Aldosterone increases cardiac vagal tone via G protein-coupled oestrogen receptor activation

A hallmark of cultured smooth muscle cells (SMCs) is the rapid down-regulation of several lineage-restricted genes that define their in vivo differentiated phenotype. Identifying factors that maintain an SMC differentiated phenotype has important implications in understanding the molecular underpinnings governing SMC differentiation and their subversion to an altered phenotype in various disease settings. Here, we show that several G-protein coupled receptors [α-thrombin, lysophosphatidic acid and angiotensin II (AII)] increase the expression of smooth muscle calponin (SM-Calp) in rat and human SMC. The increase in SM-Calp protein appears to be selective for G-protein-coupled receptors as epidermal growth factor was without effect. Studies using AII showed a 30-fold increase in SM-Calp protein, which was dose- and time-dependent and mediated by the angiotensin receptor-1 (AT1 receptor). The increase in SM-Calp protein with AII was attributable to transcriptional activation of SM-Calp based on increases in steady-state SM-Calp mRNA, increases in SM-Calp promoter activity and complete abrogation of protein induction with actinomycin D. To examine the potential role of extracellular signal-regulated kinase (Erk1/2), protein kinase B, p38 mitogen-activated protein kinase and protein kinase C in AII-induced SM-Calp, inhibitors to each of the signalling pathways were used. None of these signalling molecules appears to be crucial for AII-induced SM-Calp expression, although Erk1/2 may be partially involved. These results identify SM-Calp as a target of AII-mediated signalling, and suggest that the SMC response to AII may incorporate a novel activity of SM-Calp.

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A generic microfluidic biosensor of G protein-coupled receptor activation – impedance measurements of reversible morphological changes of reverse transfected HEK293 cells on microelectrodes

RSC Advances ◽

10.1039/c5ra04976h ◽

2015 ◽

Vol 5 (65) ◽

pp. 52563-52570 ◽

Cited By ~ 4

Author(s):

Saurabh K. Srivastava ◽

Rajesh Ramaneti ◽

Margriet Roelse ◽

Hien Duy Tong ◽

Elwin X. Vrouwe ◽

...

Keyword(s):

G Protein ◽

Morphological Changes ◽

Receptor Activation ◽

Membrane Receptors ◽

Hek293 Cells ◽

G Protein Coupled Receptor ◽

Impedance Measurements ◽

Transfected Cells ◽

Microfluidic Biosensor ◽

G Protein Coupled

Flowcell with micro-IDEs (250–500 μm) covered with both stable and reverse transfected cells overexpressing membrane receptors to demonstrate impedance responses to serial injections of analyte.

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Regulation of the Epithelial Na+ Channel by the RH Domain of G Protein-coupled Receptor Kinase, GRK2, and Gαq/11

Journal of Biological Chemistry ◽

10.1074/jbc.m111.239772 ◽

2011 ◽

Vol 286 (22) ◽

pp. 19259-19269 ◽

Cited By ~ 9

Author(s):

Il-Ha Lee ◽

Sung-Hee Song ◽

Craig R. Campbell ◽

Sharad Kumar ◽

David I. Cook ◽

...

Keyword(s):

G Protein ◽

Kinase Activity ◽

Receptor Activation ◽

Na Channel ◽

G Protein Signaling ◽

Protein Signaling ◽

Receptor Kinase ◽

G Protein Coupled Receptor ◽

G Protein Coupled ◽

Α Subunits

The G protein-coupled receptor kinase (GRK2) belongs to a family of protein kinases that phosphorylates agonist-activated G protein-coupled receptors, leading to G protein-receptor uncoupling and termination of G protein signaling. GRK2 also contains a regulator of G protein signaling homology (RH) domain, which selectively interacts with α-subunits of the Gq/11 family that are released during G protein-coupled receptor activation. We have previously reported that kinase activity of GRK2 up-regulates activity of the epithelial sodium channel (ENaC) in a Na+ absorptive epithelium by blocking Nedd4-2-dependent inhibition of ENaC. In the present study, we report that GRK2 also regulates ENaC by a mechanism that does not depend on its kinase activity. We show that a wild-type GRK2 (wtGRK2) and a kinase-dead GRK2 mutant (K220RGRK2), but not a GRK2 mutant that lacks the C-terminal RH domain (ΔRH-GRK2) or a GRK2 mutant that cannot interact with Gαq/11/14 (D110AGRK2), increase activity of ENaC. GRK2 up-regulates the basal activity of the channel as a consequence of its RH domain binding the α-subunits of Gq/11. We further found that expression of constitutively active Gαq/11 mutants significantly inhibits activity of ENaC. Conversely, co-expression of siRNA against Gαq/11 increases ENaC activity. The effect of Gαq on ENaC activity is not due to change in ENaC membrane expression and is independent of Nedd4-2. These findings reveal a novel mechanism by which GRK2 and Gq/11 α-subunits regulate the activity ENaC.

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Disabling Gβγ SNARE interaction in transgenic mice disrupts GPCR-mediated presynaptic inhibition leading to physiological and behavioral phenotypes

10.1101/280347 ◽

2018 ◽

Cited By ~ 2

Author(s):

Zack Zurawski ◽

Analisa D. Thompson Gray ◽

Lillian J. Brady ◽

Brian Page ◽

Emily Church ◽

...

Keyword(s):

Transgenic Mice ◽

Calcium Channels ◽

G Protein ◽

Presynaptic Inhibition ◽

Receptor Activation ◽

G Protein Coupled Receptors ◽

Voltage Gated ◽

Voltage Gated Calcium Channels ◽

Crucial Component ◽

G Protein Coupled

ABSTRACTGi/o-coupled G-protein coupled receptors modulate neurotransmission presynaptically through inhibition of exocytosis. Release of Gβγ subunits decreases the activity of voltage-gated calcium channels (VGCC), decreasing excitability. A less understood Gβγ–mediated mechanism downstream of calcium entry is the binding of Gβγ to SNARE complexes. Here, we create a mouse partially deficient in this interaction. SNAP25Δ3 homozygote animals are developmentally normalbut impaired gait and supraspinal nociception. They also have elevated stress-induced hyperthermia and impaired inhibitory postsynaptic responses to α2A-AR, but normal inhibitory postsynaptic responses to Gi/o-coupled GABAB receptor activation. SNAP25Δ3 homozygotes have deficits in inhibition of hippocampal postsynaptic responses to 5 HT1b agonists that affect hippocampal learning. These data suggest that Gi/o-coupled GPCR inhibition of exocytosis through the Gβγ-SNARE interaction is a crucial component of numerous physiological and behavioral processes.

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An Efficient Strategy to Estimate Thermodynamics and Kinetics of G Protein-Coupled Receptor Activation Using Metadynamics and Maximum Caliber

10.1101/367888 ◽

2018 ◽

Author(s):

Derya Meral ◽

Davide Provasi ◽

Marta Filizola

Keyword(s):

G Protein ◽

Adaptive Sampling ◽

Receptor Activation ◽

Conformational Space ◽

Kinetic Properties ◽

G Protein Coupled Receptor ◽

Efficient Strategy ◽

Gpcr Activation ◽

Kinetic Rates ◽

G Protein Coupled

ABSTRACTComputational strategies aimed at unveiling the thermodynamic and kinetic properties of G Protein-Coupled Receptor (GPCR) activation require extensive molecular dynamics simulations of the receptor embedded in an explicit lipid-water environment. A possible method for efficiently sampling the conformational space of such a complex system is metadynamics (MetaD) with path collective variables (CV). Here, we applied well-tempered MetaD with path CVs to one of the few GPCRs for which both inactive and fully active experimental structures are available, the μ-opioid receptor (MOR), and assessed the ability of this enhanced sampling method to estimate thermodynamic properties of receptor activation in line with those obtained by more computationally expensive adaptive sampling protocols. While n-body information theory (nBIT) analysis of these simulations confirmed that MetaD can efficiently characterize ligand-induced allosteric communication across the receptor, standard MetaD cannot be used directly to derive kinetic rates because transitions are accelerated by a bias potential. Applying the principle of Maximum Caliber (MaxCal) to the free-energy landscape of morphine-bound MOR reconstructed from MetaD, we obtained Markov State Models (MSMs) that yield kinetic rates of MOR activation in agreement with those obtained by adaptive sampling. Taken together, these results suggest that the MetaD-MaxCal combination creates an efficient strategy for estimating thermodynamic and kinetic properties of GPCR activation at an affordable computational cost.

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