GS Activation Is Time-limiting in Initiating Receptor-mediated Signaling

Hein, Peter; Rochais, Francesca; Hoffmann, Carsten; Dorsch, Sandra; Nikolaev, Viacheslav O.; Engelhardt, Stefan; Berlot, Catherine H.; Lohse, Martin J.; Bünemann, Moritz

doi:10.1074/jbc.m606713200

Cited by 118 publications

(160 citation statements)

References 32 publications

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“…These signals amount to up to 15% of the initial F YFP /F CFP ratio. Compared with the speed of effects measured with the ␤ 2 AR-369 FRET sensor, these effects were considerably slower, in line with earlier findings showing that G-protein activation and deactivation are much slower than receptor activation and deactivation (31,38).…”

Section: Expression Of the ␤ 2 Ar Fret Sensors In Hek293supporting

confidence: 88%

“…Molecular Biology-The pcDNA3 plasmid encoding for the human ␤ 2 AR as well as pcDNA3 plasmids containing the cDNA sequence for G␣ s -YFP, CFP-G␥ 2 , or ␤ 2 AR-YFP have been described previously (31,33). The plasmids encoding bovine ␤-arrestin2 fused to YFP (5), as well as GRK2 and GRK5 (49) have been described previously.…”

Section: Methodsmentioning

confidence: 99%

“…CHO cells were co-transfected with the following amounts of plasmids/well: 2 g of G␣ s -YFP, 0.2 g of G␥ 2 -CFP, and 0.5 g of G␤ 1 in a mixture of 2.5 g of polyethylenimine and 100 l of Opti-MEM, whereas for HEK293 cells the amounts were 0.17 g of ␤ 2 AR-YFP and 0.15 g of ␤-arrestin2-CFP (FRET measurements) or 0.17 g of ␤ 2 AR and 0.15 g of ␤-arrestin2-YFP (confocal microscopy) per well using Effectene according to the manufacturer's instructions. These amounts were chosen to give equimolar expression of G-proteins or receptors versus ␤-arrestin2, respectively, as determined by fluorescence or Western blotting (31).…”

Section: Methodsmentioning

confidence: 99%

“…Determination of G s Activation-G-protein activation by GPCRs in intact cells can also be investigated with FRET-based approaches, either by studying the interaction between receptors and G-proteins (31,38) or by measuring changes between the G-protein subunits (25,30,39,40). Because it produces a more robust signal and also because it reflects G-protein activation, we chose the latter technique to compare the effects of different ligands on G s activation by ␤ 2 ARs in intact cells.…”

Section: Expression Of the ␤ 2 Ar Fret Sensors In Hek293mentioning

confidence: 99%

“…yellow fluorescent protein (YFP)), and the binding of agonists to these receptors causes a change in FRET that reports the agonistinduced conformational change (29). Similar technologies based on FRET between interacting proteins have been developed to monitor G-protein activation (30,31), cAMP generation (32), and ␤-arrestin binding to receptors (5) in intact cells and in real time.…”

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confidence: 99%

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Differential Signaling of the Endogenous Agonists at the β2-Adrenergic Receptor

Reiner

Ambrosio

Hoffmann

et al. 2010

Journal of Biological Chemistry

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103

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The concept of "functional selectivity" or "biased signaling" suggests that a ligand can have distinct efficacies with regard to different signaling pathways. We have investigated the question of whether biased signaling may be related to distinct agonist-induced conformational changes in receptors using the ␤ 2 -adrenergic receptor (␤ 2 AR) and its two endogenous ligands epinephrine and norepinephrine as a model system. Agonist-induced conformational changes were determined in a fluorescently tagged ␤ 2 AR FRET sensor. In this ␤ 2 AR sensor, norepinephrine caused signals that amounted to only ≈50% of those induced by epinephrine and the standard "full" agonist isoproterenol. Furthermore, norepinephrine-induced changes in the ␤ 2 AR FRET sensor were slower than those induced by epinephrine (rate constants, 47 versus 128 ms). A similar partial ␤ 2 AR activation signal was revealed for the synthetic agonists fenoterol and terbutaline. However, norepinephrine was almost as efficient as epinephrine (and isoproterenol) in causing activation of G s and adenylyl cyclase. In contrast, fenoterol was quite efficient in triggering ␤-arrestin2 recruitment to the cell surface and its interaction with ␤ 2 AR, as well as internalization of the receptors, whereas norepinephrine caused partial and slow changes in these assays. We conclude that partial agonism of norepinephrine at the ␤ 2 AR is related to the induction of a different active conformation and that this conformation is efficient in signaling to G s and less efficient in signaling to ␤-arrestin2. These observations extend the concept of biased signaling to the endogenous agonists of the ␤ 2 AR and link it to distinct conformational changes in the receptor.

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Section: Expression Of the ␤ 2 Ar Fret Sensors In Hek293supporting

confidence: 88%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%