Beta-Arrestins Methods and Protocols

This volume looks at various approaches to study the pleiotropic roles of b-arrestins (b-arrs) in the control of signal transduction, and the resulting cellular and in vivo consequences that arise. The chapters in this book cover diverse topics around b-arrs such as their established roles in GPCR regulation and trafficking; regulatory scaffolding functions of b-arrs in  MAPK signaling, cAMP hydrolysis and cytoskeletal dynamics; proteomic analysis of the b-arr interactome; mathematical modelling of b-arr signaling networks; functional selectivity involving biased ligands; nucleocytoplasmic trafficking and primary cilia-associated functions of b-arrs; conformational plasticity of b-arrs; and the roles of b-arrs in allergic inflammation, Type 2 Diabetes, Parkinson’s disease, and cancer. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents,step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

A Brief History of the b-Arrestins.- b-Arrestins: Multitask Scaffolds Orchestrating the Where and When In Cell Signaling.- Methods to Monitor the Trafficking of ß-Arrestin/G Protein-Coupled Receptor Complexes using Enhanced Bystander BRET.- Methods to Investigate b-Arrestin-Mediated Regulation of GPCR Function in Human Airway Smooth Muscle.- Measuring Recruitment of b-Arrestin to G Protein-Coupled Heterodimers using Bioluminescence Resonance Energy Transfer.- Detection of ß-Arrestin-Mediated G Protein-Coupled Receptor Ubiquitination using BRET.- Using In Vitro Pull-Down and In Cell Overexpression Assays to Study Protein Interactions with Arrestin.- Methods to Investigate Arrestins in Complex with Phosphodiesterases.- Methods to Characterize Protein Interactions with ß-arrestin in cellulo.- Methods to Investigate the ß-Arrestin-Mediated control of ARF6 Activation to Regulate Trafficking and Actin Cytoskeleton Remodeling.- Methods to Investigate the Roles of b-Arrestin-Dependent RalGDS Activation in GPCR-Stimulated Membrane Blebbing.- Methods to Determine Interaction Interfaces between b-Arrestins and Their Protein Partners.- Workflow Description to Dynamically Model ß-Arrestin Signaling Networks.- Proteomic Analysis of the ß-Arrestin Interactome.- Quantitating Ligand Bias using the Competitive Model of Ligand Activity.-     Methods to Investigate the Nucleocytoplasmic Shuttling Properties of b-Arrestins.- Monitoring ß-Arrestin2 Targeting to the Centrosome, Basal Body and Primary Cilium by Fluorescence Microscopy.- A Mass Spectrometry-Based Structural Assay for Activation-Dependent Conformational Changes in ß-Arrestins.- Probing Arrestin Function using Intramolecular FlAsH-BRET Biosensors.- Methods to Study the Roles of b-Arrestins in Meningococcal Signaling.- Methods to Investigate the Roles for ß-Arrestin2 in Allergic Inflammatory Airway Disease.- Methods to Study Roles of ß-Arrestins in the Regulation of Pancreatic ß-Cell Function.- Methods to Investigate ß-Arrestin Function in Metabolic Regulation.- Methods to Investigate the Role of b-Arrestin Signaling in Parkinson's Ddisease.- Methods to Investigate ß-Arrestin-1/ß-Catenin Signaling in Ovarian Cancer Cells.