Discovery of antibodies that bind G protein-coupled receptors

描述(申请人提供)：G蛋白偶联受体(GPCRs)是跨细胞膜传递细胞外信号的完整膜蛋白，与癌症、糖尿病和艾滋病毒感染等多种疾病直接相关。GPCRs组成了哺乳动物细胞中最大的一类膜蛋白--人类基因组编码了800多种--最近的估计表明，GPCRs占所有药物靶标的60%-70%。然而，尽管GPCRs广泛存在并具有潜在的医学意义，但由于蛋白质表达、纯化和结晶的困难，以及在当地环境中普遍缺乏操纵它们的试剂，大多数GPCRs的结构、功能和生物学仍然难以捉摸。因此，目前只有四种独特的gpr结构被报道。在其中几种情况下，通过特异性结合抗体和抗体片段(例如，Fab或Fv)，GPCR结构的确定得到了极大的帮助。不幸的是，用常规方法很难产生与完整膜蛋白特异性和紧密结合的抗体。因此，该项目的目标是开发新的基因和蛋白质工程工具，允许快速获得结合GPCRs的高亲和力抗体。具体地说，将在大肠杆菌中设计两种新的蛋白质片段互补分析(PCA)来检测涉及GPCRs的蛋白质-蛋白质相互作用。这些PCA策略是基于分裂蛋白互补的原理，在该原理中，来自一个酶的两个非活性片段融合到一对相互作用的伙伴上。当两个伴侣相互作用时，两个不活跃的片段被带到附近，一个完整的酶被重组。第一种策略使用了TEM-1 2-内酰胺酶PCA来检测胞浆内表达的抗体片段与胞膜中表达的GPCRs天然表位之间的相互作用(特异性目标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二聚体阻断(特异性目标2)。在这些选择策略中的每一种策略中，将感兴趣的GPCR的编码序列与来自可选择标记酶的蛋白质片段融合。因此，标记的重组和在选择介质上的存活依赖于GPCR和融合到可选择标记的另一半的伙伴蛋白之间的相互作用。这种方法的优点是，通过简单的克隆选择，可以在活的大肠杆菌细胞中直接和快速地检测到涉及GPCRs的分子相互作用。这里的研究将集中在以下哺乳动物的视紫红质受体家族的GPCRs：腺苷A2A，大麻素CB1和CB2，多巴胺D2和神经降压素受体-1的长剪接变体。开发方便的基因工具，以高通量和成本效益的方式快速分离GPCR特异性抗体，将解决阻碍GPCR领域和整个NIH科学界的一个重大技术差距。

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.