Melatonin Receptor Type 1 Signals to Extracellular Signal-Regulated Kinase 1 and 2 via Giand GsDually Coupled Pathways in HEK-293 Cells

Biochemistry ◽  
2014 ◽  
Vol 53 (17) ◽  
pp. 2827-2839 ◽  
Author(s):  
Linjie Chen ◽  
Xiaobai He ◽  
Yaping Zhang ◽  
Xiaopan Chen ◽  
Xiangru Lai ◽  
...  
Keyword(s):  
Receptor Type ◽  
Melatonin Receptor ◽  
Hek 293 ◽  
Extracellular Signal Regulated Kinase ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Extracellular Signal

Extracellular signal-regulated kinase activation by parathyroid hormone in distal tubule cells

2007 ◽  
Vol 292 (3) ◽  
pp. F1028-F1034 ◽  
Author(s):  
W. Bruce Sneddon ◽  
Yanmei Yang ◽  
Jianming Ba ◽  
Lisa M. Harinstein ◽  
Peter A. Friedman
Keyword(s):  
Conditioned Media ◽  
Kidney Cells ◽  
Wild Type ◽  
Egfr Transactivation ◽  
Hek 293 ◽  
Erk Activation ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Extracellular Signal

The PTH receptor (PTH1R) activates multiple signaling pathways, including extracellular signal-regulated kinases 1 and 2 (ERK1/2). The role of epidermal growth factor receptor (EGFR) transactivation in ERK1/2 activation by PTH in distal kidney cells, a primary site of PTH action, was characterized. ERK1/2 phosphorylation was stimulated by PTH and blocked by the EGFR inhibitor, AG1478. Upon PTH stimulation, metalloprotease cleavage of membrane-bound heparin-binding fragment (HB-EGF) induced EGFR transactivation of ERK. Conditioned media from PTH-treated distal kidney cells activated ERK in HEK-293 cells. AG1478 added to HEK-293 cells ablated transactivation by conditioned media. HB-EGF directly activated ERK1/2 in HEK-293 cells. Pretreatment of distal kidney cells with the metalloprotease inhibitor GM-6001 abolished transactivation of ERK1/2 by PTH. The role of the PTH1R COOH terminus in PTX-sensitive ERK1/2 activation was characterized in HEK-293 cells transfected with wild-type PTH1R, with a PTH1R mutated at its COOH terminus, or with PTH1R truncated at position 480. PTH stimulated ERK by wild-type, mutated and truncated PTH1Rs 21-, 27- and 57-fold, respectively. Thus, the PTH1R COOH terminus exerts an inhibitory effect on ERK activation. EBP50, a scaffolding protein that binds to the PDZ recognition domain of the PTH1R, impaired PTH but not isoproterenol or calcitonin-induced ERK activation. Pertussis toxin inhibited PTH-stimulated ERK1/2 by mutated and truncated PTH1Rs and abolished ERK1/2 activation by wild-type PTH1R. We conclude that ERK phosphorylation in distal kidney cells by PTH requires PTH1R activation of Gi, which leads to stimulation of metalloprotease-mediated cleavage of HB-EGF and transactivation of the EGFR and is regulated by EBP50.

scholarly journals Characterization of a human 5-hydroxytryptamine3 receptor type A (h5-HT3R-AS) subunit stably expressed in HEK 293 cells

1996 ◽  
Vol 118 (5) ◽  
pp. 1237-1245 ◽  
Author(s):  
Anthony G. Hope ◽  
John A. Peters ◽  
Angus M. Brown ◽  
Jeremy J. Lambert ◽  
Thomas P. Blackburn
Keyword(s):  
Type A ◽  
Receptor Type ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

scholarly journals P2X7 receptor activates extracellular signal-regulated kinases ERK1 and ERK2 independently of Ca2+ influx

2003 ◽  
Vol 374 (1) ◽  
pp. 51-61 ◽  
Author(s):  
Jan AMSTRUP ◽  
Ivana NOVAK
Keyword(s):  
P2x7 Receptor ◽  
Fluorescent Protein ◽  
Expression System ◽  
Receptor Expression ◽  
Nucleotide Receptors ◽  
P2x7 Receptors ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Extracellular Signal

P2X7 nucleotide receptors modulate a spectrum of cellular events in various cells including epithelia, such as exocrine pancreas. Although the pharmacology and channel properties of the P2X7 receptors have been studied intensively, signal transduction pathways are relatively unknown. In this study we applied a heterologous expression system of rat P2X7 receptors in HEK-293 cells. We followed the receptor expression and function using the enhanced green fluorescent protein (EGFP) tag, activation of intracellular proteins and increases in cellular Ca2+. EGFP-P2X7 receptors localized to the plasma membrane, clusters within the membrane and intracellularly. Stimulation of P2X7 receptors in HEK-293 cells led to an activation of extracellular signal-regulated kinases ERK1 and ERK2 and this activation was seen after just 1 min of stimulation with ATP. Using C- and N-terminal P2X7-receptor mutants we show that the N-terminus is important in activation of ERKs, whereas deletion of the last 230 amino acids in the C-terminus did not effect ERK activation. On the other hand, Ca2+ entry was impaired in C-terminal but not in N-terminal mutants. In cell suspensions prepared from rat pancreas we show that P2X7 receptors also activate ERK1 and ERK2, indicating that these signalling pathways are also turned on in native epithelium.

scholarly journals Activation of vanilloid receptor type I in the endoplasmic reticulum fails to activate store-operated Ca2+ entry

2003 ◽  
Vol 372 (2) ◽  
pp. 517-528 ◽  
Author(s):  
Brian J. WISNOSKEY ◽  
William G. SINKINS ◽  
William P. SCHILLING
Keyword(s):  
Endoplasmic Reticulum ◽  
Recombinant Baculovirus ◽  
Vanilloid Receptor ◽  
Confocal Imaging ◽  
Receptor Type ◽  
Type I ◽  
Hek 293 ◽  
293 Cells ◽  
Endoplasmic Reticulum Targeting

To evaluate interaction of vanilloid receptor type 1 (TRPV1) with endogenous Ca2+ signalling mechanisms, TRPV1 was expressed in Spodoptera frugiperda (Sf 9) insect cells using recombinant baculovirus. Stimulation of TRPV1-expressing cells, but not control Sf 9 cells, with resiniferatoxin (RTX), capsaicin or anandamide, produced an increase in cytosolic free Ca2+ concentration ([Ca2+]i), with EC50 values of 166 pM, 24.5 nM and 3.89 μM respectively. In the absence of extracellular Ca2+, both capsaicin and RTX caused an increase in [Ca2+]i with EC50 values of approx. 10 μM and 10 nM respectively. This TRPV1-induced release of Ca2+ from intracellular stores was not blocked by U73122, suggesting that phospholipase C was not involved. Substantial overlap was found between the thapsigargin- and RTX-sensitive internal Ca2+ pools, and confocal imaging showed that intracellular TRPV1 immunofluorescence co-localized with the endoplasmic reticulum targeting motif KDEL. To determine if TRPV1-induced mobilization of intracellular Ca2+ activates endogenous store-operated Ca2+ entry, the effect of 2-aminoethoxydiphenyl borate (2-APB) on Ba2+ influx was examined. 2-APB blocked thapsigargin-induced Ba2+ influx, but not RTX-induced Ba2+ entry. In the combined presence of thapsigargin and a store-releasing concentration of RTX, the 2-APB-sensitive component was essentially identical with the thapsigargin-induced component. Similar results were obtained in HEK-293 cells stably expressing TRPV1. These results suggest that TRPV1 forms agonist-sensitive channels in the endoplasmic reticulum, which when activated, release Ca2+ from internal stores, but fail to activate endogenous store-operated Ca2+ entry. Selective activation of intracellular TRPV1, without concomitant involvement of plasmalemmal Ca2+ influx mechanisms, could play an important role in Ca2+ signalling within specific subcellular microdomains.

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

Genetic Variability of the CLEC4M Endothelial Lectin Receptor Modulates Binding and Internalization of Von Willebrand Factor and Contributes to Variance in Plasma VWF Levels

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 16-16
Author(s):  
Laura L. Swystun ◽  
Natalia Rydz ◽  
Colleen Notley ◽  
Jonathan J. Riches ◽  
Andrew D Paterson ◽  
...  
Keyword(s):  
Genetic Variability ◽  
Tandem Repeats ◽  
Type I ◽  
Dependent Manner ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Abstract 16 Type 1 von Willebrand's Disease (VWD) can result from decreased synthesis or accelerated clearance of von Willebrand Factor (VWF), resulting in partial quantitative deficiency. Approximately 35% of individuals with Type 1 VWD do not have a putative mutation in their VWF gene, suggesting that genes other than VWF may contribute to the pathophysiology of this disease. Recently, the CHARGE GWAS meta-analysis identified single nucleotide polymorphisms in the gene encoding the C-type lectin domain family 4 member M (CLEC4M) as being significantly associated with plasma VWF levels in normal individuals. CLEC4M is a pathogen recognition receptor with a polymorphic extracellular neck region consisting of a variable number of tandem repeats (VNTR) (3 – 9 repeats). We hypothesize that CLEC4M binds to and clears VWF from the circulation, and that different CLEC4M VNTR alleles may contribute to differences in plasma levels of VWF in normal subjects and patients with Type 1 VWD. Previously, genotyping of 555 subjects (196 cases with type 1 VWD, and 362 family members) for the CLEC4M VNTR number showed that the most frequently documented alleles were VNTR 5 (29%), 6 (15%), and 7 (53%). Family-based association analysis on kindreds with type I VWD has demonstrated a significant excess transmission of VNTR 6 to the type I VWD phenotype (p=0.005) and an association of this VNTR allele with VWF:RCo (p=0.037). In the present studies, we have complemented this genetic association data with experiments to directly evaluate the ability of CLEC4M to bind and internalize VWF. Further, we characterized the ability of different CLEC4M VNTR alleles to facilitate VWF clearance. Binding of VWF to CLEC4M was assessed with a modified ELISA using a recombinant CLEC4M-Fc chimera. CLEC4M-Fc bound to Humate P (plasma-derived VWF-FVIII) in a dose-dependent manner. CLEC4M-Fc also bound to recombinant human VWF, and factor VIII-free plasma-derived VWF. CLEC4M-Fc demonstrated a 70% increase in binding to de-O-glycosylated Humate P (p=0.041), and a 75% decrease in binding to de-N-glycosylated Humate P (p=0.046) relative to controls. Additionally, pre-incubation of CLEC4M-Fc with the polysaccharide mannan attenuated binding to all VWF preparations by approximately 50%. Binding and internalization of VWF by HEK 293 cells stably expressing CLEC4M (VNTR allele 7) was assessed with immunofluorescence and ELISA. Binding of VWF co-localized with CLEC4M expression on HEK 293 cells. CLEC4M and VWF co-localized with early endosomal antigen-1, suggesting that CLEC4M participates in receptor-mediated endocytosis of VWF. CLEC4M-expressing cells bound and internalized VWF in a dose- and time-dependent manner relative to controls. Preincubation of CLEC4M expressing cells with mannan inhibited VWF binding and internalization by 50% (p=0.0088). The contribution of CLEC4M genetic variability to VWF binding and internalization was measured using HEK 293 cells expressing CLEC4M with 4, 6, 7, and 9 tandem repeats. Decreased binding and internalization of VWF was observed with cells expressing CLEC4M 4 and 9 tandem repeat constructs as compared to CLEC4M with 7 tandem repeats (CLEC4M 4 – 60% reduction, p < 0.001; CLEC4M 9 – 45% reduction, p=0.006). Cells expressing the CLEC4M VNTR combination 4 and 9, had a 55% decrease in binding and internalization of VWF relative to cells expressing CLEC4M with 7 VNTRs (p < 0.001). These VNTR associated differences in VWF binding/internalization were not accounted for by variances in the CLEC4M expression levels in the transfected HEK 293 cells. These studies demonstrate that the C-type lectin CLEC4M binds to and internalizes VWF through an N-glycan-dependent mechanism. Additionally, it provides further evidence that polymorphisms in the CLEC4M gene contribute to quantitative VWF deficiency. Disclosures: Montgomery: Gen-Probe/GTI Diagnostics: Consultancy; CSL Behring: Consultancy; Octapharma: Consultancy. James:CSL-Behring, Baxter, Bayer: Honoraria, Research Funding.

scholarly journals c-Src Regulates Clathrin Adapter Protein 2 Interaction with β-Arrestin and the Angiotensin II Type 1 Receptor during Clathrin- Mediated Internalization

2005 ◽  
Vol 19 (2) ◽  
pp. 491-503 ◽  
Author(s):  
Delphine Fessart ◽  
May Simaan ◽  
Stéphane A. Laporte
Keyword(s):  
Angiotensin Ii ◽  
Src Kinase ◽  
Receptor Internalization ◽  
Ang Ii ◽  
Coated Pits ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

Abstract β-Arrestins are multifunctional adapters involved in the internalization and signaling of G protein-coupled receptors (GPCRs). They target receptors to clathrin-coated pits (CCPs) through binding with clathrin and clathrin adapter 2 (AP-2) complex. They also act as transducers of signaling by recruiting c-Src kinase to certain GPCRs. Here we sought to determine whether c-Src regulates the recruitment of AP-2 to β-arrestin and the angiotensin II (Ang II) type 1 receptor (AT1R) during internalization. We show that the agonist stimulation of native AT1R in vascular smooth muscle cells (VSMCs) induces the formation of an endogenous complex containing c-Src, β-arrestins and AP-2. In vitro studies using coimmunoprecipitation experiments and a yeast three-hybrid assay reveal that c-Src stabilizes the agonist-independent association between β-arrestin2 and the β-subunit of AP-2 independently of the kinase activity of c-Src. However, although c-Src expression promoted the rapid dissociation of AP-2 from both β-arrestin and AT1R after receptor stimulation, a kinase-inactive mutant of c-Src failed to induce the dissociation of AP-2 from the agonist-occupied receptor. Thus, the consequence of c-Src in regulating the dissociation of AP-2 from the receptor was also examined on the internalization of AT1R by depleting c-Src in human embryonic kidney (HEK) 293 cells using a small interfering RNA strategy. Experiments in c-Src depleted cells reveal that AT1R remained mostly colocalized with AP-2 at the plasma membrane after Ang II stimulation, consistent with the observed delay in receptor internalization. Moreover, coimmunoprecipitation experiments in c-Src depleted HEK 293 cells and VSMCs showed an increased association of AP-2 to the agonist-occupied AT1R and β-arrestin, respectively. Together, our results support a role for c-Src in regulating the dissociation of AP-2 from agonist-occupied AT1R and β-arrestin during the clathrin-mediated internalization of receptors and suggest a novel function for c-Src kinase in the internalization of AT1R.

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