scholarly journals Dominance of Gs in doubly Gs/Gi-coupled chimaeric A1/A2A adenosine receptors in HEK-293 cells

2000 ◽  
Vol 352 (1) ◽  
pp. 203-210 ◽  
Author(s):  
Amy L. TUCKER ◽  
LiGuo JIA ◽  
Diane HOLETON ◽  
Allen J. TAYLOR ◽  
Joel LINDEN
Keyword(s):  
Adenylate Cyclase ◽  
G Proteins ◽  
Adenosine Receptors ◽  
Wild Type ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
A2a Receptors ◽  
Recombinant Receptors ◽  
C Terminus ◽  
293 Cells

A1 adenosine receptors inhibit adenylate cyclase by activating Gi/Go, whereas A2A receptors activate Gs. We examined how regions of A1 and A2A receptors regulate coupling to G-proteins by constructing chimaeras in which the third intracellular loops (3ICL or L) and/or the C-termini (or T) were switched. Pertussis toxin (PTX) was used in membrane radioligand binding assays to calculate the fraction of recombinant receptors coupled to Gi/Go and in whole cells to differentially influence agonist-stimulated cAMP accumulation. Switching A1/A2A 3ICL domains results in receptors that maintain binding selectivity for ligands but are doubly coupled. Receptor chimaeras with an A1 3ICL sequence (A2A/A1L or A2A/A1LT) respond to agonist stimulation with elevated cAMP despite being coupled predominantly to Gi/Go. These chimaeras have basal cAMP levels lower than those of wild-type A2A receptors, similar to wild-type A1 receptors. The A1 C-terminus modulates the coupling of receptors with A1 3ICL such that A2A/A1LT is better coupled to Gi/Go than A2A/A1L. The C-terminus has little impact on coupling to receptors containing A2A 3ICL sequence. Our results show that the C-terminus sequence selectively facilitates coupling to Gi/Go mediated by A1 3ICL and not by other intracellular domains that favour Gi coupling. The C-terminus sequence has little or no effect on coupling to Gs. For doubly Gs/Gi-coupled adenosine receptors in HEK-293 cells, Gs-mediated stimulation predominates over Gi/Go-mediated inhibition of adenylate cyclase. We discuss the signalling consequences of simultaneously activating opposing G-proteins within single cells.

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.

Effect of human acetylcholinesterase subunit assembly on its circulatory residence

2001 ◽  
Vol 354 (3) ◽  
pp. 613-625 ◽  
Author(s):  
Theodor CHITLARU ◽  
Chanoch KRONMAN ◽  
Baruch VELAN ◽  
Avigdor SHAFFERMAN
Keyword(s):  
Laser Desorption Ionization ◽  
Wild Type ◽  
Ionization Time ◽  
Hek 293 ◽  
Enzyme Preparations ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Differential Behaviour ◽  
Oligomeric Forms

Sialylated recombinant human acetylcholinesterase (rHuAChE), produced by stably transfected cells, is composed of a mixed population of monomers, dimers and tetramers and manifests a time-dependent circulatory enrichment of the higher-order oligomeric forms. To investigate this phenomenon further, homogeneous preparations of rHuAChE differing in their oligomerization statuses were generated: (1) monomers, represented by the oligomerization-impaired C580A-rHuAChE mutant, (2) wild-type (WT) dimers and (3) tetramers of WT-rHuAChE generated in vitro by complexation with a synthetic ColQ-derived proline-rich attachment domain (‘PRAD’) peptide. Three different series of each of these three oligoform preparations were produced: (1) partly sialylated, derived from HEK-293 cells; (2) fully sialylated, derived from engineered HEK-293 cells expressing high levels of sialyltransferase; and (3) desialylated, after treatment with sialidase to remove sialic acid termini quantitatively. The oligosaccharides associated with each of the various preparations were extensively analysed by matrix-assisted laser desorption ionization–time-of-flight MS. With the enzyme preparations comprising the fully sialylated series, a clear linear relationship between oligomerization and circulatory mean residence time (MRT) was observed. Thus monomers, dimers and tetramers exhibited MRTs of 110, 195 and 740min respectively. As the level of sialylation decreased, this differential behaviour became less pronounced; eventually, after desialylation all oligoforms had the same MRT (5min). These observations suggest that multiple removal systems contribute to the elimination of AChE from the circulation. Here we also demonstrate that by the combined modulation of sialylation and tetramerization it is possible to generate a rHuAChE displaying a circulatory residence exceeding that of all other known forms of native or recombinant human AChE.

scholarly journals β-Arrestin mediates oxytocin receptor signaling, which regulates uterine contractility and cellular migration

2011 ◽  
Vol 300 (3) ◽  
pp. E468-E477 ◽  
Author(s):  
Chad A. Grotegut ◽  
Liping Feng ◽  
Lan Mao ◽  
R. Phillips Heine ◽  
Amy P. Murtha ◽  
...  
Keyword(s):  
Knockout Mice ◽  
Mapk Signaling ◽  
Oxytocin Receptor ◽  
Cellular Migration ◽  
Wild Type ◽  
Uterine Contractility ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
Uterine Muscle ◽  
293 Cells

Desensitization of the oxytocin receptor (OXTR) in the setting of prolonged oxytocin exposure may lead to dysfunctional labor, which increases the risk for cesarean delivery, and uterine atony, which may result in postpartum hemorrhage. The molecular mechanism for OXTR desensitization is through the agonist-mediated recruitment of the multifunctional protein β-arrestin. In addition to its desensitizing function, β-arrestins have recently been shown to simultaneously activate downstream signaling. We tested whether oxytocin stimulation promotes β-arrestin-mediated OXTR desensitization in vivo and activates β-arrestin-mediated mitogen-activated protein kinase (MAPK) growth signaling. Uterine muscle strips isolated from wild-type mice exhibited diminished uterine contractility following repeated exposure to oxytocin, whereas uterine muscle strips from β-arrestin-1 and β-arrestin-2 knockout mice showed no desensitization. Utilizing siRNA knockdown of β-arrestin-1 and β-arrestin-2 in HEK-293 cells expressing the OXTR, we demonstrated oxytocin-mediated MAPK signaling that was dependent on β-arrestin-1 and β-arrestin-2. Wild-type and β-arrestin-1 and β-arrestin-2 knockout mice receiving intravenous oxytocin also demonstrated oxytocin-mediated MAPK signaling that was dependent on β-arrestin-1 and β-arrestin-2. Finally, to test the significance of β-arrestin-mediated signaling from the OXTR, HEK-293 cells expressing the OXTR showed β-arrestin-dependent proliferation in a cell migration assay following oxytocin treatment. In conclusion, β-arrestin is a multifunctional scaffold protein that mediates both desensitization of the OXTR, leading to decreases in uterine contractility, and MAPK growth signaling following stimulation by oxytocin. The development of unique OXTR ligands that prevent receptor desensitization may be a novel approach in the treatment of adverse clinical events secondary to prolonged oxytocin therapy.

scholarly journals Dominance of Gs in doubly Gs/Gi-coupled chimaeric A1/A2A adenosine receptors in HEK-293 cells

2000 ◽  
Vol 352 (1) ◽  
pp. 203 ◽  
Author(s):  
Amy L. TUCKER ◽  
LiGuo JIA ◽  
Diane HOLETON ◽  
Allen J. TAYLOR ◽  
Joel LINDEN
Keyword(s):  
Adenosine Receptors ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
A2a Adenosine Receptors ◽  
293 Cells

scholarly journals K201 (JTV519) suppresses spontaneous Ca2+ release and [3H]ryanodine binding to RyR2 irrespective of FKBP12.6 association

2007 ◽  
Vol 404 (3) ◽  
pp. 431-438 ◽  
Author(s):  
Donald J. Hunt ◽  
Peter P. Jones ◽  
Ruiwu Wang ◽  
Wenqian Chen ◽  
Jeff Bolstad ◽  
...  
Keyword(s):  
Ventricular Myocytes ◽  
Dependent Manner ◽  
Wild Type ◽  
Concentration Dependent Manner ◽  
Hek 293 ◽  
Human Embryonic Kidney Cells ◽  
Hek 293 Cells ◽  
Fk506 Binding Protein ◽  
293 Cells

K201 (JTV519), a benzothiazepine derivative, has been shown to possess anti-arrhythmic and cardioprotective properties, but the mechanism of its action is both complex and controversial. It is believed to stabilize the closed state of the RyR2 (cardiac ryanodine receptor) by increasing its affinity for the FKBP12.6 (12.6 kDa FK506 binding protein) [Wehrens, Lehnart, Reiken, Deng, Vest, Cervantes, Coromilas, Landry and Marks (2004) Science 304, 292–296]. In the present study, we investigated the effect of K201 on spontaneous Ca2+ release induced by Ca2+ overload in rat ventricular myocytes and in HEK-293 cells (human embryonic kidney cells) expressing RyR2 and the role of FKBP12.6 in the action of K201. We found that K201 abolished spontaneous Ca2+ release in cardiac myocytes in a concentration-dependent manner. Treating ventricular myocytes with FK506 to dissociate FKBP12.6 from RyR2 did not affect the suppression of spontaneous Ca2+ release by K201. Similarly, K201 was able to suppress spontaneous Ca2+ release in FK506-treated HEK-293 cells co-expressing RyR2 and FKBP12.6. Furthermore, K201 suppressed spontaneous Ca2+ release in HEK-293 cells expressing RyR2 alone and in cells co-expressing RyR2 and FKBP12.6 with the same potency. In addition, K201 inhibited [3H]ryanodine binding to RyR2-wt (wild-type) and an RyR2 mutant linked to ventricular tachycardia and sudden death, N4104K, in the absence of FKBP12.6. These observations demonstrate that FKBP12.6 is not involved in the inhibitory action of K201 on spontaneous Ca2+ release. Our results also suggest that suppression of spontaneous Ca2+ release and the activity of RyR2 contributes, at least in part, to the anti-arrhythmic properties of K201.

scholarly journals Wild-type and mutant ferroportins do not form oligomers in transfected cells

2006 ◽  
Vol 396 (2) ◽  
pp. 265-275 ◽  
Author(s):  
Ana Sofia Gonçalves ◽  
Françoise Muzeau ◽  
Rand Blaybel ◽  
Gilles Hetet ◽  
Fathi Driss ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Human Kidney ◽  
Cell Types ◽  
Dominant Negative ◽  
Wild Type ◽  
Dominant Form ◽  
Hek 293 ◽  
C Terminus ◽  
293 Cells ◽  
Iron Export

Ferroportin [FPN; Slc40a1 (solute carrier family 40, member 1)] is a transmembrane iron export protein expressed in macrophages and duodenal enterocytes. Heterozygous mutations in the FPN gene result in an autosomal dominant form of iron overload disorder, type-4 haemochromatosis. FPN mutants either have a normal iron export activity but have lost their ability to bind hepcidin, or are defective in their iron export function. The mutant protein has been suggested to act as a dominant negative over the wt (wild-type) protein by multimer formation. Using transiently transfected human epithelial cell lines expressing mouse FPN modified by the addition of a haemagglutinin or c-Myc epitope at the C-terminus, we show that the wtFPN is found at the plasma membrane and in Rab5-containing endosomes, as are the D157G and Q182H mutants. However, the delV162 mutant is mostly intracellular in HK2 cells (human kidney-2 cells) and partially addressed at the cell surface in HEK-293 cells (human embryonic kidney 293 cells). In both cell types, it is partially associated with the endoplasmic reticulum and with Rab5-positive vesicles. However, this mutant is complex-glycosylated like the wt protein. D157G and G323V mutants have a defective iron export capacity as judged by their inability to deplete the intracellular ferritin content, whereas Q182H and delV162 have normal iron export function and probably have lost their capacity to bind hepcidin. In co-transfection experiments, the delV162 mutant does not co-localize with the wtFPN, does not prevent its normal targeting to the plasma membrane and cannot be immunoprecipitated in the same complex, arguing against the formation of FPN hetero-oligomers.

The human 5-ht5A receptor couples to Gi/Go proteins and inhibits adenylate cyclase in HEK 293 cells

1998 ◽  
Vol 361 (2-3) ◽  
pp. 299-309 ◽  
Author(s):  
Bart J.B. Francken ◽  
Mirek Jurzak ◽  
Jurgen F.M. Vanhauwe ◽  
Walter H.M.L. Luyten ◽  
Josée E. Leysen
Keyword(s):  
Adenylate Cyclase ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

scholarly journals Rare mutations in the human Na-K-Cl cotransporter (NKCC2) associated with lower blood pressure exhibit impaired processing and transport function

2011 ◽  
Vol 300 (4) ◽  
pp. F840-F847 ◽  
Author(s):  
Michelle Y. Monette ◽  
Jesse Rinehart ◽  
Richard P. Lifton ◽  
Biff Forbush
Keyword(s):  
Blood Pressure ◽  
Thick Ascending Limb ◽  
Transport Function ◽  
Lower Blood Pressure ◽  
Wild Type ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
Hek Cells ◽  
293 Cells ◽  
Lower Blood

The Na-K-Cl cotransporter (NKCC2) is the major salt transport pathway in the thick ascending limb of Henle's loop and is part of the molecular mechanism for blood pressure regulation. Recent screening of ∼3,000 members of the Framingham Heart Study identified nine rare independent mutations in the gene encoding NKCC2 (SLC12A1) associated with clinically reduced blood pressure and protection from hypertension (Ji WZ, Foo JN, O'Roak BJ, Zhao H, Larson MG, Simon DB, Newton-Cheh C, State M, Levy D, Lifton RP. Nat Genet 40: 592–599, 2008). To investigate their functional consequences, we introduced the nine mutations in human NKCC2A and examined protein function, expression, localization, regulation, and ion transport kinetics using heterologous expression in Xenopus laevis oocytes and HEK-293 cells. When expressed in oocytes, four of the mutants (T235M, R302W, L505V, and P569H) exhibited reduced transport function compared with wild-type. In HEK-293 cells, the same four mutants exhibited reduced function, and in addition N399S and P1083A had significantly lower activity than wild-type. The two most functionally impaired mutants (R302W and L505V) exhibited dramatically diminished production of complex-glycosylated protein and a decrease in or absence of plasma membrane localization, indicative of a processing defect. All of the functional human (h) NKCC2A variants were regulated by changes in oocyte volume and intracellular chloride in HEK cells, but P254A and N399S exhibited a lower constitutive activity in HEK cells. The P569H mutant exhibited a 50% reduction in sodium affinity compared with wild-type, predicting lower transport activity at lower intratubular salt concentrations, while the P254A mutant exhibited a 35% increase in rubidium affinity. We conclude that defects in NKCC2 processing, transport turnover rate, regulation, and ion affinity contribute to impaired transport function in six of the nine identified mutants, providing support for the predictive approach of Ji et al. to identify functionally important residues by sequence conservation. Such mutations in hNKCC2A are likely to reduce renal salt reabsorption, providing a mechanism for lower blood pressure.

scholarly journals The M3Muscarinic Acetylcholine Receptor Expressed in HEK-293 Cells Signals to Phospholipase D via G12but Not Gq-type G Proteins

2000 ◽  
Vol 276 (4) ◽  
pp. 2474-2479 ◽  
Author(s):  
Ulrich Rümenapp ◽  
Melanie Asmus ◽  
Helge Schablowski ◽  
Markus Woznicki ◽  
Li Han ◽  
...  
Keyword(s):  
G Proteins ◽  
Acetylcholine Receptor ◽  
Phospholipase D ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

Intracellular calcium measurements as a method in studies on activity of purinergic P2X receptor channels

2003 ◽  
Vol 285 (2) ◽  
pp. C467-C479 ◽  
Author(s):  
Mu-Lan He ◽  
Hana Zemkova ◽  
Taka-aki Koshimizu ◽  
Melanija Tomić ◽  
Stanko S. Stojilkovic
Keyword(s):  
Purinergic Receptors ◽  
Host Cells ◽  
P2x Receptor ◽  
Wild Type ◽  
Hek 293 ◽  
Human Embryonic Kidney Cells ◽  
Voltage Gated ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Intracellular Ca

Extracellular nucleotide-activated purinergic receptors (P2XRs) are a family of cation-permeable channels that conduct small cations, including Ca2+, leading to the depolarization of cells and subsequent stimulation of voltage-gated Ca2+ influx in excitable cells. Here, we studied the spatiotemporal characteristics of intracellular Ca2+ signaling and its dependence on current signaling in excitable mouse immortalized gonadotropin-releasing hormone-secreting cells (GT1) and nonexcitable human embryonic kidney cells (HEK-293) cells expressing wild-type and chimeric P2XRs. In both cell types, P2XR generated depolarizing currents during the sustained ATP stimulation, which desensitized in order (from rapidly desensitizing to nondesensitizing): P2X3R > P2X2b + X4R > P2X2bR > P2X2a + X4R > P2X4R > P2X2aR > P2X7R. HEK-293 cells were not suitable for studies on P2XR-mediated Ca2+ influx because of the coactivation of endogenously expressed Ca2+-mobilizing purinergic P2Y receptors. However, when expressed in GT1 cells, all wild-type and chimeric P2XRs responded to agonist binding with global Ca2+ signals, which desensitized in the same order as current signals but in a significantly slower manner. The global distribution of Ca2+ signals was present independently of the rate of current desensitization. The temporal characteristics of Ca2+ signals were not affected by voltage-gated Ca2+ influx and removal of extracellular sodium. Ca2+ signals reflected well the receptor-specific EC50 values for ATP and the extracellular Zn2+ and pH sensitivities of P2XRs. These results indicate that intracellular Ca2+ measurements are useful for characterizing the pharmacological properties and messenger functions of P2XRs, as well as the kinetics of channel activity, when the host cells do not express other members of purinergic receptors.

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