Bresnick Janine N, Skynner Heather A, Chapman Kerry L, Jack Andrew D, Zamiara Elize, Negulescu Paul, Beaumont Kevin, Patel Smita, McAllister George
Department of Biochemistry and Molecular Biology, Merck Sharp and Dohme Ltd., Harlow, Essex, United Kingdom.
Assay Drug Dev Technol. 2003 Apr;1(2):239-49. doi: 10.1089/15406580360545053.
The superfamily of GPCRs have diverse biological roles, transducing signals from a range of stimuli, from photon recognition by opsins to neurotransmitter regulation of neuronal function. Of the many identified genes encoding GPCRs, >130 are orphan receptors ( i.e., their endogenous ligands are unknown), and this subset represents putative novel therapeutic targets for pharmaceutical intervention in a variety of diseases. As an initial step toward drug discovery, determining a biological function for these newly identified receptors is of vital importance, and thus identification of a natural ligand(s) is a primary aim. There are several established methods for doing this, but many have drawbacks and usually require some in-depth knowledge about how the receptor functions. The technique described here utilizes a transcription-based reporter assay in live cells. This allows the determination of the signal transduction pathway any given oGPCR uses, without any prior knowledge of the endogenous ligand. This can therefore reduce the redundancy of effort involved in screening ligands at a given receptor in multiple formats (i.e., Galpha(s), Galpha(i/0), and Galpha(q) assays), as well as ensuring that the receptor targeted is capable of signaling if appropriately activated. Such knowledge is often laboriously obtained, and for almost all oGPCRs, this kind of information is not yet available. This technology can also be used to develop inverse agonist as well as agonist sensitive high throughput assays for oGPCRs. The veracity of this approach is demonstrated, using a number of known GPCRs. The likely signaling pathways of the GPR3, GPR12, GPR19, GPR21, and HG55 oGPCRs are shown, and a high throughput assay for GPR26 receptors developed. The methods outlined here for elucidation of the signal transduction pathways for oGPCRs and development of functional assays should speed up the process of identification of ligands for this potentially therapeutically useful group of receptors.
G蛋白偶联受体（GPCR）超家族具有多种生物学作用，可转导来自一系列刺激的信号，从视蛋白对光子的识别到神经递质对神经元功能的调节。在众多已鉴定的编码GPCR的基因中，超过130个是孤儿受体（即其内源配体未知），这一子集代表了多种疾病药物干预的潜在新型治疗靶点。作为药物发现的第一步，确定这些新鉴定受体的生物学功能至关重要，因此鉴定天然配体是主要目标。有几种既定方法可用于此，但许多方法都有缺点，通常需要对受体功能有一些深入了解。这里描述的技术利用活细胞中的基于转录的报告基因检测。这使得能够确定任何给定的孤儿GPCR所使用的信号转导途径，而无需事先了解内源配体。因此，这可以减少以多种形式（即Gαs、Gαi/0和Gαq检测）在给定受体上筛选配体所涉及的重复工作，同时确保靶向的受体在适当激活时能够发出信号。此类知识通常很难获得，而且几乎所有孤儿GPCR都还没有此类信息。该技术还可用于开发孤儿GPCR的反向激动剂以及激动剂敏感的高通量检测方法。使用一些已知的GPCR证明了该方法的准确性。展示了GPR3、GPR12、GPR19、GPR21和HG55孤儿GPCR可能的信号转导途径，并开发了GPR26受体的高通量检测方法。这里概述的用于阐明孤儿GPCR信号转导途径和开发功能检测方法的方法，应能加快识别这一潜在具有治疗用途的受体组配体的过程。
