻素CB 1和CB 2，多巴胺D2和神经降压素受体的长剪接变体-1。以高通量和成本效益的方式快速分离GPCR特异性抗体的方便遗传工具的开发将解决阻碍GPCR领域和整个NIH科学界的主要技术差距。 公共卫生相关性：抗体已被广泛用于研究和操纵可溶性的药理学蛋白质靶标，但也有可能克服与膜蛋白的功能和结构表征相关的许多瓶颈。不幸的是，使用常规手段产生针对膜蛋白如G蛋白偶联受体（GPCR）的高质量抗体具有挑战性。因此，在本项目中，将开发用于分离针对GPCR的重组抗体的创新新方法，这将为与GPCR和许多其他膜蛋白系统相关的诊断、治疗和研究提供基于有效抗体的试剂的丰富来源。