Development of sensitive cellular assay systems and their application to the identification of "orphan" seven-transmembrane receptors

University dissertation from Knut Kotarsky; Tordmulevägen 3c; 22735 LUND Sweden

Abstract: Seven-transmembrane, G-protein coupled receptors play a central role in physiology since they facilitate cell communication in multicellular organisms by recognition of a broad range of ligands. They also represent important drug targets. Unfortunately, for many of these receptors the endogenous ligands, and, hence, their physiological functions, remain to be identified. These receptors are usually referred to as “orphan” receptors. A pre-requisite for the identification of ligands activating “orphan” receptors is powerful assay systems displaying a high assay quality as specified by a high Z-value. Until now, reporter gene assays have not been in common use in this process. Therefore, we aimed to develop improved reporter gene assays. This aim was accomplished by optimizing the promoter region of the construct, the reporter enzyme, and the assay procedure. Furthermore, a fluorescence-based clone selection step was introduced, that allowed the selection of the most sensitive reporter cell clones. The established test cell lines responded sensitively upon stimulation of various cell surface receptors as demonstrated by several receptors. In the first approach, transcription of the reporter gene was under control of a synthetic promoter consisting of 9 TPA responsive elements. In a further improved construct, the promoter was extended with six NF-kB and six STAT motifs. The used reporter gene was designed as a fusion gene coding for green fluorescent protein and Photinus luciferase. The amplification of reporter enzyme activity was substantially larger than in any other described system, and the high assay quality made it suitable as a primary screening tool. The development of efficient assays allowed the screening for hitherto unknown ligands to orphan seven-transmembrane receptors. Natural ligands for two recently unknown receptors were identified. Thus, we identified the second leukotriene B4 receptor, BLT2, and the first cell surface, free fatty acid receptor, FFA1. The BLT2 receptor was first identified in silico, cloned, and subsequently functionally expressed in HeLa cells. The identification of FFA1 was accomplished using a reverse pharmacology approach. The FFA1 receptor (previously known as GPR40) responded to medium to long chain free fatty acids, including compounds like ±9-hydroxy-octadecadienoic acid (9-HODE) and a conjugated linoleic acid (10,12-CLA). Receptor expression was detected in heart, skeletal muscle, liver and in pancreatic b-cells. Most importantly, the identification of anti-diabetic drugs (thiazolidinediones and MEDICA16) as agents acting on this receptor implies an important connection between FFA1R and type II diabetes.

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