Beta-Arrestin2 as a Competitor for GRK2 Interaction with the GLP-1 Receptor upon Receptor Activation

Jørgensen, Rasmus; Roed, Sarah Norklit; Heding, Anders; Elling, Christian

doi:10.1159/000330742

Cited by 16 publications

(13 citation statements)

References 58 publications

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“…GLP-1 stimulated GRK2 recruitment to GLP-1R with faster kinetics than Arr3 (k = 0.040 s −1 and k = 0.017 s −1 , respectively). Previously, studies using BRET to investigate the kinetics of GRK2 and Arr3 interaction with GLP-1R produced similar results, with a faster time course being observed for GRK2 than Arr3 [34]. The authors propose a model whereby Arr3 competes with GRK2 for interaction with the phosphorylated receptor.…”

Section: Discussionmentioning

confidence: 67%

The GIP Receptor Displays Higher Basal Activity than the GLP-1 Receptor but Does Not Recruit GRK2 or Arrestin3 Effectively

et al. 2014

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Background and ObjectivesGlucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are important regulators of insulin secretion, and their functional loss is an early characteristic of type 2 diabetes mellitus (T2DM). Pharmacological levels of GLP-1, but not GIP, can overcome this loss. GLP-1 and GIP exert their insulinotropic effects through their respective receptors expressed on pancreatic β-cells. Both the GLP-1 receptor (GLP-1R) and the GIP receptor (GIPR) are members of the secretin family of G protein-coupled receptors (GPCRs) and couple positively to adenylate cyclase. We compared the signalling properties of these two receptors to gain further insight into why GLP-1, but not GIP, remains insulinotropic in T2DM patients.MethodsGLP-1R and GIPR were transiently expressed in HEK-293 cells, and basal and ligand-induced cAMP production were investigated using a cAMP-responsive luciferase reporter gene assay. Arrestin3 (Arr3) recruitment to the two receptors was investigated using enzyme fragment complementation, confocal microscopy and fluorescence resonance energy transfer (FRET).ResultsGIPR displayed significantly higher (P<0.05) ligand-independent activity than GLP-1R. Arr3 displayed a robust translocation to agonist-stimulated GLP-1R but not to GIPR. These observations were confirmed in FRET experiments, in which GLP-1 stimulated the recruitment of both GPCR kinase 2 (GRK2) and Arr3 to GLP-1R. These interactions were not reversed upon agonist washout. In contrast, GIP did not stimulate recruitment of either GRK2 or Arr3 to its receptor. Interestingly, arrestin remained at the plasma membrane even after prolonged (30 min) stimulation with GLP-1. Although the GLP-1R/arrestin interaction could not be reversed by agonist washout, GLP-1R and arrestin did not co-internalise, suggesting that GLP-1R is a class A receptor with regard to arrestin binding.ConclusionsGIPR displays higher basal activity than GLP-1R but does not effectively recruit GRK2 or Arr3.

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Section: Discussionmentioning

confidence: 67%

The GIP Receptor Displays Higher Basal Activity than the GLP-1 Receptor but Does Not Recruit GRK2 or Arrestin3 Effectively

et al. 2014

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“…␤-Arrestin-2 Recruitment Assay-Bioluminescence resonance energy transfer saturation experiments for measuring ␤-arrestin-2 recruitment were carried out as described previously (19). In short, HEK293 cells were transiently cotransfected in 25-cm 2 culture flasks with ␤-arrestin-2-GFP, GLP-1R-RLuc8, and pcDNA3.1, GCGR, GIPR, or HA-␤ 2 AR plasmid in a 6:1:1 (8.5:1.5:1.5 g) DNA ratio.…”

Section: Methodsmentioning

confidence: 99%

Functional Consequences of Glucagon-like Peptide-1 Receptor Cross-talk and Trafficking

Roed

Nøhr

Wismann

et al. 2015

Journal of Biological Chemistry

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“…Interestingly, the kinetics of GRK2 recruitment to OTR preceded β-arrestin2 recruitment to the same receptor suggesting that in this case, GRK2-mediated phosphorylation of OTR occurred before β-arrestin2 was recruited to the receptor. Other studies using BRET to investigate the interaction of GRKs with various GPCRs can be found in the following references (Huttenrauch et al, 2005; Small et al, 2006; Jorgensen et al, 2008, 2011; Namkung et al, 2009). …”

Section: β-Arrestin-based Bret Sensorsmentioning

confidence: 99%

BRET biosensors to study GPCR biology, pharmacology, and signal transduction

Salahpour¹

2012

Front. Endocrin.

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Bioluminescence resonance energy transfer (BRET)-based biosensors have been extensively used over the last decade to study protein–protein interactions and intracellular signal transduction in living cells. In this review, we discuss the various BRET biosensors that have been developed to investigate biology, pharmacology, and signaling of G protein-coupled receptors (GPCRs). GPCRs form two distinct types of multiprotein signal transduction complexes based upon their inclusion of G proteins or β-arrestins that can be differentially affected by drugs that exhibit functional selectivity toward G protein or β-arrestin signaling. BRET has been especially adept at illuminating the dynamics of protein–protein interactions between receptors, G proteins, β-arrestins, and their many binding partners in living cells; as well as measuring the formation and accumulation of second messengers following receptor activation. Specifically, we discuss in detail the application of BRET to study dopamine and trace amine receptors signaling, presenting examples of an exchange protein activated by cAMP biosensor to measure cAMP, β-arrestin biosensors to determine β-arrestin recruitment to the receptor, and dopamine D2 receptor and trace amine-associated receptor 1 biosensors to investigate heterodimerization between them. As the biochemical spectrum of BRET biosensors expands, the number of signaling pathways that can be measured will concomitantly increase. This will be particularly useful for the evaluation of functional selectivity in which the real-time BRET capability to measure distinct signaling modalities will dramatically shorten the time to characterize new generation of biased drugs. These emerging approaches will further expand the growing application of BRET in the screening for novel pharmacologically active compounds.

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Beta-Arrestin2 as a Competitor for GRK2 Interaction with the GLP-1 Receptor upon Receptor Activation

Cited by 16 publications

References 58 publications

The GIP Receptor Displays Higher Basal Activity than the GLP-1 Receptor but Does Not Recruit GRK2 or Arrestin3 Effectively

The GIP Receptor Displays Higher Basal Activity than the GLP-1 Receptor but Does Not Recruit GRK2 or Arrestin3 Effectively

Functional Consequences of Glucagon-like Peptide-1 Receptor Cross-talk and Trafficking

BRET biosensors to study GPCR biology, pharmacology, and signal transduction

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