An intrabody sensor to monitor conformational activation of β-arrestins.

Hemlata Dwivedi-Agnihotri, Parishmita Sarma, S Deeksha, Kouki Kawakami, Asuka Inoue, Arun K Shukla

Methods in cell biology 2022

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Agonist-induced interaction of β-arrestins with GPCRs is critically involved in downstream signaling and regulation. This interaction is associated with activation and major conformational changes in β-arrestins. Although there are some assays available to monitor the conformational changes in β-arrestins in cellular context, additional sensors to report β-arrestin activation, preferably with high-throughput capability, are likely to be useful considering the structural and functional diversity in GPCR-β-arrestin complexes. We have recently developed an intrabody-based sensor as an integrated approach to monitor GPCR-β-arrestin interaction and conformational change, and generated a luminescence-based reporter using NanoBiT complementation technology. This sensor is derived from a synthetic antibody fragment referred to as Fab30 that selectively recognizes activated and receptor-bound conformation of β-arrestin1. Here, we present a step-by-step protocol to employ this intrabody sensor to measure the interaction and conformational activation of β-arrestin1 upon agonist-stimulation of a prototypical GPCR, the complement C5a receptor (C5aR1). This protocol is potentially applicable to other GPCRs and may also be leveraged to deduce qualitative differences in β-arrestin1 conformations induced by different ligands and receptor mutants. Copyright © 2022 Elsevier Inc. All rights reserved.

Citation

Hemlata Dwivedi-Agnihotri, Parishmita Sarma, S Deeksha, Kouki Kawakami, Asuka Inoue, Arun K Shukla. An intrabody sensor to monitor conformational activation of β-arrestins. Methods in cell biology. 2022;169:267-278