Abstract
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 β(2)-adrenergic receptor (β(2)AR) and its two endogenous ligands epinephrine and norepinephrine as a model system. Agonist-induced conformational changes were determined in a fluorescently tagged β(2)AR FRET sensor. In this β(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 β(2)AR FRET sensor were slower than those induced by epinephrine (rate constants, 47 versus 128 ms). A similar partial β(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 β-arrestin2 recruitment to the cell surface and its interaction with β(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 β(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 β-arrestin2. These observations extend the concept of biased signaling to the endogenous agonists of the β(2)AR and link it to distinct conformational changes in the receptor.
Highlights
It has become apparent over recent years that the situation is far more complex and that ligands may cause differ
It has been shown for several receptor systems that ligands may differentially activate two major pathways: “classical” G-protein-mediated signals versus “nonclassical” activation via -arrestins and often involving MAPKs. -Arrestins are recruited to receptors in response to agonist activation and agonist-induced phosphorylation by G-proteincoupled receptor kinases (GRKs)2 [3,4,5]
A growing body of experiments provides evidence that this classical view of receptor function is incomplete and that ligands may cause distinct responses for different downstream effects, including most notably G-protein-dependent versus -arrestin-dependent pathways [9]. Such data have been obtained for many receptors, including serotonin, opioid, vasopressin, dopamine, and -adrenergic receptors [1, 2, 10]. These observations include ligands that differentially affect G-protein activation versus receptor internalization [11] as well as compounds that differentially activate the MAPK cascade compared with G-protein-dependent signaling [12,13,14]
Summary
Materials—All of the 2AR agonists were purchased from Sigma-Aldrich, except for (Ϫ)-isoproterenol hydrochloride, which was from Tocris/Biozol (Eching, Germany). Transient transfection of HEK293 cells with the 2AR FRET sensors were performed with Lipofectamine 2000 according to the manufacturer’s instructions. Opti-MEM, whereas for HEK293 cells the amounts were 0.17 g of 2AR-YFP and 0.15 g of -arrestin2-CFP (FRET measurements) or 0.17 g of 2AR 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 -arrestin, respectively, as determined by fluorescence or Western blotting [31]. C, maximal rate constants of the signals of epinephrine and norepinephrine recorded as in B but with 1 mM norepinephrine and 0.1
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