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

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

Identification and characterization of murine glycoprotein 2‐expressing intestinal dendritic cells

The intestinal lamina propria (LP) contains distinct subsets of classical dendritic cells (cDC), each playing key non-redundant roles in intestinal immune homeostasis. Here, we show that glycoprotein 2 (GP2), a GPI-anchored protein and receptor for bacterial type-I fimbriae, is selectively expressed by CD103+CD11b+ cDC in the murine small intestine (SI). GP2 expression was induced on CD103+CD11b+ cDC within the SI-LP and was regulated by IRF4, TGFβR1- and retinoic acid signalling. Mice selectively lacking Gp2 on CD103+CD11b+ cDC (huLang-Cre.gp2fl/fl mice) had normal numbers and proportions of innate and adaptive immune cells in the SI-LP suggesting that GP2 expression by CD103+CD11b+ cDC is not required for intestinal immune homoeostasis

Reverse pharmacology and the de-orphanization of 7TM receptors

Approximately 800 genes coding for seven-transmembrane, G-protein-coupled receptors have so far been recognized. In spite of this, many of these receptors are defined by their sequence only, and are therefore classified as orphan receptors. Without knowing what their endogenous ligands are, we lack the information needed to understand their physiological role and hence cannot make use of them as drug targets. In this communication, we discuss different strategies, as well as difficulties in the deorphanizing process

A chimeric reporter gene allowing for clone selection and high-throughput screening of reporter cell lines expressing G-protein-coupled receptors

Efficient screening of ligands interacting with G-protein-coupled receptors is central for modern drug development. Here, we describe an optimized reporter vector primarily intended for use in reporter cell lines expressing such receptors. The construct consists of a synthetic enhancer containing 9x TRE (12-O-tetradecanoylphorbol-13-acetate-responsive elements) fused to a minimal CMV (cytomegalovirus) promoter. Activation of the promoter construct leads to the expression of a chimeric reporter protein based on the genes for enhanced green fluorescent protein and Photinus luciferase. The chimeric protein allows for both clonal selection by fluorescence, which facilitates the selection of optimal reporter cell lines and high-throughput screening by luminescens. In designing the vector, increasing numbers of TRE motifs were tested in front of two different minimal promoters. The reporter gene was more strongly inducible with increasing numbers of TRE motifs. The constructs were tested in two cell lines, CHO and HeLa. The latter regulated reporter gene activity stronger in response to PMA (phorbol 12-myristate 13-acetate) stimulation and were used to construct HF1 reporter cell lines. Model experiments were carried out on these reporter cells transfected with the human BLTR, human CCR5, or the rat alpha(1b) receptor. After maximal agonist stimulation reporter gene activity was increased 200-, 15-, and 50-fold, respectively

Chronic ethanol exposure enhances activating protein-1 transcriptional activity in human neuroblastoma cells

This study demonstrates a method for studying the effects of ethanol on transcription mediated by activating protein-1 (AP-1). The effects of ethanol on AP-1 activity and on the signaling cascades in this process were investigated by using a reporter gene technique with secreted alkaline phosphatase as the reporter gene coupled to nine DNA AP-1-binding elements. Long-term ethanol exposure (48-72 h) dose dependently enhanced AP-1 transcriptional activity in SH-SY5Y cells. Shorter exposure periods with ethanol did not influence AP-1 transcriptional activity compared with findings for control cells. Inhibition of protein kinase C (PKC) dramatically decreased AP-1 activity in both control and ethanol-exposed cells and abolished the ethanol enhancement. This finding suggests a pivotal role for PKC-coupled signaling in AP-1 transcriptional activity. Phorbol ester stimulation of AP-1 transcriptional activity was not influenced by long-term ethanol exposure. This finding indicates that signaling events upstream of PKC are the targets for ethanol. Mitogen-activated protein kinases ERK and p38 may play a role in ethanol-enhanced AP-1 activity because inhibitors of both enzymes partly reduced the enhancement. The inhibitors also partly blocked phorbol ester-induced AP-1 activation, which demonstrates a function of these mitogen-activated protein kinases downstream of PKC

Progress in methodology improved reporter gene assays used to identify ligands acting on orphan seven-transmembrane receptors.

Seven-transmembrane G-protein-coupled receptors play a central role in physiology by facilitating cell communication through recognition of a wide range of ligands. Even more important, they represent important drug targets. Unfortunately, for many of these receptors the endogenous ligands, and hence their functions, remain to be identified. These receptors are referred to as "orphan" receptors. A pre-requisite for the identification of ligands activating orphan receptors is powerful assay systems. Until now, reporter gene assays have not been in common use in this process. Here, we summarize our development of improved reporter gene assays. We optimized reporter gene assays in respect of (i) the promoter region of the construct, (ii) the reporter enzyme used, (iii) and the assay procedure. Furthermore, an unique fluorescence-based clone selection step was introduced, allowing rapid selection of the most sensitive reporter cell clones when establishing stable reporter cell lines. Mathematical formulae are provided to enable a simple and reliable comparison between different cell lines, when tested with a compound of interest. The resulting reporter cell lines responded in a very sensitive way to the stimulation of various test receptors. The reporter system was termed HighTRACE® (high-throughput reporter assay with clone election). Its high assay quality makes it suitable as a primary screening tool. Ligands for two recently unknown 7TM receptors were identified using the HighTRACE® system i.e., two cell surface free fatty acid receptors, GPR40 (FFA1R) and GPR43 (FFA2R). The identification was accomplished using a reverse pharmacology approach