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      The cooperative roles of the dopamine receptors, D 1R and D 5R, on the regulation of renal sodium transport

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          Determining the individual roles of the two dopamine D 1-like receptors (D 1R and D 5R) on sodium transport in the human renal proximal tubule has been complicated by their structural and functional similarity. Here we used a novel D 5R-selective antagonist (LE-PM436) and D 1R or D 5R-specific gene silencing to determine second messenger coupling pathways and heterologous receptor interaction between the two receptors. D 1R and D 5R co-localized in renal proximal tubule cells and physically interact, as determined by co-immunoprecipitation and FRET microscopy. Stimulation of renal proximal tubule cells with fenoldopam (D 1R/D 5R agonist) led to both adenylyl cyclase and phospholipase C (PLC) activation using real-time FRET biosensors ICUE3 and CYPHR, respectively. Fenoldopam increased cAMP accumulation and PLC activity and inhibited both NHE3 and NaKATPase activities. LE-PM436 and D 5R siRNA blocked the fenoldopam-stimulated PLC pathway but not cAMP accumulation, while D 1R siRNA blocked both fenoldopam-stimulated cAMP accumulation and PLC signaling. Either D 1R or D 5R siRNA, or LE-PM436 blocked the fenoldopam dependent inhibition of sodium transport. Further studies using the cAMP-selective D 1R/D 5R agonist SKF83822 and PLC-selective D 1R/D 5R agonist SKF83959 confirmed the cooperative influence of the two pathways on sodium transport. Thus, D 1R and D 5R interact in the inhibition of NHE3 and NaKATPase activity, the D 1R primarily by cAMP, while the D 1R/D 5R heteromer modulates the D 1R effect through a PLC pathway.

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          Most cited references 48

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          A genetically encoded fluorescent reporter reveals oscillatory phosphorylation by protein kinase C

          Signals transduced by kinases depend on the extent and duration of substrate phosphorylation. We generated genetically encoded fluorescent reporters for PKC activity that reversibly respond to stimuli activating PKC. Specifically, phosphorylation of the reporter expressed in mammalian cells causes changes in fluorescence resonance energy transfer (FRET), allowing real time imaging of phosphorylation resulting from PKC activation. Targeting of the reporter to the plasma membrane, where PKC is activated, reveals oscillatory phosphorylation in HeLa cells in response to histamine. Each oscillation in substrate phosphorylation follows a calcium oscillation with a lag of ∼10 s. Novel FRET-based reporters for PKC translocation, phosphoinositide bisphosphate conversion to IP3, and diacylglycerol show that in HeLa cells the oscillatory phosphorylations correlate with Ca2+-controlled translocation of conventional PKC to the membrane without oscillations of PLC activity or diacylglycerol. However, in MDCK cells stimulated with ATP, PLC and diacylglycerol fluctuate together with Ca2+ and phosphorylation. Thus, specificity of PKC signaling depends on the local second messenger-controlled equilibrium between kinase and phosphatase activities to result in strict calcium-controlled temporal regulation of substrate phosphorylation.
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            Cloning of the gene for a human dopamine D5 receptor with higher affinity for dopamine than D1.

            Dopamine receptors belong to a superfamily of receptors that exert their biological effects through guanine nucleotide-binding (G) proteins. Two main dopamine receptor subtypes have been identified, D1 and D2, which differ in their pharmacological and biochemical characteristics. D1 stimulates adenylyl cyclase activity, whereas D2 inhibits it. Both receptors are primary targets for drugs used to treat many psychomotor diseases, including Parkinson's disease and schizophrenia. Whereas the dopamine D1 receptor has been cloned, biochemical and behavioural data indicate that dopamine D1-like receptors exist which either are not linked to adenylyl cyclase or display different pharmacological activities. We report here the cloning of a gene encoding a 477-amino-acid protein with strong homology to the cloned D1 receptor. The receptor, called D5, binds drugs with a pharmacological profile similar to that of the cloned D1 receptor, but displays a 10-fold higher affinity for the endogenous agonist, dopamine. As with D1, the dopamine D5 receptor stimulates adenylyl cyclase activity. Northern blot and in situ hybridization analyses reveal that the receptor is neuron-specific, localized primarily within limbic regions of the brain; no messenger RNA was detected in kidney, liver, heart or parathyroid gland. The existence of a dopamine D1-like receptor with these characteristics had not been predicted and may represent an alternative pathway for dopamine-mediated events and regulation of D2 receptor activity.
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              beta2-adrenergic receptor signaling and desensitization elucidated by quantitative modeling of real time cAMP dynamics.

              G protein-coupled receptor signaling is dynamically regulated by multiple feedback mechanisms, which rapidly attenuate signals elicited by ligand stimulation, causing desensitization. The individual contributions of these mechanisms, however, are poorly understood. Here, we use an improved fluorescent biosensor for cAMP to measure second messenger dynamics stimulated by endogenous beta(2)-adrenergic receptor (beta(2)AR) in living cells. beta(2)AR stimulation with isoproterenol results in a transient pulse of cAMP, reaching a maximal concentration of approximately 10 microm and persisting for less than 5 min. We investigated the contributions of cAMP-dependent kinase, G protein-coupled receptor kinases, and beta-arrestin to the regulation of beta(2)AR signal kinetics by using small molecule inhibitors, small interfering RNAs, and mouse embryonic fibroblasts. We found that the cAMP response is restricted in duration by two distinct mechanisms in HEK-293 cells: G protein-coupled receptor kinase (GRK6)-mediated receptor phosphorylation leading to beta-arrestin mediated receptor inactivation and cAMP-dependent kinase-mediated induction of cAMP metabolism by phosphodiesterases. A mathematical model of beta(2)AR signal kinetics, fit to these data, revealed that direct receptor inactivation by cAMP-dependent kinase is insignificant but that GRK6/beta-arrestin-mediated inactivation is rapid and profound, occurring with a half-time of 70 s. This quantitative system analysis represents an important advance toward quantifying mechanisms contributing to the physiological regulation of receptor signaling.

                Author and article information

                Kidney Int
                Kidney Int.
                Kidney international
                22 February 2014
                19 February 2014
                July 2014
                01 January 2015
                : 86
                : 1
                : 118-126
                [1 ]The University of Virginia Health System, Department of Pathology, Charlottesville, VA
                [2 ]Institut für Pharmazie, Lehrstuhl für Pharmazeutische/Medizinische Chemie, FriedrichSchiller-Universität Jena, Jena, Germany
                [3 ]University of Maryland School of Medicine, Departments of Medicine and Physiology, Baltimore, MD
                Author notes
                Correspondence to be sent to: Robin A. Felder, Ph.D. The University of Virginia, P.O. Box 801400, Charlottesville, VA 22908, rfelder@ 123456virginia.edu , Ph 434-466-1131, Fax 434-924-5718



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