For GPCRs with an unknown crystal structure time-consuming and cost-intensive high-throughput screenings of small compounds are commonly performed in vivo. For these receptors a structural-based virtual ligand screening is possible, that models new derivatives of known ligands to improve its characteristics. Recently the crystal structure of the ET-B receptor was solved as well 5. So far, over 100 GPCR structures such as bovine rhodopsin receptor 2, β 2-adrenergic receptor 3 and CXCR4 chemokine receptor 4 could be crystallized and solved at atomic resolution. As a consequence, the characterization of GPCRs has become a research topic of enormous economic importance. Furthermore, GPCRs serve as targets for more than 50% of all modern pharmaceutical drugs 1. GPCRs are key elements regulating intracellular signaling cascades activated by extracellular stimuli such as odorants, light, peptides, neurotransmitters and hormones. Currently more than 1000 annotated members of GPCRs are characterized by a general topology composed of seven transmembrane-spanning helices (TM1-7) connected by three extracellular (E1-3) and three intracellular (C1-3) loops. G protein-coupled receptors (GPCRs) are known to form the largest class of transmembrane proteins in humans. To determine the functionality of the cell-free synthesized ET-B receptor, we analyzed the binding of its ligand endothelin-1 (ET-1) in a qualitative fluorescence-based assay and in a quantitative radioligand binding assay. In this study we present CFPS in combination with fast fluorescence-based screening methods to determine the localization, orientation and ligand-binding properties of the endothelin B (ET-B) receptor upon expression in an insect-based cell-free system. This method is based on eukaryotic cell lysates containing translocationally active endogenous endoplasmic reticulum-derived microsomes where the insertion of GPCRs into biologically active membranes is supported. Here, we describe a simply portable method to synthesize GPCRs and analyze their ligand-binding properties without the requirement of additional supplements such as liposomes or nanodiscs.
A promising approach for membrane protein synthesis and analysis has emerged during the last years and is known as cell-free protein synthesis (CFPS). Up to date, there still exist a number of GPCRs that has not been structurally and functionally analyzed due to difficulties in cell-based membrane protein production. Furthermore, mutations in GPCRs can cause acquired and inheritable diseases. In particular, G protein-coupled receptors (GPCRs) have attracted increasing interest since they affect cellular signaling.
Membrane proteins are key elements in cell-mediated processes.
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