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A rapid fluorogenic GPCR-β-arrestin interaction assay.

ABSTRACT: Detection of protein-protein interactions involved in signal transduction in live cells and organisms has a variety of important applications. We report a fluorogenic assay for G protein-coupled receptor (GPCR)-β-arrestin interaction that is genetically encoded, generalizes to multiple GPCRs, and features high signal-to-noise because fluorescence is absent until its components interact upon GPCR activation. Fluorescence after protease-activated receptor-1 activation developed in minutes and required specific serine-threonine residues in the receptor carboxyl tail, consistent with a classical G protein-coupled receptor kinase dependent β-arrestin recruitment mechanism. This assay provides a useful complement to other in vivo assays of GPCR activation.

SUBMITTER: Zhang Q

PROVIDER: S-EPMC5866937 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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A rapid fluorogenic GPCR-β-arrestin interaction assay.

Zhang Qiang Q Zheng Yao-Wu YW Coughlin Shaun R SR Shu Xiaokun X

Protein science : a publication of the Protein Society 20180223 4

Detection of protein-protein interactions involved in signal transduction in live cells and organisms has a variety of important applications. We report a fluorogenic assay for G protein-coupled receptor (GPCR)-β-arrestin interaction that is genetically encoded, generalizes to multiple GPCRs, and features high signal-to-noise because fluorescence is absent until its components interact upon GPCR activation. Fluorescence after protease-activated receptor-1 activation developed in minutes and requ ...[more]

PMID: 29411438

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Project description:β-arrestins regulate many cellular functions including intracellular signaling and desensitization of G protein-coupled receptors (GPCRs). Previous studies show that β-arrestin signaling and receptor endocytosis are modulated by the plasma membrane phosphoinositide lipid phosphatidylinositol-(4, 5)-bisphosphate (PI(4,5)P2). We found that β-arrestin also helped promote synthesis of PI(4,5)P2 and up-regulated GPCR endocytosis. We studied these questions with the Gq-coupled protease-activated receptor 2 (PAR2), which activates phospholipase C, desensitizes quickly, and undergoes extensive endocytosis. Phosphoinositides were monitored and controlled in live cells using lipid-specific fluorescent probes and genetic tools. Applying PAR2 agonist initiated depletion of PI(4,5)P2, which then recovered during rapid receptor desensitization, giving way to endocytosis. This endocytosis could be reduced by various manipulations that depleted phosphoinositides again right after phosphoinositide recovery: PI(4)P, a precusor of PI(4,5)P2, could be depleted at either the Golgi or the plasma membrane (PM) using a recruitable lipid 4-phosphatase enzyme and PI(4,5)P2 could be depleted at the PM using a recruitable 5-phosphatase. Endocytosis required the phosphoinositides. Knock-down of β-arrestin revealed that endogenous β-arrestin normally doubles the rate of PIP5-kinase (PIP5K) after PAR2 desensitization, boosting PI(4,5)P2-dependent formation of clathrin-coated pits (CCPs) at the PM. Desensitized PAR2 receptors were swiftly immobilized when they encountered CCPs, showing a dwell time of ∼90 s, 100 times longer than for unactivated receptors. PAR2/β-arrestin complexes eventually accumulated around the edges or across the surface of CCPs promoting transient binding of PIP5K-Iγ. Taken together, β-arrestins can coordinate potentiation of PIP5K activity at CCPs to induce local PI(4,5)P2 generation that promotes recruitment of PI(4,5)P2-dependent endocytic machinery.

Dataset Information

A rapid fluorogenic GPCR-β-arrestin interaction assay.

Publications

A rapid fluorogenic GPCR-β-arrestin interaction assay.

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