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.
Subject(s)
Arrestins/metabolism , Bioluminescence Resonance Energy Transfer Techniques/methods , Receptors, Chemokine/metabolism , Arrestins/analysis , Chemokines/metabolism , Chemokines/pharmacology , Dose-Response Relationship, Drug , Humans , Ligands , Mutation , Protein Interaction Mapping/methods , Receptors, Chemokine/analysis , Receptors, Chemokine/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Signal-To-Noise RatioABSTRACT
In January 2008, fatal anaphylactoid reaction (AR) was found to be associated with oversulfated chondroitin sulphate (OSCS) contaminated heparin. Although attributed to bradykinin released during contact system activation by OSCS, no final evidence until now exists for a bradykinin release during incubation of contaminated heparin with human plasma. The first objective of our study was to measure and to characterize the kinetic profile of bradykinin release in human plasma incubated with OSCS and contaminated heparin. As these AR occurred mainly in the first minutes of the dialysis session, we examine the different factors likely to influence the kinin-forming capacity of OSCS: dilution of plasma, presence of an angiotensin converting enzyme inhibitor, capacity of the patient to metabolise kinins.
Subject(s)
Anaphylaxis/chemically induced , Anticoagulants/adverse effects , Chondroitin Sulfates/adverse effects , Drug Contamination , Heparin/adverse effects , Aminopeptidases/metabolism , Anaphylaxis/metabolism , Angiotensin-Converting Enzyme Inhibitors/analysis , Anticoagulants/chemistry , Anticoagulants/therapeutic use , Bradykinin/blood , China , Chondroitin Sulfates/analysis , Dialysis , Heparin/chemistry , Heparin/therapeutic use , Humans , Kinins/metabolism , Sulfates/analysisABSTRACT
Severe hypersensitivity reactions have been reported with bolus injection of heparin contaminated with oversulfated chondroitin sulphate, which has been shown to activate the plasma contact system. In this paper, we parallel the pathophysiology of these acute side effects with this of hypersensitivity reaction during hemodialysis or blood product transfusion in patients treated with an angiotensin converting enzyme inhibitor.
