ABSTRACT
G protein-coupled receptors (GPCRs) are membrane-located proteins and, therefore, are exposed to changes in membrane potential (V(M)) in excitable tissues. These changes have been shown to alter receptor activation of certain Gi-and Gq-coupled GPCRs. By means of a combination of whole-cell patch-clamp and Förster resonance energy transfer (FRET) in single cells, we demonstrate that the activation of the Gs-coupled ß1-adrenoreceptor (ß1-AR) by the catecholamines isoprenaline (Iso) and adrenaline (Adr) is regulated by V(M). This voltage-dependence is also transmitted to G protein and arrestin 3 signaling. Voltage-dependence of ß2-AR activation, however, was weak compared with ß1-AR voltage-dependence. Drug efficacy is a major target of ß1-AR voltage-dependence as depolarization attenuated receptor activation, even under saturating concentrations of agonists, with significantly faster kinetics than the deactivation upon agonist withdrawal. Also the efficacy of the endogenous full agonist adrenaline was reduced by depolarization. This is a unique finding since reports of natural full agonists at other voltage-dependent GPCRs only show alterations in affinity during depolarization. Based on a Boltzmann function fit to the relationship of V(M) and receptor-arrestin 3 interaction we determined the voltage-dependence with highest sensitivity in the physiological range of membrane potential. Our data suggest that under physiological conditions voltage regulates the activity of agonist-occupied ß1-adrenoceptors on a very fast time scale.
Subject(s)
Catecholamines/pharmacology , Membrane Potentials , Receptors, Adrenergic, beta-1/metabolism , Adrenergic beta-1 Receptor Agonists/pharmacology , Arrestins/metabolism , Epinephrine/pharmacology , Fluorescence Resonance Energy Transfer , GTP-Binding Protein alpha Subunits, Gs/metabolism , HEK293 Cells , Humans , Isoproterenol/pharmacology , Patch-Clamp Techniques , Receptors, Adrenergic, beta-2/metabolism , Signal Transduction , Single-Cell AnalysisABSTRACT
Many different neurotransmitters and hormones control intracellular signaling by regulating the production of the second messenger cAMP. The function of the broadly expressed adenylyl cyclases (ACs) 5 and 6 is regulated by either stimulatory or inhibitory G proteins. By analyzing a well-known rebound stimulation phenomenon after withdrawal of Gi protein in atrial myocytes, we discovered that AC5 and -6 are tightly regulated by the second messenger PIP3. By monitoring cAMP levels in real time by means of Förster resonance energy transfer (FRET)-based biosensors, we reproduced the rebound stimulation in a heterologous expression system specifically for AC5 or -6. Strikingly, this cAMP rebound stimulation was completely blocked by the PI3K inhibitor wortmannin, both in atrial myocytes and in transfected human embryonic kidney cells. Similar effects were observed by heterologous expression of the PIP3 phosphatase and tensin homolog (PTEN). However, general kinase inhibitors or inhibitors of Akt had no effect, suggesting a PIP3-dependent mechanism. These findings demonstrate the existence of a novel general pathway for regulation of AC5 and -6 activity via PIP3 that leads to pronounced alterations of cytosolic cAMP levels.
Subject(s)
Adenylyl Cyclases/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Cell Line , Cell Line, Tumor , Cyclic AMP/metabolism , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , HEK293 Cells , HeLa Cells , Humans , PTEN Phosphohydrolase/metabolism , Signal Transduction/physiologyABSTRACT
The present study demonstrates that agonist-mediated activation of α2A adrenergic receptors (α(2A)AR) is voltage-dependent. By resolving the kinetics of conformational changes of α(2A)AR at defined membrane potentials, we show that negative membrane potentials in the physiological range promote agonist-mediated activation of α(2A)AR. We discovered that the conformational change of α(2A)AR by voltage is independent from receptor-G protein docking and regulates receptor signaling, including ß-arrestin binding, activation of G proteins, and G protein-activated inwardly rectifying K(+) currents. Comparison of the dynamics of voltage-dependence of clonidine- vs. norepinephrine-activated receptors uncovers interesting mechanistic insights. For norepinephrine, the time course of voltage-dependent deactivation reflected the deactivation kinetics of the receptor after agonist withdrawal and was strongly attenuated at saturating concentrations. In contrast, clonidine-activated α(2A)AR were switched by voltage even under fully saturating concentrations, and the kinetics of this switch was notably faster than dissociation of clonidine from α(2A)AR, indicating voltage-dependent regulation of the efficacy. We conclude that adrenergic receptors exhibit a unique, agonist-dependent mechanism of voltage-sensitivity that modulates downstream receptor signaling.
