Activation of ERK, JNK, Akt, and G-protein coupled signaling by hybrid angiotensin II AT1/bradykinin B2 receptors expressed in HEK-293 cells

2007 ◽  
Vol 101 (1) ◽  
pp. 192-204 ◽  
Author(s):  
Jun Yu ◽  
David Lubinsky ◽  
Natia Tsomaia ◽  
Zhenhua Huang ◽  
Linda Taylor ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
G Protein ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
B2 Receptors ◽  
G Protein Coupled

scholarly journals Activation of the Novel Estrogen Receptor G Protein-Coupled Receptor 30 (GPR30) at the Plasma Membrane

Endocrinology ◽  
2007 ◽  
Vol 148 (7) ◽  
pp. 3236-3245 ◽  
Author(s):  
E. Filardo ◽  
J. Quinn ◽  
Y. Pang ◽  
C. Graeber ◽  
S. Shaw ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
G Protein ◽  
Intracellular Camp ◽  
Calcium Stores ◽  
G Protein Coupled Receptor ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
G Protein Coupled

G protein-coupled receptor 30 (GPR30), a seven-transmembrane receptor (7TMR), is associated with rapid estrogen-dependent, G protein signaling and specific estrogen binding. At present, the subcellular site of GPR30 action is unclear. Previous studies using antibodies and fluorochrome-labeled estradiol (E2) have failed to detect GPR30 on the cell surface, suggesting that GPR30 may function uniquely among 7TMRs as an intracellular receptor. Here, we show that detectable expression of GPR30 on the surface of transfected HEK-293 cells can be selected by fluorescence-activated cell sorting. Expression of GPR30 on the cell surface was confirmed by confocal microscopy using the lectin concanavalin A as a plasma membrane marker. Stimulation of GPR30-expressing HEK-293 cells with 17β-E2 caused sequestration of GPR30 from the cell surface and resulted in its codistribution with clathrin and mobilization of intracellular calcium stores. Evidence that GPR30 signals from the cell surface was obtained from experiments demonstrating that the cell-impermeable E2-protein conjugates E2-BSA and E2-horseradish peroxidase promote GPR30-dependent elevation of intracellular cAMP concentrations. Subcellular fractionation studies further support the plasma membrane as a site of GPR30 action with specific [3H]17β-E2 binding and G protein activation associated with plasma membrane but not microsomal, or other fractions, prepared from HEK-293 or SKBR3 breast cancer cells. These results suggest that GPR30, like other 7TMRs, functions as a plasma membrane receptor.

G protein-coupled receptor kinase (GRK) specificity of endogenous type 1 vasoactive intestinal polvpeptide (VIP) receptor expressed on the surface of HEK 293 cells

2000 ◽  
Vol 118 (4) ◽  
pp. A309
Author(s):  
Michael A. Shetzline ◽  
Julia K. Walker ◽  
Brian M. Curtin ◽  
Richard T. Premont ◽  
Marc G Caron
Keyword(s):  
G Protein ◽  
Receptor Kinase ◽  
G Protein Coupled Receptor ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
G Protein Coupled ◽  
Vip Receptor

scholarly journals Adenine nucleotides inhibit recombinant N-type calcium channels via G protein-coupled mechanisms in HEK 293 cells; involvement of the P2Y13 receptor-type

2004 ◽  
Vol 141 (1) ◽  
pp. 141-151 ◽  
Author(s):  
Kerstin Wirkner ◽  
Joana Schweigel ◽  
Zoltan Gerevich ◽  
Heike Franke ◽  
Clemens Allgaier ◽  
...  
Keyword(s):  
Calcium Channels ◽  
G Protein ◽  
Adenine Nucleotides ◽  
Receptor Type ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
G Protein Coupled

Quantification of G-Protein Coupled Receptor Internalization Using G-Protein Coupled Receptor-Green Fluorescent Protein Conjugates with the ArrayScan™ High-Content Screening System

1999 ◽  
Vol 4 (2) ◽  
pp. 75-86 ◽  
Author(s):  
Bruce R. Conway ◽  
Lisa K. Minor ◽  
Jun Z. Xu ◽  
Joseph W. Gunnet ◽  
Robbin DeBiasio ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
G Protein ◽  
Fluorescent Protein ◽  
Receptor Internalization ◽  
Hek 293 ◽  
Whole Cells ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Green Fluorescent ◽  
G Protein Coupled

Many G-protein coupled receptors (GPCRs) undergo ligand-dependent homologous desensitization and internalization. Desensitization, defined as a decrease in the responsiveness to ligand, is accompanied by receptor aggregation on the cell surface and internalization via clathrin-coated pits to an intracellular endosomal compartment. In this study, we have taken advantage of the trafficking properties of GPCRs to develop a useful screening method for the identification of receptor mimetics. A series of studies were undertaken to evaluate the expression, functionality, and ligand-dependent trafficking of GPCR-green fluorescent protein (GFP) fusion conjugates stably transfected into HEK 293 cells. These GPCR-GFP expressing cells were then utilized in the validation of the ArrayScan™ (Cellomics™, Pittsburgh, PA), a microtiter plate imaging system that permits cellular and subcellular quantitation of fluorescence in whole cells. These studies demonstrated our ability to measure the internalization of a parathy-roid hormone (PTH) receptor-GFP conjugate after ligand treatment by spatially resolving internalized receptors. Internalization was time- and dose-dependent and appeared to be selective for PTH. Similar results were obtained for a β2-adrenergic receptor (β2 AR)-GFP conjugate stably expressed in HEK 293 cells. The internalized GFP-labeled receptors were visualized as numerous punctate "spots" within the cell interior. An algorithm has been developed that identifies and collects information about these spots, allowing quantification of the internalization process. Variables such as the receptor-GFP expression level, plating density, cell number per field, number of fields scanned per well, spot size, and spot intensity were evaluated during the development of this assay. The method represents a valuable tool to screen for receptor mimetics and antagonists of receptor internalization in whole cells rapidly.

scholarly journals Bursts of action potential waveforms relieve G-protein inhibition of recombinant P/Q-type Ca2+ channels in HEK 293 cells.

1997 ◽  
Vol 499 (3) ◽  
pp. 637-644 ◽  
Author(s):  
D L Brody ◽  
P G Patil ◽  
J G Mulle ◽  
T P Snutch ◽  
D T Yue
Keyword(s):  
Action Potential ◽  
G Protein ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
Protein Inhibition ◽  
293 Cells ◽  
Ca2 Channels

scholarly journals BRET Analysis of GPCR Dimers in Neurons and Non-Neuronal Cells: Evidence for Inactive, Agonist, and Constitutive Conformations

2021 ◽  
Vol 22 (19) ◽  
pp. 10638
Author(s):  
Chayma El Khamlichi ◽  
Laetitia Cobret ◽  
Jean-Michel Arrang ◽  
Séverine Morisset-Lopez
Keyword(s):  
Cortical Neurons ◽  
G Protein Coupled Receptors ◽  
Constitutive Activity ◽  
Western Blots ◽  
Inverse Agonists ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Functional Consequences ◽  
G Protein Coupled

G-protein-coupled receptors (GPCRs) are dimeric proteins, but the functional consequences of the process are still debated. Active GPCR conformations are promoted either by agonists or constitutive activity. Inverse agonists decrease constitutive activity by promoting inactive conformations. The histamine H3 receptor (H3R) is the target of choice for the study of GPCRs because it displays high constitutive activity. Here, we study the dimerization of recombinant and brain H3R and explore the effects of H3R ligands of different intrinsic efficacy on dimerization. Co-immunoprecipitations and Western blots showed that H3R dimers co-exist with monomers in transfected HEK 293 cells and in rodent brains. Bioluminescence energy transfer (BRET) analysis confirmed the existence of spontaneous H3R dimers, not only in living HEK 293 cells but also in transfected cortical neurons. In both cells, agonists and constitutive activity of the H3R decreased BRET signals, whereas inverse agonists and GTPγS, which promote inactive conformations, increased BRET signals. These findings show the existence of spontaneous H3R dimers not only in heterologous systems but also in native tissues, which are able to adopt a number of allosteric conformations, from more inactive to more active states.

Voltage-dependent facilitation of cardiac L-type Ca channels expressed in HEK-293 cells requires β-subunit

2000 ◽  
Vol 278 (1) ◽  
pp. H126-H136 ◽  
Author(s):  
Timothy J. Kamp ◽  
Hai Hu ◽  
Eduardo Marban
Keyword(s):  
G Protein ◽  
Somatostatin Receptors ◽  
Ca Channel ◽  
Β Subunit ◽  
Ca Channels ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
Protein Inhibition ◽  
293 Cells ◽  
Voltage Dependent

The activity of native L-type Ca channels can be facilitated by strong depolarizations. The cardiac Ca channel α1C-subunit was transiently expressed in human embryonic kidney (HEK-293) cells, but these channels did not exhibit voltage-dependent facilitation. Coexpression of the Ca channel β1a- or β2a-subunit with the α1C-subunit enabled voltage-dependent facilitation in 40% of cells tested. The onset of facilitation in α1C + β1a-expressing HEK-293 cells was rapid after a depolarization to +100 mV (τ = 7.0 ms). The kinetic features of the facilitated currents were comparable to those observed for voltage-dependent relief of G protein inhibition demonstrated for many neuronal Ca channels; however, intracellular dialysis with guanosine 5′- O-(2-thiodiphosphate) and guanosine 5′- O-(3-thiotriphosphate) in the patch pipette had no effect on facilitation. Stimulation of G protein-coupled receptors, either endogenous (somatostatin receptors) or coexpressed (adenosine A1receptors), did not affect voltage-dependent facilitation. These results indicate that the cardiac Ca channel α1C-subunit can exhibit voltage-dependent facilitation in HEK-293 cells only when coexpressed with an auxiliary β-subunit and that this facilitation is independent of G protein pathways.

The constitutively active N111G-AT1 receptor for angiotensin II modifies the morphology and cytoskeletal organization of HEK-293 cells

2005 ◽  
Vol 308 (1) ◽  
pp. 188-195 ◽  
Author(s):  
Mannix Auger-Messier ◽  
Eric S. Turgeon ◽  
Richard Leduc ◽  
Emanuel Escher ◽  
Gaetan Guillemette
Keyword(s):  
Angiotensin Ii ◽  
At1 Receptor ◽  
Cytoskeletal Organization ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Constitutively Active
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