title: G protein-independent cell-based assays for drug discovery on seven-transmembrane receptors.
abstract: Conventional cell-based assays for seven-transmembrane receptors, also known as G protein-coupled receptors, rely on the coupling of the ligand-bound receptor to heterotrimeric G proteins. New assay methods have become available that are not based on G protein activation, but that apply the molecular mechanism underlying the attenuation of G protein signaling mediated by beta-arrestin. beta-arrestin is a cytoplasmic protein that targets receptors to clathrin-coated endocytotic vesicles for degradation or recycling. This process has been visualized and quantified in high-content imaging assays using receptor- or beta-arrestin-chimeras with green fluorescent protein. Other assay methods use bioluminescence resonance energy transfer, enzyme fragment complementation, or a protease-activated transcriptional reporter gene, to measure receptor-beta-arrestin proximity. beta-arrestin recruitment assays have been applied successfully for receptors coupling to Galpha(q), Galpha(s) and Galpha(i) proteins, thus providing a generic assay platform for drug discovery on G protein-coupled receptors. The best understood signal transduction pathway elicited by the seven-transmembrane Frizzled receptors does not involve G proteins. The activation of Frizzleds by their cognate ligands of the Wnt family recruits the phosphoprotein dishevelled. Dishevelled regulates a protein complex involved in the destruction of beta-catenin. Activation of Frizzled blocks degradation of beta-catenin, which translocates to the nucleus to activate transcription of Wnt-responsive genes. The cytoplasm-to-nuclear translocation of beta-catenin forms the basis of several high-content assays to measure Wnt/Frizzled signal transduction. Interestingly, Frizzled receptors have recently been shown to internalize and to recruit beta-arrestin. This suggests that beta-arrestin recruitment assays may be applied for drug discovery on seven-transmembrane receptors beyond G protein-coupled receptors.
authors.affiliation: Molecular Pharmacology Unit, Organon BioSciences, Oss, The Netherlands.
authors.forename: Folkert
authors.initials: F
authors.lastname: Verkaar
authors.forename: Jos W G
authors.initials: JW
authors.lastname: van Rosmalen
authors.forename: Marion
authors.initials: M
authors.lastname: Blomenröhr
authors.forename: Chris J
authors.initials: CJ
authors.lastname: van Koppen
authors.forename: W Matthijs
authors.initials: WM
authors.lastname: Blankesteijn
authors.forename: Jos F M
authors.initials: JF
authors.lastname: Smits
authors.forename: Guido J R
authors.initials: GJ
authors.lastname: Zaman
mesh_terms: D019390:Arrestins / Q000378:metabolism*; D001681:Biological Assay / Q000379:methods*; D015374:Biosensing Techniques / Q000379:methods; D015195:Drug Design; D019204:GTP-Binding Proteins / Q000378:metabolism*; D004364:Pharmaceutical Preparations / Q000008:administration & dosage*; D011956:Receptors, Cell Surface / Q000187:drug effects* / Q000378:metabolism*; D000071557:beta-Arrestins
chemical_list: D019390:Arrestins; D004364:Pharmaceutical Preparations; D011956:Receptors, Cell Surface; D000071557:beta-Arrestins; D019204:GTP-Binding Proteins