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New Insights into Arrestin Recruitment to GPCRs.

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 advantages of our system for probing receptor interaction with effector proteins will help elucidate the molecular basis of GPCR signaling efficacy and specificity in different cell types.

SUBMITTER: Spillmann M

PROVIDER: S-EPMC7404097 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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New Insights into Arrestin Recruitment to GPCRs.

Spillmann Martin M Thurner Larissa L Romantini Nina N Zimmermann Mirjam M Meger Benoit B Behe Martin M Waldhoer Maria M Schertler Gebhard F X GFX Berger Philipp P

International journal of molecular sciences 20200713 14

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 cl ...[more]

PMID: 32668755

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Project description:G-protein-coupled receptors (GPCRs) are critically regulated by ?-arrestins, which not only desensitize G-protein signalling but also initiate a G-protein-independent wave of signalling. A recent surge of structural data on a number of GPCRs, including the ?2 adrenergic receptor (?2AR)-G-protein complex, has provided novel insights into the structural basis of receptor activation. However, complementary information has been lacking on the recruitment of ?-arrestins to activated GPCRs, primarily owing to challenges in obtaining stable receptor-?-arrestin complexes for structural studies. Here we devised a strategy for forming and purifying a functional human ?2AR-?-arrestin-1 complex that allowed us to visualize its architecture by single-particle negative-stain electron microscopy and to characterize the interactions between ?2AR and ?-arrestin 1 using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and chemical crosslinking. Electron microscopy two-dimensional averages and three-dimensional reconstructions reveal bimodal binding of ?-arrestin 1 to the ?2AR, involving two separate sets of interactions, one with the phosphorylated carboxy terminus of the receptor and the other with its seven-transmembrane core. Areas of reduced HDX together with identification of crosslinked residues suggest engagement of the finger loop of ?-arrestin 1 with the seven-transmembrane core of the receptor. In contrast, focal areas of raised HDX levels indicate regions of increased dynamics in both the N and C domains of ?-arrestin 1 when coupled to the ?2AR. A molecular model of the ?2AR-?-arrestin signalling complex was made by docking activated ?-arrestin 1 and ?2AR crystal structures into the electron microscopy map densities with constraints provided by HDX-MS and crosslinking, allowing us to obtain valuable insights into the overall architecture of a receptor-arrestin complex. The dynamic and structural information presented here provides a framework for better understanding the basis of GPCR regulation by arrestins.

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New Insights into Arrestin Recruitment to GPCRs.

Publications

New Insights into Arrestin Recruitment to GPCRs.

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