GPCR signaling complexes in living cells

描述(由申请人提供)：G蛋白偶联受体(GPCRs)是最大的细胞表面受体家族，是所有处方和滥用药物的相当大一部分的靶标。人们普遍认为，最大的GPCR家族(A类受体)的成员以二聚体或更高阶低聚物的形式自组装，GPCR二聚体已被认为是新型治疗药物的潜在靶点。然而，二聚化的功能后果只对少数几个受体进行了描述，还没有发现与二聚体特异结合的配体。这个项目的主要目标是检验这一假设，即A类原核体之间的大多数相互作用既是暂时的，又是结构上的非特异性的。如果是这样的话，这就解释了为什么二聚化很少会导致公开的功能变化或唯一的结合位点。该项目的目标是使用荧光共振能量转移(FRET)、生物发光共振能量转移(BRET)、时间分辨荧光共振能量转移(TRRET)和基于亲和力的细胞共募集试验来确定大样本A类受体与跨膜控制蛋白之间相互作用的物理稳定性。包括大样本的非GPCR控制蛋白将使我们能够确定A类原核糖体之间的物理相互作用是特殊的，还是一般多角体跨膜蛋白之间的典型相互作用。这些实验将更好地定义GPCRs最大亚家族的四元结构，并可能迫使目前激励寻找二聚体选择性药物的标准模型进行修订。

DESCRIPTION (provided by applicant): G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors, and are the targets of a substantial fraction of all prescribed ad abused drugs. It is widely accepted that members of the largest GPCR family (class A receptors) self-assemble as dimers or higher-order oligomers, and GPCR dimers have been proposed as potential targets for novel therapeutic drugs. However, functional consequences of dimerization have been described for only a few receptors, and ligands that bind specifically to dimers have not been found. The main goal of this project is to test the hypothesis that most interactions between classes A protomers are both transient and structurally nonspecific. If this is the case, it would explain why dimerization is rarely leads to overt functional changes or unique binding sites. The objective of the proposed project is to determine the physical stability of interactions between a large sample of class A receptors and transmembrane control proteins using fluorescence resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET), time-resolved fluorescence resonance energy transfer (TR-FRET), and an affinity-based on-cell corecruitment assay. Inclusion of a large sample of non-GPCR control proteins will allow us to determine if physical interactions between classes A protomers are special, or are typical of interactions between polytopic transmembrane proteins in general. These experiments will better define the quaternary structure of the largest subfamily of GPCRs, and may force a revision of the standard model that currently motivates the search for dimer-selective drugs.