FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2022294588> ?p ?o ?g. }

• W2022294588 endingPage "27860" @default.
• W2022294588 startingPage "27849" @default.
• W2022294588 abstract "The vasopressin type 2 receptor (V2R) is a critical G protein-coupled receptor (GPCR) for vertebrate physiology, including the balance of water and sodium ions. It is unclear how its two native hormones, vasopressin (VP) and oxytocin (OT), both stimulate the same cAMP/PKA pathway yet produce divergent antinatriuretic and antidiuretic effects that are either strong (VP) or weak (OT). Here, we present a new mechanism that differentiates the action of VP and OT on V2R signaling. We found that vasopressin, as opposed to OT, continued to generate cAMP and promote PKA activation for prolonged periods after ligand washout and receptor internalization in endosomes. Contrary to the classical model of arrestin-mediated GPCR desensitization, arrestins bind the VP-V2R complex yet extend rather than shorten the generation of cAMP. Signaling is instead turned off by the endosomal retromer complex. We propose that this mechanism explains how VP sustains water and Na+ transport in renal collecting duct cells. Together with recent work on the parathyroid hormone receptor, these data support the existence of a novel “noncanonical” regulatory pathway for GPCR activation and response termination, via the sequential action of β-arrestin and the retromer complex.Background: It remains unclear why vasopressin induces greater antidiuresis through V2R than does oxytocin.Results: Vasopressin sustains cAMP signaling during V2R internalization, a process promoted by β-arrestins, and is halted by the retromer complex.Conclusion: This new noncanonical model of GPCR signaling differentiates the actions of vasopressin and oxytocin.Significance: This emerging model may explain the physiological bias between ligands. The vasopressin type 2 receptor (V2R) is a critical G protein-coupled receptor (GPCR) for vertebrate physiology, including the balance of water and sodium ions. It is unclear how its two native hormones, vasopressin (VP) and oxytocin (OT), both stimulate the same cAMP/PKA pathway yet produce divergent antinatriuretic and antidiuretic effects that are either strong (VP) or weak (OT). Here, we present a new mechanism that differentiates the action of VP and OT on V2R signaling. We found that vasopressin, as opposed to OT, continued to generate cAMP and promote PKA activation for prolonged periods after ligand washout and receptor internalization in endosomes. Contrary to the classical model of arrestin-mediated GPCR desensitization, arrestins bind the VP-V2R complex yet extend rather than shorten the generation of cAMP. Signaling is instead turned off by the endosomal retromer complex. We propose that this mechanism explains how VP sustains water and Na+ transport in renal collecting duct cells. Together with recent work on the parathyroid hormone receptor, these data support the existence of a novel “noncanonical” regulatory pathway for GPCR activation and response termination, via the sequential action of β-arrestin and the retromer complex. Background: It remains unclear why vasopressin induces greater antidiuresis through V2R than does oxytocin. Results: Vasopressin sustains cAMP signaling during V2R internalization, a process promoted by β-arrestins, and is halted by the retromer complex. Conclusion: This new noncanonical model of GPCR signaling differentiates the actions of vasopressin and oxytocin. Significance: This emerging model may explain the physiological bias between ligands. Arrestin is thought to desensitize heterotrimeric G protein signaling at the plasma membrane by physically interacting with activated GPCR, 3The abbreviations used are: GPCRG protein-coupled receptorV2Rvasopressin type 2 receptorVPvasopressinOToxytocinPTHRparathyroid hormone type 1 receptorENaCepithelial sodium channelAQP2aquaporin 2GTPγSguanosine 5′-3-O-(thio)triphosphateTIRFtotal internal reflection fluorescenceCFPcyan fluorescence proteinBisTris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolTMRtetramethylrhodamineMDCKMadin-Darby canine kidney cell. thus preventing receptor-G protein coupling (1.Lohse M.J. Benovic J.L. Codina J. Caron M.G. Lefkowitz R.J. β-Arrestin: a protein that regulates β-adrenergic receptor function.Science. 1990; 248: 1547-1550Crossref PubMed Scopus (909) Google Scholar), promoting receptor internalization (2.Laporte S.A. Miller W.E. Kim K.M. Caron M.G. β-Arrestin/AP-2 interaction in G protein-coupled receptor internalization: identification of a β-arrestin binding site in β2-adaptin.J. Biol. Chem. 2002; 277: 9247-9254Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar), and promoting the recruitment of enzymes such as cAMP-specific phosphodiesterase type 4D or diacylglycerol kinases to enhance cAMP or diacylglycerol degradation, respectively (3.Nelson C.D. Perry S.J. Regier D.S. Prescott S.M. Topham M.K. Lefkowitz R.J. Targeting of diacylglycerol degradation to M1 muscarinic receptors by β-arrestins.Science. 2007; 315: 663-666Crossref PubMed Scopus (124) Google Scholar, 4.Perry S.J. Baillie G.S. Kohout T.A. McPhee I. Magiera M.M. Ang K.L. Miller W.E. McLean A.J. Conti M. Houslay M.D. Lefkowitz R.J. Targeting of cyclic AMP degradation to β2-adrenergic receptors by β-arrestins.Science. 2002; 298: 834-836Crossref PubMed Scopus (410) Google Scholar). This model of arrestin-mediated desensitization has been supported with extensive work on several GPCRs such as rhodopsin and the β2-adrenergic receptor, among others, and is considered a universal and “canonical” mechanism for GPCR desensitization (5.Luttrell L.M. Lefkowitz R.J. The role of β-arrestins in the termination and transduction of G-protein-coupled receptor signals.J. Cell Sci. 2002; 115: 455-465Crossref PubMed Google Scholar). This model has been challenged by recent studies revealing that β-arrestins prolong rather than attenuate cAMP stimulation triggered by the parathyroid hormone type 1 receptor (6.Feinstein T.N. Wehbi V.L. Ardura J.A. Wheeler D.S. Ferrandon S. Gardella T.J. Vilardaga J.P. Retromer terminates the generation of cAMP by internalized PTH receptors.Nat. Chem. Biol. 2011; 7: 278-284Crossref PubMed Scopus (174) Google Scholar). This new finding coupled with the recent observations that the PTHR along with other GPCRs can sustain heterotrimeric G protein signaling after internalization to endosomes (6.Feinstein T.N. Wehbi V.L. Ardura J.A. Wheeler D.S. Ferrandon S. Gardella T.J. Vilardaga J.P. Retromer terminates the generation of cAMP by internalized PTH receptors.Nat. Chem. Biol. 2011; 7: 278-284Crossref PubMed Scopus (174) Google Scholar, 7.Calebiro D. Nikolaev V.O. Gagliani M.C. de Filippis T. Dees C. Tacchetti C. Persani L. Lohse M.J. Persistent cAMP-signals triggered by internalized G-protein-coupled receptors.PLoS Biol. 2009; 7: e1000172Crossref PubMed Scopus (406) Google Scholar, 8.Ferrandon S. Feinstein T.N. Castro M. Wang B. Bouley R. Potts J.T. Gardella T.J. Vilardaga J.-P. Sustained cyclic AMP production by parathyroid hormone receptor endocytosis.Nat. Chem. Biol. 2009; 5: 734-742Crossref PubMed Scopus (413) Google Scholar, 9.Kotowski S.J. Hopf F.W. Seif T. Bonci A. von Zastrow M. Endocytosis promotes rapid dopaminergic signaling.Neuron. 2011; 71: 278-290Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 10.Mullershausen F. Zecri F. Cetin C. Billich A. Guerini D. Seuwen K. Persistent signaling induced by FTY720-phosphate is mediated by internalized S1P1 receptors.Nat. Chem. Biol. 2009; 5: 428-434Crossref PubMed Scopus (285) Google Scholar) point to an emerging noncanonical signaling model of GPCR where β-arrestins prolong rather than attenuate ligand actions when a receptor internalizes in endosomes (11.Vilardaga J.P. Gardella T.J. Wehbi V.L. Feinstein T.N. Non-canonical signaling of the PTH receptor.Trends Pharmacol. Sci. 2012; 33: 423-431Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). G protein-coupled receptor vasopressin type 2 receptor vasopressin oxytocin parathyroid hormone type 1 receptor epithelial sodium channel aquaporin 2 guanosine 5′-3-O-(thio)triphosphate total internal reflection fluorescence cyan fluorescence protein 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol tetramethylrhodamine Madin-Darby canine kidney cell. We do not know how many GPCRs exhibit this noncanonical form of signaling. Several factors led us to investigate the vasopressin type 2 receptor (V2R), a GS-coupled receptor that controls water and sodium ion homeostasis by regulating their reuptake in the collecting duct of the kidney (12.Brown D. Breton S. Ausiello D.A. Marshansky V. Sensing, signaling and sorting events in kidney epithelial cell physiology.Traffic. 2009; 10: 275-284Crossref PubMed Scopus (52) Google Scholar). These include the following: 1) a high affinity complex between V2R and its native ligand, vasopressin (VP), which remains stable and active (cAMP production) under acidic conditions, pH 5.5, characteristic of early endosomes (13.Zalyapin E.A. Bouley R. Hasler U. Vilardaga J.P. Lin H.Y. Brown D. Ausiello D.A. Effects of the renal medullary pH and ionic environment on vasopressin binding and signaling.Kidney Int. 2008; 74: 1557-1567Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar); 2) the redistribution of VP-V2R-arrestin complexes to early endosomes (14.Chen S. Webber M.J. Vilardaga J.P. Khatri A. Brown D. Ausiello D.A. Lin H.Y. Bouley R. Visualizing microtubule-dependent vasopressin type 2 receptor trafficking using a new high-affinity fluorescent vasopressin ligand.Endocrinology. 2011; 152: 3893-3904Crossref PubMed Scopus (6) Google Scholar); 3) distinct physiological responses to its two native and structurally similar ligands, vasopressin (VP, strong antidiuretic and antinatriuretic effects) versus oxytocin (OT, weak to no effect) (15.Balment R.J. Brimble M.J. Forsling M.L. Kelly L.P. Musabayane C.T. A synergistic effect of oxytocin and vasopressin on sodium excretion in the neurohypophysectomized rat.J. Physiol. 1986; 381: 453-464Crossref PubMed Scopus (43) Google Scholar, 16.Chou C.L. DiGiovanni S.R. Luther A. Lolait S.J. Knepper M.A. Oxytocin as an antidiuretic hormone. II. Role of V2 vasopressin receptor.Am. J. Physiol. 1995; 269: F78-F85PubMed Google Scholar, 17.Chou C.L. DiGiovanni S.R. Mejia R. Nielsen S. Knepper M.A. Oxytocin as an antidiuretic hormone. I. Concentration dependence of action.Am. J. Physiol. 1995; 269: F70-F77PubMed Google Scholar); and 4) evidence that a mutant of V2R associated with nephrogenic diabetes insipidus remains in the endoplasmic reticulum yet generates cAMP when challenged with a membrane permeant agonist (18.Robben J.H. Kortenoeven M.L. Sze M. Yae C. Milligan G. Oorschot V.M. Klumperman J. Knoers N.V. Deen P.M. Intracellular activation of vasopressin V2 receptor mutants in nephrogenic diabetes insipidus by nonpeptide agonists.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 12195-12200Crossref PubMed Scopus (66) Google Scholar). These observations raised the hypothesis that internalized VP-V2R complexes could access the machinery for signaling from membranes inside the cell to continue to stimulate the cAMP/PKA pathway via a noncanonical signaling model of GPCR. To address this hypothesis, we used a series of biochemical, optical, and physiological assays to study V2R signaling in response to its two native ligands: vasopressin and oxytocin (13.Zalyapin E.A. Bouley R. Hasler U. Vilardaga J.P. Lin H.Y. Brown D. Ausiello D.A. Effects of the renal medullary pH and ionic environment on vasopressin binding and signaling.Kidney Int. 2008; 74: 1557-1567Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 14.Chen S. Webber M.J. Vilardaga J.P. Khatri A. Brown D. Ausiello D.A. Lin H.Y. Bouley R. Visualizing microtubule-dependent vasopressin type 2 receptor trafficking using a new high-affinity fluorescent vasopressin ligand.Endocrinology. 2011; 152: 3893-3904Crossref PubMed Scopus (6) Google Scholar). These experiments employed cultured human embryonic kidney (HEK293) cells stably expressing recombinant V2R and a line derived from renal principal cells of the mouse collecting duct, mpkCCDC14, which expresses V2R and the epithelial sodium channel (ENaC) (19.Gumz M.L. Cheng K.Y. Lynch I.J. Stow L.R. Greenlee M.M. Cain B.D. Wingo C.S. Regulation of αENaC expression by the circadian clock protein Period 1 in mpkCCD(c14) cells.Biochim. Biophys. Acta. 2010; 1799: 622-629Crossref PubMed Scopus (59) Google Scholar, 20.Robert-Nicoud M. Flahaut M. Elalouf J.M. Nicod M. Salinas M. Bens M. Doucet A. Wincker P. Artiguenave F. Horisberger J.D. Vandewalle A. Rossier B.C. Firsov D. Transcriptome of a mouse kidney cortical collecting duct cell line: effects of aldosterone and vasopressin.Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 2712-2716Crossref PubMed Scopus (185) Google Scholar). We found that vasopressin induces a V2R-active state that promotes cAMP generation not only at the plasma membrane but also after β-arrestin1/2-mediated receptor internalization to endosomes. Oxytocin, however, did not induce significant β-arrestins binding or V2R internalization, and cAMP generation ended rapidly after ligand washout. Sustained cAMP generation triggered by VP was inhibited by the endosomal retromer complex. As in HEK293 cells, VP induced greater cAMP generation than OT in mpkCCDC14 cells and caused a sustained activation of ENaC on the apical plasma membrane, whereas OT did not. In both HEK293 and mpkCCDC14 cells, increased expression of β-arrestins augmented rather than attenuated the duration of cAMP production. These results indicate that noncanonical actions of β-arrestins and retromer can be extended to the regulation of V2R signaling in kidney cells, which may account for the biological differences between VP and OT on water and electrolyte homeostasis. Vasopressin type 2 receptor (V2R) and hemagglutinin (HA)-tagged V2R (HA-V2R) cDNAs were purchased from Missouri S&T cDNA Resource Center (Rolla, MO). V2R was amplified using AccuPrime Taq polymerase (Invitrogen) to introduce flanking 5′-BglII and 3′-AgeI restriction enzyme sites. The digested product was ligated into pECFP-N1 (Clontech) vector to generate V2R C-terminally tagged with enhanced CFP (V2RCFP). Cell culture reagents were obtained from Invitrogen. Human embryonic kidney cells (HEK293) (ATCC, Manassas, VA) were cultured in DMEM supplemented with 10% fetal bovine serum at 37 °C in a humidified atmosphere containing 5% CO2. HEK293 cells stably expressing HA-V2R were grown in selection medium (DMEM, 10% FBS, 500 μg/ml neomycin). Culturing of mpkCCD-C14 collecting duct-derived cells was done as described previously (21.Bens M. Vallet V. Cluzeaud F. Pascual-Letallec L. Kahn A. Rafestin-Oblin M.E. Rossier B.C. Vandewalle A. Corticosteroid-dependent sodium transport in a novel immortalized mouse collecting duct principal cell line.J. Am. Soc. Nephrol. 1999; 10: 923-934Crossref PubMed Google Scholar, 22.Carattino M.D. Edinger R.S. Grieser H.J. Wise R. Neumann D. Schlattner U. Johnson J.P. Kleyman T.R. Hallows K.R. Epithelial sodium channel inhibition by AMP-activated protein kinase in oocytes and polarized renal epithelial cells.J. Biol. Chem. 2005; 280: 17608-17616Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). AQP2-MDCK cells were cultured in DMEM supplemented with 10% fetal bovine serum and G418 (500 μg/ml) (23.Yui N. Lu H.A. Chen Y. Nomura N. Bouley R. Brown D. Basolateral targeting and microtubule-dependent transcytosis of the aquaporin-2 water channel.Am. J. Physiol. Cell Physiol. 2012; 304: C38-C48Crossref PubMed Scopus (47) Google Scholar). For transient expression, cells were transfected with the appropriate cDNAs using FuGENE 6 (Roche Applied Science) according to the manufacturer's instructions unless otherwise mentioned. We have optimized expression conditions to ensure moderate expression levels of fluorescent arrestins. We performed Western blots to verify the levels of fluorescent β-arrestin1 or β-arrestin2 expression relative to native β-arrestin1/2 in our experiments. We also ensured that the expression of fluorescent-labeled β-arrestin1 or β-arrestin2 was similar in the examined cells by performing experiments in cells displaying comparable fluorescence levels. We depleted the retromer subunit Vps35 from HEK293 cells using a pre-validated quartet of siRNA duplex nucleotides (SmartPool, Dharmacon). Control transfections were done with a scrambled siRNA (OriGene). Briefly, 24 h after plating, cells were transferred to serum- and antibiotic-free medium and transfected with X-TremeGene reagent (Roche Applied Science) according to the manufacturer's directions. Medium was supplemented with serum and antibiotics 24 h after transfection, and experiments were performed between 72 and 96 h after transfection. Plasmid transfections were done using FuGENE (Roche Applied Science) according to the manufacturer's directions. Cells plated on poly-d-lysine-coated glass coverslips and maintained in FRET buffer were imaged using a Nikon Ti-E inverted microscope equipped with an oil immersion ×60 NA 1.49 plan-apo TIRF objective and a TIRF excitation arm with motorized critical angle control (Nikon). Critical angles for CFP and YFP excitation were determined in advance, and identical angles were used in every experiment. In-line neutral density filters were used to minimize bleaching. Excitation light came from a 442-nm solid-state laser (CFP, Melles Griot) and the 514-nm line of an argon gas laser (YFP, Melles Griot) using appropriate dichroic filters. YFP was detected using 500 ± 20 nm (excitation) and 535 ± 30 nm (emission) filters; CFP was detected using 436 ± 20 nm (excitation) and 480 ± 40 nm (emission); and FRET was detected using a 436 ± 20 mm (excitation) and a 535 ± 30 nm (emission). The experiments were performed from membrane preparations of HEK293 cells stably expressing HA-V2R. Briefly, confluent cells were washed with ice-cold PBS and incubated with hypotonic buffer (10 mm Hepes, 0.5 mm EDTA, pH 7.4) for 15 min on ice. Swollen cells were harvested, collected by centrifugation (100 × g for 10 min), and resuspended in 5 volumes of 10 mm Tris, 1 mm EDTA, pH 7.4, with a mixture of proteinase inhibitors. Cells were disrupted with 30–40 strokes with a tissue grinder on ice. Lysates were centrifuged at 1000 × g for 10 min to remove unbroken cells and large cell debris. The supernatant was further centrifuged at 30,000 × g for 20 min at 4 °C. The membrane pellet was resuspended in ice-cold buffer (10 mm Hepes, 0.1 mm EDTA, pH 7.4) and rapidly frozen in liquid nitrogen. Membranes were stored at −80 °C until used. A small volume of membrane preparation was kept to determine protein concentration. Frozen membrane aliquots (50 μg/tube) were incubated with 100 μl of assay buffer (10 mm Hepes, 100 mm NaCl, 5 mm MgCl2, pH 7.4) containing 5 μm GDP, with or without 100 nm purified β-arrestin2 (see below) for 45 min at room temperature. Then, either 100 nm vasopressin (AVP) and 10 nm [35S]GTPγS were added for different periods of time at 30 °C. Incubations were terminated by addition of 800 μl of ice-cold assay buffer and 100 μm GTPγS and transferred on ice. Cell membranes were recovered from the reaction mixture by centrifugation at 20,000 × g for 10 min. The resulting supernatant was removed. Membrane pellets were solubilized, and 2 μl of anti-GαS antibody (Millipore) was added to immunoprecipitate [35S]GTPγS-bound GαS. Samples were under agitation for 1 h at 4 °C. Then 20 μl of protein G-Sepharose (Santa Cruz Biotechnology) was added to each sample and incubated overnight at 4 °C. The beads were washed three times, suspended with 200 μl of 0.5% SDS, and incubated at 90 °C for 2–3 min. The entire contents of each tube were transferred to a vial containing 5 ml of scintillation mixture, and radioactivity was counted by β-emission spectrometry. Note that purified β-arrestin2 used in these experiments was prepared as described previously (24.Wehbi V.L. Stevenson H.P. Feinstein T.N. Calero G. Romero G. Vilardaga J.P. Noncanonical GPCR signaling arising from a PTH receptor-arrestin-Gβγ complex.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: 1530-1535Crossref PubMed Scopus (114) Google Scholar). mpkCCDc14 cells grown on Transwell filter supports (Costar) (21.Bens M. Vallet V. Cluzeaud F. Pascual-Letallec L. Kahn A. Rafestin-Oblin M.E. Rossier B.C. Vandewalle A. Corticosteroid-dependent sodium transport in a novel immortalized mouse collecting duct principal cell line.J. Am. Soc. Nephrol. 1999; 10: 923-934Crossref PubMed Google Scholar) were mounted in modified Costar Ussing chambers, and the cultures were continuously short circuited with an automatic voltage clamp (Dept. of Bioengineering, University of Iowa, Iowa City). Transepithelial resistance was measured by periodically applying a 2.5-mV bipolar pulse and calculated by Ohm's law. The bathing Ringer's solution composition, gassing, and washing techniques have been described previously (25.Butterworth M.B. Edinger R.S. Johnson J.P. Frizzell R.A. Acute ENaC stimulation by cAMP in a kidney cell line is mediated by exocytic insertion from a recycling channel pool.J. Gen. Physiol. 2005; 125: 81-101Crossref PubMed Scopus (144) Google Scholar). AQP2 stably transfected MDCK cells (AQP2-MDCK) were plated on 6-well filt ers (32 × 104 cells/well). Cells were grown on filters for 5 days to be fully polarized and treated with 50 mm indomethacin overnight to lower endogenous cAMP levels (26.van Balkom B.W. Graat M.P. van Raak M. Hofman E. van der Sluijs P. Deen P.M. Role of cytoplasmic termini in sorting and shuttling of the aquaporin-2 water channel.Am. J. Physiol. Cell Physiol. 2004; 286: C372-C379Crossref PubMed Scopus (30) Google Scholar). Culture medium was replaced with serum- and antibiotic-free DMEM for 2 h before stimulation. Then cells were treated with or without VP or OT for 10 min. For washout experiments, cells were washed and cultured in serum- and antibiotic-free DMEM for 20 min. Filters were cut from plastic supports and incubated in lysis buffer (150 mm NaCl, 20 mm Tris-HCl, 5 mm EDTA, 1% Triton X-100 containing 10 mm NaF and 1 mm Na3VO4) for 20 min. Cell were scraped, and cell lysate was then transferred into Eppendorf tubes, rotated for 30 min at 4 °C, centrifuged for 10 min at 10,000 × g, and 12 μl of reduced samples (6 μg of protein) per lane were run on a NuPAGE 1.0-mm 15-well 4–12% BisTris gel and then transferred onto a PVDF membrane (Invitrogen). Transferred PVDF membranes were incubated in 5% skimmed milk in PBS/Tween 0.05% buffer for blocking, then incubated with primary antibody, diluted 1:5000–1:10,000 in PBS/Tween 0.05% buffer overnight (Ser(P)-256 and Ser(P)-269 AQP2 antibody) for 1 h (AQP2 polyclonal antibody). Then PVDF membranes were washed four times for 15 min in PBS/Tween 0.05% buffer (PBST), incubated with HRP-conjugated secondary antibody diluted 1:10,000 for 30 min, and washed in 0.05% PBST four times for 15 min. Signals were visualized using Western Lightning ECL and Biomax XAR film. The same PVDF membranes were stripped in stripping buffer (0.2 m glycine, 0.05% Tween 20, pH was adjusted to 2.5 by adding HCl) for 1 h, and then re-probed with the next antibody. Complete removal of the first primary antibody was confirmed by incubating the stripped membrane with secondary antibody (donkey anti rabbit IgG HRP conjugated). The order of detection was Ser(P)-269, Ser(P)-256, and then total AQP2. HEK293 cells stably expressing HA-V2R were studied by a confocal microscope using methods described in previous studies (6.Feinstein T.N. Wehbi V.L. Ardura J.A. Wheeler D.S. Ferrandon S. Gardella T.J. Vilardaga J.P. Retromer terminates the generation of cAMP by internalized PTH receptors.Nat. Chem. Biol. 2011; 7: 278-284Crossref PubMed Scopus (174) Google Scholar, 8.Ferrandon S. Feinstein T.N. Castro M. Wang B. Bouley R. Potts J.T. Gardella T.J. Vilardaga J.-P. Sustained cyclic AMP production by parathyroid hormone receptor endocytosis.Nat. Chem. Biol. 2009; 5: 734-742Crossref PubMed Scopus (413) Google Scholar). In brief, cell imaging was performed at room temperature in Hepes buffer containing 0.1% BSA using a Nikon A1s confocal microscope attached to a Ti-E inverted base using a ×60 1.45 NA plan-apo objective. Live cell time-lapse confocal imaging of transfected cells treated with tetramethylrhodamine (TMR)-labeled vasopressin (VPTMR) (14.Chen S. Webber M.J. Vilardaga J.P. Khatri A. Brown D. Ausiello D.A. Lin H.Y. Bouley R. Visualizing microtubule-dependent vasopressin type 2 receptor trafficking using a new high-affinity fluorescent vasopressin ligand.Endocrinology. 2011; 152: 3893-3904Crossref PubMed Scopus (6) Google Scholar) was performed at 37 °C using a Nikon A1-Rs confocal system attached to a Ti-E inverted base and an apo ×60 1.49 NA TIRF objective. The microscope was encased in a microscope cage incubator to maintain the temperature at 37 °C. HEK293 cells expressing HA-V2R were grown in a Multiwell 12-well plate (BD Biosciences). Upon reaching 70% confluency, cells were transfected using 3.75 μl of Lipofectamine 2000 and 1 μg of cDNA of green fluorescent protein (GFP)-tagged β-arrestin 2 (βarr2-GFP), GαS (GαS-GFP), or retromer (Vps29-YFP). 24 h later, cells were trypsinized and seeded in glass bottom FluoroDishes (World Precision Instruments, Sarasota, FL). 24–48 h later, the cells were washed and imaged in Hanks' Buffer (Invitrogen) with 10 mm Hepes, 25 mm sodium bicarbonate, and 10 mm glucose added to the solution. After the control picture was taken, 1 μm VPTMR was incubated with cells for 10 min, and then ligand was washed out, and images were taken at the designated time intervals. FRET analysis was performed as described previously (8.Ferrandon S. Feinstein T.N. Castro M. Wang B. Bouley R. Potts J.T. Gardella T.J. Vilardaga J.-P. Sustained cyclic AMP production by parathyroid hormone receptor endocytosis.Nat. Chem. Biol. 2009; 5: 734-742Crossref PubMed Scopus (413) Google Scholar). Cells plated on poly-d-lysine-coated glass coverslips and maintained in FRET buffer were placed on a Nikon Ti-E inverted microscope equipped with an oil immersion ×60 NA 1.49 plan-apo objective and a dichroic beam splitter to allow simultaneous imaging of CFP and YFP fluorescence channels (DualView2, Photometrics, Tucson, AZ). The emission fluorescence intensities were determined at 535 ± 15 nm (YFP) and 480 ± 20 nm (CFP) with a beam splitter DCLP of 505 nm. The FRET ratio for single experiments was corrected according to Equation 1, Ratio(FYEPFCFP)=FYEPex436/em535×a−FCEPex436/em480−b×FYEPex500/em535FCEPex436/em480(Eq. 1) where FYFPex436/em535 and FCFPex436/em480 represent, respectively, the emission (em) intensities of YFP (recorded at 535 nm) and CFP (recorded at 480 nm) upon excitation (ex) at 436 nm; a and b represent correction factors for the bleed through of CFP into the 535-nm channel (a = 0.35) and the cross-talk due to the direct YFP excitation by light at 436 nm (b = 0.06). FYFPex500/em535 represents the emission intensity of YFP (recorded at 535 nm) upon direct excitation at 500 nm, and was recorded at the beginning of each experiment. Note that bleed through of YFP into the 480-nm channel was negligible. For each measurement, changes in fluorescence emissions due to photobleaching were subtracted. To ensure that CFP- and YFP-labeled molecule expression were similar in examined cells, we performed experiments in cells displaying comparable fluorescence levels. The means of intermolecular FRET experiments were calculated according to Equation 2, which normalizes for different expression levels of CFP and YFP molecules, NFRET=FYEPex436/em535−a×FCEPex436/em480−b×FYEPex500/em535FCEPex436/em480×FYEPex500/em535(Eq. 2) LLC-PK1 cells expressing V2R C-terminally tagged with enhanced GFP (V2R-GFP) (28.Bouley R. Lin H.Y. Raychowdhury M.K. Marshansky V. Brown D. Ausiello D.A. Downregulation of the vasopressin type 2 receptor after vasopressin-induced internalization: involvement of a lysosomal degradation pathway.Am. J. Physiol. Cell Physiol. 2005; 288: C1390-C1401Crossref PubMed Scopus (42) Google Scholar) were grown in 6-cm dishes and transfected with 4 μg of both c-Myc-tagged Vps29 and Vps26 using 15 μl of Lipofectamine 2000. After 48 h, cells were incubated with 1 μm VP for different times of incubation (30, 60, 120, or 240 min). Cells were then lysed for 20 min at 4 °C in lysis buffer containing 50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS supplemented with protease inhibitors (Roche Applied Science). Before Western blot analysis, protein concentrations were measured with the bicinchoninic acid protein assay as recommended by the manufacturer (Invitrogen). Reduced samples from cell lysates (20 μg) were loaded onto each lane of a standard NUPAGE 4–12% BisTris gel and then electroblotted on Immobilon transfer membranes (Millipore, Billerica, MA). The filters were blocked with Li-Cor blocking buffer by shaking for 1 h at room temperature before the incubation (1 h) of the polyclonal anti-GFP (0.4 μg/ml, Molecular Probes) and monoclonal anti-actin antibody in Li-Cor blocking buffer, Tween 0.1%. After being washed three times in PBS/Tween 0.1% buffer, the membranes were incubated with goat anti-mouse IRdye 800CW and goat anti-rabbit IRDye 680 diluted in Li-Cor blocking buffer. After 1 h of incubation, membranes were washed three times with PBS/Tween 0.1% buffer. After two rinses in distilled water, fluorescence in the membranes was recorded by using a Li-Cor infrared imaging system (Li-Cor Biosciences). Density of protein bands was quantified using IP Lab Spectrum software (Scanalytics, Vienna, VA). Western blot analyses were performed according to standard procedures. Briefly, HEK293 cells were transfected and harvested 48 h after transfection. The total protein extract was run on an SDS-PAGE and blotted onto 0.45-mm PVDF membr" @default.
• W2022294588 created "2016-06-24" @default.
• W2022294588 creator A5018572753 @default.
• W2022294588 creator A5021697510 @default.
• W2022294588 creator A5037580161 @default.
• W2022294588 creator A5040397891 @default.
• W2022294588 creator A5046080800 @default.
• W2022294588 creator A5048970112 @default.
• W2022294588 creator A5069204789 @default.
• W2022294588 creator A5075288934 @default.
• W2022294588 creator A5081576808 @default.
• W2022294588 creator A5087672907 @default.
• W2022294588 date "2013-09-01" @default.
• W2022294588 modified "2023-10-12" @default.
• W2022294588 title "Noncanonical Control of Vasopressin Receptor Type 2 Signaling by Retromer and Arrestin" @default.
• W2022294588 cites W1727091879 @default.
• W2022294588 cites W1933410007 @default.
• W2022294588 cites W1967965496 @default.
• W2022294588 cites W1968578345 @default.
• W2022294588 cites W1971748110 @default.
• W2022294588 cites W1980257483 @default.
• W2022294588 cites W1986177696 @default.
• W2022294588 cites W1987672555 @default.
• W2022294588 cites W1991800350 @default.
• W2022294588 cites W1996193406 @default.
• W2022294588 cites W2003500217 @default.
• W2022294588 cites W2011486404 @default.
• W2022294588 cites W2016930096 @default.
• W2022294588 cites W2018544658 @default.
• W2022294588 cites W2020620553 @default.
• W2022294588 cites W2023629636 @default.
• W2022294588 cites W2029309701 @default.
• W2022294588 cites W203306182 @default.
• W2022294588 cites W2039668638 @default.
• W2022294588 cites W2055951748 @default.
• W2022294588 cites W2057772810 @default.
• W2022294588 cites W2058407325 @default.
• W2022294588 cites W2058555785 @default.
• W2022294588 cites W2058746068 @default.
• W2022294588 cites W2060207881 @default.
• W2022294588 cites W2061098391 @default.
• W2022294588 cites W2061111237 @default.
• W2022294588 cites W2061286602 @default.
• W2022294588 cites W2067080420 @default.
• W2022294588 cites W2071103368 @default.
• W2022294588 cites W2074486707 @default.
• W2022294588 cites W2075988095 @default.
• W2022294588 cites W2082817154 @default.
• W2022294588 cites W2091150954 @default.
• W2022294588 cites W2099258590 @default.
• W2022294588 cites W2107907493 @default.
• W2022294588 cites W2113753551 @default.
• W2022294588 cites W2117954730 @default.
• W2022294588 cites W2122680576 @default.
• W2022294588 cites W2130536801 @default.
• W2022294588 cites W2131717455 @default.
• W2022294588 cites W2137092708 @default.
• W2022294588 cites W2137743691 @default.
• W2022294588 cites W2156064425 @default.
• W2022294588 cites W2156212279 @default.
• W2022294588 cites W2160121812 @default.
• W2022294588 cites W2166522627 @default.
• W2022294588 cites W2166726745 @default.
• W2022294588 cites W2166748873 @default.
• W2022294588 cites W2239676257 @default.
• W2022294588 cites W2394975842 @default.
• W2022294588 cites W2409431293 @default.
• W2022294588 doi "https://doi.org/10.1074/jbc.m112.445098" @default.
• W2022294588 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/3784700" @default.
• W2022294588 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/23935101" @default.
• W2022294588 hasPublicationYear "2013" @default.
• W2022294588 type Work @default.
• W2022294588 sameAs 2022294588 @default.
• W2022294588 citedByCount "168" @default.
• W2022294588 countsByYear W20222945882014 @default.
• W2022294588 countsByYear W20222945882015 @default.
• W2022294588 countsByYear W20222945882016 @default.
• W2022294588 countsByYear W20222945882017 @default.
• W2022294588 countsByYear W20222945882018 @default.
• W2022294588 countsByYear W20222945882019 @default.
• W2022294588 countsByYear W20222945882020 @default.
• W2022294588 countsByYear W20222945882021 @default.
• W2022294588 countsByYear W20222945882022 @default.
• W2022294588 countsByYear W20222945882023 @default.
• W2022294588 crossrefType "journal-article" @default.
• W2022294588 hasAuthorship W2022294588A5018572753 @default.
• W2022294588 hasAuthorship W2022294588A5021697510 @default.
• W2022294588 hasAuthorship W2022294588A5037580161 @default.
• W2022294588 hasAuthorship W2022294588A5040397891 @default.
• W2022294588 hasAuthorship W2022294588A5046080800 @default.
• W2022294588 hasAuthorship W2022294588A5048970112 @default.
• W2022294588 hasAuthorship W2022294588A5069204789 @default.
• W2022294588 hasAuthorship W2022294588A5075288934 @default.
• W2022294588 hasAuthorship W2022294588A5081576808 @default.
• W2022294588 hasAuthorship W2022294588A5087672907 @default.
• W2022294588 hasBestOaLocation W20222945881 @default.
• W2022294588 hasConcept C102747710 @default.
• W2022294588 hasConcept C135285700 @default.

• next