New Insights into Arrestin Recruitment to GPCRs.

International Journal of Molecular Sciences
Martin SpillmannPhilipp Berger

Abstract

G protein-coupled receptors (GPCRs) are cellular master regulators that translate extracellular stimuli such as light, small molecules or peptides into a cellular response. Upon ligand binding, they bind intracellular proteins such as G proteins or arrestins, modulating intracellular signaling cascades. Here, we use a protein-fragment complementation approach based on nanoluciferase (split luciferase assay) to assess interaction of all four known human arrestins with four different GPCRs (two class A and two class B receptors) in live cells. Besides directly tagging the 11S split-luciferase subunit to the receptor, we also could demonstrate that membrane localization of the 11S subunit with a CAAX-tag allowed us to probe arrestin recruitment by endogenously expressed GPCRs. Varying the expression levels of our reporter constructs changed the dynamic behavior of our assay, which we addressed with an advanced baculovirus-based multigene expression system. Our detection assay allowed us to probe the relevance of each of the two arrestin binding sites in the different GPCRs for arrestin binding. We observed remarkable differences between the roles of each arresting binding site in the tested GPCRs and propose that the distinct adva...Continue Reading

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Citations

Feb 5, 2021·The FEBS Journal·Jeffrey S Smith, Thomas F Pack

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Methods Mentioned

BETA
bioluminescence
fluorescence microscopy
BRET
transfection
Assay
PCR
FCS
bioluminescence resonance energy transfer

Software Mentioned

Transfluor assay
GraphPad Prism
PathHunter

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