Apr 10, 2020

A novel luminescence-based β-arrestin membrane recruitment assay for unmodified GPCRs

BioRxiv : the Preprint Server for Biology
M. Hauge PedersenJonathan A Javitch

Abstract

G protein-coupled receptors (GPCRs) signal through activation of G proteins and subsequent modulation of downstream effectors. More recently, G protein-independent signaling via the arrestin pathway has also been implicated in important physiological functions. This has led to great interest in the identification of biased ligands that favor either the G protein or arrestin-signaling pathways. Currently available screening techniques that measure arrestin recruitment have required C-terminal receptor modifications that can in principle alter protein interactions and thus signaling. Here, we have developed a novel luminescence-based assay to measure arrestin recruitment to any unmodified receptor. NanoLuc, an engineered luciferase from ophlorus gracilirostris (deep sea shrimp), is smaller and brighter than other well-established luciferases. Recently, several publications have explored functional NanoLuc split sites for use in complementation assays. Here, we have identified a novel split site and have fused the N-terminal fragment to a membrane tether and the C-terminal fragment to the N-terminus of either {beta}-arrestin 1 or 2. Upon receptor activation, arrestin is recruited to the plasma membrane in an agonist concentration-...Continue Reading

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Mentioned in this Paper

Study
Brain
Face
Participant
Literature
Recognition (Psychology)
Molecular Recognition
Catabolism
Prosopagnosia, Developmental
Impaired Health

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