title: Analysis of Arrestin Recruitment to Chemokine Receptors by Bioluminescence Resonance Energy Transfer.
abstract: Chemokine receptors recruit the multifunctional scaffolding protein beta arrestin in response to binding of their chemokine ligands. Given that arrestin recruitment represents a signaling axis that is in part independent from G-protein signaling, it has become a hallmark of G protein-coupled receptor functional selectivity. Therefore, quantification of arrestin recruitment has become a requirement for the delineation of chemokine and drug candidate activity along different signaling axes. Bioluminescence resonance energy transfer (BRET) techniques provide methodology for such quantification that can reveal differences between nonredundant chemokines binding the same receptor, and that can be upscaled for high-throughput testing. We here provide protocols for the careful setup of BRET-based arrestin recruitment assays, and examples for the application of such systems in dose-response or time-course experiments. Suggestions are given for troubleshooting, optimizing test systems, and the interpretation of results obtained with BRET-based assays, which indeed yield an intricate blend of quantitative and qualitative information.
authors.affiliation: Department of Biochemistry and Research Centre, Sainte-Justine Hospital, Université de Montréal, Montreal, Quebec, Canada.
authors.forename: J
authors.initials: J
authors.lastname: Bonneterre
authors.affiliation: Department of Biochemistry and Research Centre, Sainte-Justine Hospital, Université de Montréal, Montreal, Quebec, Canada.
authors.forename: N
authors.initials: N
authors.lastname: Montpas
authors.affiliation: Institut des Maladies Métaboliques et Cardiovasculaires, Institut National de la Santé et de la Recherche Médicale, U1048, Université Toulouse III Paul Sabatier, Toulouse, France.
authors.forename: C
authors.initials: C
authors.lastname: Boularan
authors.affiliation: Institut des Maladies Métaboliques et Cardiovasculaires, Institut National de la Santé et de la Recherche Médicale, U1048, Université Toulouse III Paul Sabatier, Toulouse, France.
authors.forename: C
authors.initials: C
authors.lastname: Galés
authors.affiliation: Department of Biochemistry and Research Centre, Sainte-Justine Hospital, Université de Montréal, Montreal, Quebec, Canada; Institut des Maladies Métaboliques et Cardiovasculaires, Institut National de la Santé et de la Recherche Médicale, U1048, Université Toulouse III Paul Sabatier, Toulouse, France. Electronic address: nikolaus.heveker@recherche-ste-justine.qc.ca.
authors.forename: N
authors.initials: N
authors.lastname: Heveker
mesh_terms: D019390:Arrestins / Q000032:analysis / Q000378:metabolism*; D060807:Bioluminescence Resonance Energy Transfer Techniques / Q000379:methods*; D018925:Chemokines / Q000378:metabolism / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D006801:Humans; D008024:Ligands; D009154:Mutation; D025941:Protein Interaction Mapping / Q000379:methods; D019707:Receptors, Chemokine / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D059629:Signal-To-Noise Ratio
chemical_list: D019390:Arrestins; D018925:Chemokines; D008024:Ligands; D019707:Receptors, Chemokine; D011994:Recombinant Proteins
keywords: Arrestin; BRET; Bioluminescence resonance energy transfer; CCR2; CXCR4; CXCR7; Chemokine; Chemokine receptor; Protein conformation