FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2035917312> ?p ?o ?g. }

• W2035917312 endingPage "46980" @default.
• W2035917312 startingPage "46969" @default.
• W2035917312 abstract "To identify molecules that might contribute to V2 vasopressin receptor (V2R) trafficking or signaling, we searched for novel interacting proteins with this receptor. Preliminary data, using the V2R C terminus as bait in a yeast two-hybrid screen, revealed calmodulin as a binding partner. Because calmodulin interacts with other G protein-coupled receptors, we explored this interaction and its possible functional relevance in greater detail. A Ca2+-dependent interaction occurs between calmodulin-linked agarose and the holo-V2R as well as the V2R C terminus. Truncation and site-directed mutagenesis of the V2R C terminus revealed an involvement of an RGR sequence in this interaction. NMR studies showed that a peptide fragment of the V2R C terminus containing the RGR sequence binds to calmodulin in a Ca2+-dependent manner with a Kd ≤1.5 μm; concentration-dependent binding of the V2R C terminus to calmodulin-agarose was used to estimate a Kd value of ∼200 nm for this entire C-terminal sequence as expressed in mammalian cells. Madin-Darby canine kidney II cells stably expressing either wild type or a mutant V2R, in which the RGR C-terminal sequence was mutated to alanines (AAA V2R), revealed that the steady-state localization and agonist-induced internalization of the AAA V2R resembled that of the wild type V2R in polarized Madin-Darby canine kidney II cells. V2R binding of agonist similarly was unchanged in the AAA V2R, as was the concentration response for arginine vasopressin (AVP)-stimulated cAMP accumulation. Most interestingly, AVP-induced increases in intracellular Ca2+ observed for the wild type V2R were virtually eliminated for the AAA V2R. Taken together, the data suggest that a C-terminal region of the V2R important for calmodulin interaction is also important in modulation of V2R elevation of intracellular Ca2+, a prerequisite for AVP-induced fusion of aquaporin-containing vesicles with the apical surface of renal epithelial cells. To identify molecules that might contribute to V2 vasopressin receptor (V2R) trafficking or signaling, we searched for novel interacting proteins with this receptor. Preliminary data, using the V2R C terminus as bait in a yeast two-hybrid screen, revealed calmodulin as a binding partner. Because calmodulin interacts with other G protein-coupled receptors, we explored this interaction and its possible functional relevance in greater detail. A Ca2+-dependent interaction occurs between calmodulin-linked agarose and the holo-V2R as well as the V2R C terminus. Truncation and site-directed mutagenesis of the V2R C terminus revealed an involvement of an RGR sequence in this interaction. NMR studies showed that a peptide fragment of the V2R C terminus containing the RGR sequence binds to calmodulin in a Ca2+-dependent manner with a Kd ≤1.5 μm; concentration-dependent binding of the V2R C terminus to calmodulin-agarose was used to estimate a Kd value of ∼200 nm for this entire C-terminal sequence as expressed in mammalian cells. Madin-Darby canine kidney II cells stably expressing either wild type or a mutant V2R, in which the RGR C-terminal sequence was mutated to alanines (AAA V2R), revealed that the steady-state localization and agonist-induced internalization of the AAA V2R resembled that of the wild type V2R in polarized Madin-Darby canine kidney II cells. V2R binding of agonist similarly was unchanged in the AAA V2R, as was the concentration response for arginine vasopressin (AVP)-stimulated cAMP accumulation. Most interestingly, AVP-induced increases in intracellular Ca2+ observed for the wild type V2R were virtually eliminated for the AAA V2R. Taken together, the data suggest that a C-terminal region of the V2R important for calmodulin interaction is also important in modulation of V2R elevation of intracellular Ca2+, a prerequisite for AVP-induced fusion of aquaporin-containing vesicles with the apical surface of renal epithelial cells. The V2 vasopressin receptor (V2R) 1The abbreviations used are: V2R, V2 vasopressin receptor; AVP, arginine vasopressin; BAPTA-AM, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl) ester; CaM, calmodulin; GPCR, G protein-coupled receptor; HA, hemagglutinin; MDCKII, Madin-Darby canine kidney II; NHS, sulfo-N-hydroxysuccinimide; DPBS, Dulbecco's phosphate-buffered saline; RGRm, V2R C-terminal RGR motif; WT, wild type; BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; PVDF, polyvinylidene difluoride; GST, glutathione S-transferase; DMEM, Dulbecco's modified Eagle's medium; TEMED, N,N,′,N′-tetramethylethylenediamine; 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; HBSS, Hanks' buffered saline solution; PMSF, phenylmethylsulfonyl fluoride. is a member of the G protein-coupled receptor family and belongs to a subset of the neurohypophyseal peptide hormone receptors that includes the V1a, V1b, V2, and oxytocin receptors (1Schulein R. Zuhlke K. Krause G. Rosenthal W. J. Biol. Chem. 2001; 276: 8384-8392Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). The V2R binds the agonist arginine vasopressin (AVP) and signals through the heterotrimeric protein Gs to promote water reabsorption and concentration of the urine in the collecting duct of the kidney (2Morello J.P. Salahpour A. Laperriere A. Bernier V. Arthus M.F. Lonergan M. Petaja-Repo U. Angers S. Morin D. Bichet D.G. Bouvier M. J. Clin. Investig. 2000; 105: 887-895Crossref PubMed Scopus (479) Google Scholar). AVP is a cyclic nonapeptide hormone, 8-arginine vasopressin, that is secreted by the posterior pituitary in response to low urine osmolality or to decreased blood pressure (2Morello J.P. Salahpour A. Laperriere A. Bernier V. Arthus M.F. Lonergan M. Petaja-Repo U. Angers S. Morin D. Bichet D.G. Bouvier M. J. Clin. Investig. 2000; 105: 887-895Crossref PubMed Scopus (479) Google Scholar). AVP binds to the V2R on the basolateral surface of the principal cells of the renal collecting duct, couples to Gs, and stimulates adenylyl cyclase. The subsequent increase in intracellular cAMP leads to activation of cAMP-dependent protein kinase, phosphorylation of aquaporin-2, and the translocation of pre-formed aquaporin-2-containing vesicles to the apical membrane of the principal cells (3van Balkom B.W. Savelkoul P.J. Markovich D. Hofman E. Nielsen S. van der S.P. Deen P.M. J. Biol. Chem. 2002; 277: 41473-41479Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). This translocation appears to involve calcium in some (4Chou C.L. Yip K.P. Michea L. Kador K. Ferraris J.D. Wade J.B. Knepper M.A. J. Biol. Chem. 2000; 275: 36839-36846Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 5Yip K.P. J. Physiol. (Lond.). 2002; 538: 891-899Crossref Scopus (83) Google Scholar) but not other (6Lorenz D. Krylov A. Hahm D. Hagen V. Rosenthal W. Pohl P. Maric K. EMBO Rep. 2003; 4: 88-93Crossref PubMed Scopus (67) Google Scholar) experimental conditions. Because the calmodulin inhibitor W7 blocks the AVP-induced stimulation of water flow in the toad urinary bladder (7Levine S.D. Kachadorian W.A. Levin D.N. Schlondorff D. J. Clin. Investig. 1981; 67: 662-672Crossref PubMed Scopus (44) Google Scholar) and the rat inner medullary collecting duct (4Chou C.L. Yip K.P. Michea L. Kador K. Ferraris J.D. Wade J.B. Knepper M.A. J. Biol. Chem. 2000; 275: 36839-36846Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar), calmodulin may play a role in AVP-induced insertion of aquaporin 2 channels in these systems. The ultimate insertion of aquaporin 2 water channels into the apical membrane allows the renal epithelial cells to absorb water, which accounts for the antidiuretic effect of AVP. The C-terminal region of the V2R has been shown to be important for agonist-mediated phosphorylation (8Innamorati G. Sadeghi H. Eberle A.N. Birnbaumer M. J. Biol. Chem. 1997; 272: 2486-2492Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), receptor escape from the endoplasmic reticulum and transport to the plasma membrane (9Schulein R. Hermosilla R. Oksche A. Dehe M. Wiesner B. Krause G. Rosenthal W. Mol. Pharmacol. 1998; 54: 525-535Crossref PubMed Scopus (133) Google Scholar), sequestration and endocytosis of the receptor (8Innamorati G. Sadeghi H. Eberle A.N. Birnbaumer M. J. Biol. Chem. 1997; 272: 2486-2492Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), and prevention of recycling (10Innamorati G. Sadeghi H.M. Tran N.T. Birnbaumer M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2222-2226Crossref PubMed Scopus (111) Google Scholar) of the endocytosed V2R. Consequently, we decided to explore proteins that might interact with the V2R C terminus with the intent of understanding their possible role in V2R trafficking or signaling. Materials—Human wild type (WT) V2R cDNA was graciously provided by Dr. Jürgen Wess. The pEBG-SrfI mammalian GST expression vector was generously supplied by Dr. Yusen Liu (NIA, National Institutes of Health). [2,8-3H]Adenine (30.4 Ci/mmol), 8-arginine [phenylalanyl-3,4,5-3H]vasopressin (60.0 Ci/mmol), [8-14C]cAMP ammonium salt (51.2 mCi/mmol), [methoxy-3H]inulin-methoxy (430 mCi/g) were purchased from PerkinElmer Life Sciences. Additional [2,8-3H]adenine (24.2 Ci/mmol) was purchased from MP Biomedicals. Paraformaldehyde (16% solution, EM grade) was from Electron Microscopy Sciences (Washington, PA). PVDF nylon membranes were from Millipore (Bedford, MA). Dowex AG50 W-X4 resin, 40% acrylamide, TEMED, ammonium persulfate were from Bio-Rad. cAMP (sodium salt), alumina, [Arg8]vasopressin (acetate salt), bacitracin, bovine serum albumin, calmodulin-agarose (phosphodiesterase 3′:5′-cyclic nucleotide activator from bovine brain), fetal calf serum, leupeptin, phenylmethylsulfonyl fluoride (PMSF), probenecid, soybean trypsin inhibitor, and Triton X-100 were from Sigma. The mouse HA.11 monoclonal antibody (5 μg/μl) directed against the hemagglutinin (HA) epitope tag engineered into the N terminus of the various V2R structures was obtained from Berkeley Antibody Co. (Richmond, CA). Rat anti-HA monoclonal antibody (100 μg/ml, clone 3F10) against the HA epitope tag was obtained from Roche Applied Science, and the Alexafluor-488-conjugated goat anti-rat IgG (2 μg/μl) was from Molecular Probes (Eugene, OR). Protein A beads were from Vector Laboratories (Burlingame, CA). EZ-link™ sulfo-N-hydroxysuccinimide (NHS)-biotin and Immunopure™ immobilized streptavidin were from Pierce. The 12- and 24.5-mm polycarbonate membrane filters (Transwell chambers, 0.4 μm pore size) were obtained from Costar (Cambridge, MA). Aqua-Poly/Mount was from PolySciences Inc. (Warrington, PA). Dulbecco's modified Eagle's medium (DMEM) and trypsin/EDTA were prepared by the Cell Culture Core facility sponsored by the Diabetes Research and Training Center at Vanderbilt University Medical Center. All other chemicals were reagent grade. Cell Lines—Permanent clonal MDCKII cell lines expressing HA epitope-tagged WT and mutant V2Rs were developed using the CaPO4 method as described previously (7Levine S.D. Kachadorian W.A. Levin D.N. Schlondorff D. J. Clin. Investig. 1981; 67: 662-672Crossref PubMed Scopus (44) Google Scholar). Briefly, 10 μg of V2pcD-N-HA and pCMV4N-V2R-AAA (individual cDNAs encoding HA epitope-tagged human WT or mutant (AAA) V2Rs, respectively) were each co-transfected with 2 μg of pRSVneo (cDNA encoding neomycin resistance) into MDCKII cells. Colonies were selected based on resistance to G418, a neomycin analog, and isolated as described previously (7Levine S.D. Kachadorian W.A. Levin D.N. Schlondorff D. J. Clin. Investig. 1981; 67: 662-672Crossref PubMed Scopus (44) Google Scholar). G418-resistant colonies were screened for WT or AAA V2R expression by assaying binding of the radioligand [3H]AVP. Parental and stably expressing V2R MDCKII cells were maintained in DMEM supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin at 37 °C, 5% CO2. Simian kidney fibroblast (COS M6) cells were maintained in supplemented DMEM + 20 mm HEPES. The studies presented were obtained in the WT V2R, clone 61, which expresses the HA epitope-tagged WT V2R at a density of 4.3 ± 1.0 pmol/mg specific [3H]AVP binding per mg of protein, estimated in homologous competition binding studies. Studies with AAA V2R exploited two independent clonal cell lines, clone 5 (6.7 ± 1.5 pmol/mg protein) and clone 11 (3.2 ± 2.0 pmol/mg protein). Generation of cDNAs Encoding GST-V2R-C Terminus—The cDNAs encoding the GST-FLAG-V2R-C terminus constructs in the pEBG-SrfI vector were generated via overlapping PCR extension using Pfu Turbo DNA polymerase (Stratagene). The FLAG tag was inserted immediately 5′ to the V2R C-terminal sequence and downstream of the GST start site and coding sequence. The AAA V2R construct, containing the holo-V2R receptor in pCMV4, was generated using the QuickChange II site-directed mutagenesis kit (Stratagene). The cDNAs were sequenced in their entirety to confirm that the sequences were correct. Transient Expression Studies—COS M6 cells were seeded the day prior to transfection at a density of 7.5 × 105 (60-mm dish) or 1 × 106 (100-mm dish). On the day of transfection, FuGENE 6 (Roche Applied Science) was placed dropwise into a polypropylene tube containing Opti-MEM (Invitrogen) at 37 °C and incubated for 5 min at room temperature. According to the manufacturer's instructions, 1 μg of plasmid DNA was placed into an additional tube, and the FuGENE mixture was placed dropwise on top of the DNA in a ratio of 3 μl of FuGENE to 1 μg of DNA, incubated at room temperature for 20 min, and placed onto the cells in their existing culture medium. We used 1 μg of DNA per 60-mm dish and 3 μg of DNA per 100-mm dish, respectively. The cells were maintained at 37 °C, 5% CO2 prior to assessment 48 h post-transfection. Calmodulin-linked Agarose Pull-down Experiments—Cells were placed on ice and washed once with DPBS-CM (Dulbecco's phosphate-buffered saline: 137 mm NaCl, 2.7 mm KCl, 10 mm Na2HPO4, 2 mm KH2PO4,1mm MgSO4, 0.5 mm CaCl2, pH 7.3). Cells were extracted into ice-cold dodecyl-β-d-maltoside:cholesteryl hemisuccinate buffer (DβM: CHS: 4 and 0.8 mg/ml, respectively, containing 20% glycerol, 25 mm glycylglycine, 20 mm HEPES, 100 mm NaCl, 5 mm EGTA, 100 μm PMSF, 1 μg/ml soybean trypsin inhibitor, 1 μg/ml leupeptin, pH 7.4), sequentially triturated with 22- and 25-gauge needles, and centrifuged at 100,000 × g for 60 min. The detergent-solubilized supernatant was precleared with BSA linked to agarose beads and incubated overnight with calmodulin-linked agarose (Sigma) that had been precleared with parental cellular lysate that did not express the V2R or V2R-C terminus. The resin was washed three times with DβM:CHS wash buffer (DβM:CHS-wash: 0.5 and 0.1 mg/ml, respectively, containing 25 mm glycylglycine, 20 mm HEPES, 100 mm NaCl, 5 mm EDTA, pH 7.4) and eluted into 1× Laemmli buffer for 15 min at 37 °C. Samples were run on 12% SDS-PAGE gels, transferred to PVDF, and blotted for receptor using either the mouse anti-GST (Santa Cruz Biotechnology; 1:2000) or the mouse anti-HA.11 (Babco; 1:1000) monoclonal antibodies. When binding to calmodulin, agarose was assessed as a function of calcium concentration, and the amount of free calcium present in solution was calculated using the MaxChelator program, version 2.10 (www.stanford.edu/~cpatton/maxc.html). Estimation of V2R-C Terminus Affinity for Calmodulin—Supernatants from COS M6 cells transiently expressing either the WT or AAA GST-FLAG-V2R-C terminus were prepared as described above. The WT V2R or AAA V2R-expressing cells were solubilized by scraping 100-mm dishes (2Morello J.P. Salahpour A. Laperriere A. Bernier V. Arthus M.F. Lonergan M. Petaja-Repo U. Angers S. Morin D. Bichet D.G. Bouvier M. J. Clin. Investig. 2000; 105: 887-895Crossref PubMed Scopus (479) Google Scholar, 3van Balkom B.W. Savelkoul P.J. Markovich D. Hofman E. Nielsen S. van der S.P. Deen P.M. J. Biol. Chem. 2002; 277: 41473-41479Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 4Chou C.L. Yip K.P. Michea L. Kador K. Ferraris J.D. Wade J.B. Knepper M.A. J. Biol. Chem. 2000; 275: 36839-36846Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 5Yip K.P. J. Physiol. (Lond.). 2002; 538: 891-899Crossref Scopus (83) Google Scholar) into 1.5 ml of DβM-CHS extraction buffer. For some experiments, the extract of AAA V2R was concentrated further using Amicon ultracentrifugal filter devices. Serial dilutions of the WT lysate were performed to obtain a saturation binding curve for binding of the C terminus to a constant quantity of calmodulin-linked agarose, and binding to calmodulin-agarose was performed as described above. The amount of GST-C-terminal protein in the eluate and total fractions was determined quantitatively by comparison to known amounts of bacterially expressed GST. The Western blot bands were quantitated using Scion Image software. NMR Sample Preparation and Spectroscopy—A peptide fragment of the V2R C terminus (339LLSSARGRTPPSLGPQDES357 with the cysteines normally acylated in V2R (Cys-341 and Cys-342) mutated to serine) was ordered from Genemed Synthesis and subsequently purified by reversed-phase high pressure liquid chromatography. Purity of the eluted peptide was confirmed by electrospray ionization-mass spectrometry and matrix-assisted laser desorption ionization-mass spectrometry. The final peptide was lyophilized and resuspended at 10 mg/ml in 500 mm Tris, pH 7.0. A bacterial expression vector for human calmodulin was kindly provided by Eva Thulin (University of Lund, Lund, Sweden) (11Vogel H.J. Lindahl L. Thulin E. FEBS Lett. 1983; 157: 241-246Crossref Scopus (66) Google Scholar). The construct was transformed into BL21(DE3)pLysS Escherichia coli, and the bacteria were grown in M9 minimal media supplemented with 15NH4Cl. The bacteria were grown and induced with isopropyl-β-d-thiogalactopyranoside as described previously (11Vogel H.J. Lindahl L. Thulin E. FEBS Lett. 1983; 157: 241-246Crossref Scopus (66) Google Scholar). Briefly, the growth protocol calls for serial dilution of bacteria growing at 30 °C, allowing the bacteria to double prior to each dilution. Following induction at 1 mm isopropyl-β-d-thiogalactopyranoside, the bacteria are incubated at 37 °C until growth stops. The protein was purified as described previously by Ca2+-dependent binding of calmodulin to phenyl-Sepharose. Samples were loaded in 1 mm CaCl2 and eluted at 1 mm EDTA. NMR samples were prepared by three steps of concentration and resuspension (100-fold dilution at each step) to thoroughly exchange purified calmodulin into 20 mm Tris, 100 mm KCl, 1 mm dithiothreitol, pH 7.0, supplemented with either 2 mm EDTA or 20 mm CaCl2. Final samples were adjusted to contain 10% D2O. The calmodulin concentration was determined by A276. Experiments were conducted on a Bruker Avance500 spectrometer equipped with a cryoprobe. Two-dimensional 15N-1H heteronuclear single quantum correlation experiments were performed at 25 °C, acquiring four scans per increment and 512 points in the indirect (t1) dimension. NMR data were processed and analyzed in XWinNMR (Bruker). For the preliminary titration mentioned in the text (data not shown), the peptide was resuspended at 18.3 mg/ml in 100 mm bis-Tris, pH 6.5, and Ca2+-calmodulin was prepared by thoroughly exchanging 15N-calmodulin into 20 mm bis-Tris, 100 mm KCl, 1 mm dithiothreitol, 20 mm CaCl2, pH 6.5. Assessment of Transcellular Leaks of Polarized MDCKII Cells—Integrity of the cell monolayer via [3H]methoxy-inulin leak was accomplished exactly as described previously (12Keefer J.R. Limbird L.E. J. Biol. Chem. 1993; 268: 11340-11347Abstract Full Text PDF PubMed Google Scholar). Polarized Localization of V2R at Steady State—For these studies, the appropriate number of 24.5-mm Transwells of polarized (7 days growth) WT or mutant V2R were biotinylated with NHS-Biotin (1 mg/ml) on the apical or basolateral surface as described previously (12Keefer J.R. Limbird L.E. J. Biol. Chem. 1993; 268: 11340-11347Abstract Full Text PDF PubMed Google Scholar). The cells were scraped into RIPA buffer (150 mm NaCl, 50 mm Tris, pH 8.0, 5 mm EDTA, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, pH 7.4) with protease inhibitors (100 μm PMSF, 1 μg/ml soybean trypsin inhibitor, 1 μg/ml leupeptin) present, and the extracts were centrifuged at 100,000 × g. The supernatant (detergent-solubilized preparation) was incubated overnight at 4 °C with streptavidin-agarose. The streptavidin-agarose resin was washed 2× with RIPA buffer with protease inhibitors and eluted with SDS sample buffer (1.6% SDS, 8.3% glycerol, 167 mm Tris, pH 6.8) for 10 min at 95 °C, and the eluate was resolved by SDS-PAGE on 12% gels. The resolved proteins, representing the fate of the V2R at the cell surface at the time of biotinylation, were transferred to PVDF, and the biotinylated, epitope-tagged V2R was identified by Western blot analysis using mouse HA.11 antibody against the HA epitope. Cell Surface Receptor Quantitation via Intact Cell ELISA—MDCKII cells stably expressing WT V2R or AAA V2R were seeded at 1 × 104 cells per well of a 96-well plate 1–2 days prior to the experiment. The medium was replaced with serum-free DMEM 20 min before the assay. Cells were treated with either vehicle alone or with 1 μm AVP and placed in the incubator at 37 °C and 5% CO2 throughout the drug treatment. The vehicle was 50 mm Tris, pH 7.4, 3 mm MgCl2, 1 mm EDTA, pH 8.0, 0.1% BSA, 1 mg/ml bacitracin. After treatment, the medium was removed, and the cells were fixed with 4% paraformaldehyde containing 0.12 m sucrose in DPBS-CM for 20 min at room temperature. Cells were washed twice with DPBS-CM for 5 min each and treated with 3% BSA in DPBS-CM to block nonspecific antibody binding for 30 min at 37 °C. Cells were incubated with 1:500 of anti-rat HA (Roche Applied Science) in 3% BSA in DPBS-CM for 1 h at 37 °C and washed three times with DPBS-CM. Cells were incubated with 1:100 horseradish peroxidase-conjugated goat anti-rat (Amersham Biosciences) in 3% BSA in DPBS-CM for 1 h at 37 °C. The colorimetric substrate, o-phenylenediamine dihydrochloride (Pierce, 1 mg/ml), was added and incubated for 30 min to 1 h at room temperature. The reaction was stopped with the addition of an equal volume of 1 m sulfuric acid. The absorbance at 490 nm was determined for each well using a SpectraMAX 190 microtiter plate reader (Molecular Devices). Four replicates of each treatment condition were performed per experiment. Immunolocalization of the Wild Type and Mutant V2R—Stably expressing WT V2R MDCKII cells were grown in clear 12-mm Transwell chambers and maintained for 6–9 days prior to fixing with 4% paraformaldehyde for 20 min. Paraformaldehyde-fixed cells were washed in DPBS-CM, rinsed with 50 mm NH4Cl in DPBS-CM for 15 min, and permeabilized with 0.2% Triton X-100 in DPBS-CM for 15 min at room temperature. Permeabilized cells were washed once with DPBS-CM and incubated with DPBS-CM containing 2% BSA for 30 min. The Transwells were carefully cut away from their plastic support and placed into a 12-well plate for antibody labeling. The cells were incubated with a 1:500 dilution of rat anti-HA monoclonal primary antibody for 1 h at room temperature. After rinsing cells three times in DPBS-CM (10 min/wash), a 1:1000 dilution of Alexa488-conjugated goat anti-rat IgG in DPBS-CM with 2% BSA was added, and the cells were incubated in the dark for 1 h at room temperature. Cells were then washed prior to mounting on glass slides with Aqua-Poly/Mount and covered with a glass coverslip. Slides were stored in the dark until examination with a Zeiss LSM 510 confocal laser scanning inverted microscope in the Vanderbilt Cell Imaging Core Facility. For the AVP-mediated internalization studies, polarized cells were treated either with vehicle alone or with 1 μm AVP, which was added to both the apical and basolateral chambers of the Transwells, and incubated at 37 °C and 5% CO2 throughout the drug treatment. After treatment, the medium was removed, and the cells were fixed and labeled for immunofluorescence examination as described. Competition Binding—To assess the potency of AVP in competing for the WT V2R and AAA V2R, [3H]AVP binding assays were performed. Confluent 100-mm dishes of MDCKII cells stably expressing the WT V2R or AAA V2R were placed on ice and washed once with DPBS-CM. The cells were scraped into 15:5:5 buffer (15 mm HEPES, 5 mm EGTA, 5 mm EDTA, pH 7.6) containing 100 μm PMSF and centrifuged at 18,000 rpm in an SS-34 rotor for 15 min. The pellet was resuspended in 15:5:5 buffer by sequential trituration with 22- and 25-gauge needles. Centrifugation and resuspension was repeated one time. Finally, the pellet was resuspended in 50 mm Tris, pH 7.4, 3 mm MgCl2, 1 mm EDTA, pH 8.0, 0.1% BSA, and 1 mg/ml bacitracin, which was included to protect the radioligand from proteolysis. Membranes were incubated, with shaking, for 1 h at room temperature in the presence of 1.4 nm [3H]AVP. The incubation was terminated by harvesting on a Brandel cell harvester. Nonspecific binding was defined as that binding detected in the presence of 10 μm AVP. The samples were counted using a Packard 1600 TR Tri-Carb scintillation counter. Kd and Bmax values were calculated from these homologous competition binding experiments as described by Limbird and Motulsky (13Limbird L.E. Motulsky H. Conn P.M. Goodman H.M. Handbook of Physiology: The Endocrine System Cellular Endocrinology. Oxford University Press, Inc., Oxford1998: 49-67Google Scholar). cAMP Accumulation—Basal or AVP-mediated cAMP accumulation in intact cells was measured by assessing the conversion of [3H]ATP into [3H]cAMP in cells prelabeled with [3H]adenine to permit synthesis of [3H]ATP intracellularly. MDCKII cells were seeded into 24-well plates at 1 × 105 cells per well. Twenty four hours later, the cells were labeled overnight (12–16 h at 37 °C) in supplemented DMEM containing 6 μCi/ml of [3H]adenine (PerkinElmer Life Sciences or MP Biomedicals). On the day of the assay, cells were placed on a 37 °C plate and rinsed with DPBS-CM, and serum-free DMEM supplemented with 20 mm HEPES was added. Cells were treated with AVP for 15 min, with vehicle alone to obtain basal cAMP levels, or with 10 μm forskolin to assess direct activation of adenylyl cyclase. Reactions were terminated by adding 750 μl of an ice-cold solution containing 12% trichloroacetic acid, 2 mm cAMP, 2 mm ATP, and [14C]cAMP (∼1400 cpm/ml) to permit an assessment of recovery of [3H]cAMP in subsequent purification steps. Cells were then placed on ice for 10 min, after which the reactions were transferred to glass test tubes using disposable transfer pipettes. Each well was washed with 120 μl of DMEM supplemented with 20 mm HEPES and combined with its respective harvested sample for a final volume of 2 ml. After neutralization with 120 μl of 5 n NaOH, cellular debris was pelleted by centrifugation (3000 rpm, 10 min at 4 °C). The supernatants were subjected to sequential Dowex and alumina column chromatography to isolate [3H]cAMP (14Salomon Y. Londos C. Rodbell M. Anal. Biochem. 1974; 58: 541-548Crossref PubMed Scopus (3374) Google Scholar). Recovery of [3H]cAMP from other triturated nucleotides during this procedure was corrected based on the recovery of [14C]cAMP tracer in the stop solution. Measurement of Intracellular Ca2+—We used a FlexStation II (Molecular Devices) fluorometric imaging plate reader to measure changes in intracellular Ca2+. MDCKII cells stably expressing the WT V2R or AAA V2R were plated in 96-well clear bottom black microplates (Corning Glass) at ∼1 × 105 cells per well and incubated overnight at 37 °C and 5% CO2. On the day of the assay, cells were loaded with Calcium 3 dye (FLIPR Calcium 3 assay kit, Molecular Devices) for 1 h at 37 °C in Hanks' buffered saline solution (HBSS), pH 7.4, containing 0.1% BSA and 2.5 mm probenecid, in the presence (Invitrogen catalog number 14065-056) of 1.26 mm calcium. After loading, cells were washed three times with 1× HBSS, pH 7.4, containing 0.1% BSA and 2.5 mm probenecid lacking calcium (Invitrogen catalog number 14185-052, supplemented with 0.9 mm magnesium). Cells were placed in 1× HBSS with or without calcium as indicated and placed in the FlexStation II for the assay. The machine was used in Flex mode, and the fluorescent intensity was measured from the bottom with the excitation at 485 nm and emission at 525 nm for 90 s at 25 °C. Trituration upon addition of compound during the FlexStation assay had no effect on the position of the AVP concentration-response curve for the WT V2R. The V2R C Terminus Interacts with Calmodulin in Vitro in a Calcium-dependent Manner—To investigate mechanisms involved in the processing and signaling of the V2R, we searched for proteins that could associate with the C terminus of the receptor, as this region had been implicated both in trafficking (9Schulein R. Hermosilla R. Oksche A. Dehe M. Wiesner B. Krause G. Rosenthal W. Mol. Pharmacol. 1998; 54: 525-535Crossref PubMed Scopus (133) Google Scholar) and signaling (15Wess J. Pharmacol. Ther. 1998; 80: 231-264Crossref PubMed Scopus (371) Google Scholar). In preliminary studies using yeast two-hybrid technology, we identified calmodulin as a potential interacting molecule with the V2R C terminus. Given recent reports that other GPCRs also interact with calmodulin (16Turner J.H. Gelasco A.K. Raymond J.R. J. Biol. Chem. 2004; 279: 17027-17037Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 17Nakajima Y. Yamamoto T. Nakayama T. Nakanishi S. J. Biol. Chem. 1999; 274: 27573-27577Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 18Minakami R. Jinnai N. Sugiyama H. J. Biol. Chem. 1997; 272: 20291-20298Abstract Full Text Full Text PDF PubMed Scopus (124) Goo" @default.
• W2035917312 created "2016-06-24" @default.
• W2035917312 creator A5003392314 @default.
• W2035917312 creator A5029162349 @default.
• W2035917312 creator A5086900416 @default.
• W2035917312 creator A5089716121 @default.
• W2035917312 date "2004-11-01" @default.
• W2035917312 modified "2023-09-27" @default.
• W2035917312 title "Calmodulin Interacts with the V2 Vasopressin Receptor" @default.
• W2035917312 cites W1532176495 @default.
• W2035917312 cites W1767701883 @default.
• W2035917312 cites W1968890466 @default.
• W2035917312 cites W1968901680 @default.
• W2035917312 cites W1989968095 @default.
• W2035917312 cites W1990398484 @default.
• W2035917312 cites W1991977222 @default.
• W2035917312 cites W1996591851 @default.
• W2035917312 cites W2009504616 @default.
• W2035917312 cites W2012353885 @default.
• W2035917312 cites W2016595934 @default.
• W2035917312 cites W2020828783 @default.
• W2035917312 cites W2023917526 @default.
• W2035917312 cites W2027567709 @default.
• W2035917312 cites W2028405047 @default.
• W2035917312 cites W2029474878 @default.
• W2035917312 cites W2038422963 @default.
• W2035917312 cites W2049170665 @default.
• W2035917312 cites W2049174331 @default.
• W2035917312 cites W2052198628 @default.
• W2035917312 cites W2074936851 @default.
• W2035917312 cites W2089334699 @default.
• W2035917312 cites W2090664128 @default.
• W2035917312 cites W2099529253 @default.
• W2035917312 cites W2117815723 @default.
• W2035917312 cites W2120385383 @default.
• W2035917312 cites W2124334538 @default.
• W2035917312 cites W2157907012 @default.
• W2035917312 cites W2159672893 @default.
• W2035917312 cites W2164177319 @default.
• W2035917312 cites W2169503005 @default.
• W2035917312 cites W2217392873 @default.
• W2035917312 doi "https://doi.org/10.1074/jbc.m407351200" @default.
• W2035917312 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/15319442" @default.
• W2035917312 hasPublicationYear "2004" @default.
• W2035917312 type Work @default.
• W2035917312 sameAs 2035917312 @default.
• W2035917312 citedByCount "31" @default.
• W2035917312 countsByYear W20359173122012 @default.
• W2035917312 countsByYear W20359173122013 @default.
• W2035917312 countsByYear W20359173122014 @default.
• W2035917312 countsByYear W20359173122015 @default.
• W2035917312 countsByYear W20359173122020 @default.
• W2035917312 countsByYear W20359173122021 @default.
• W2035917312 crossrefType "journal-article" @default.
• W2035917312 hasAuthorship W2035917312A5003392314 @default.
• W2035917312 hasAuthorship W2035917312A5029162349 @default.
• W2035917312 hasAuthorship W2035917312A5086900416 @default.
• W2035917312 hasAuthorship W2035917312A5089716121 @default.
• W2035917312 hasBestOaLocation W20359173121 @default.
• W2035917312 hasConcept C134018914 @default.
• W2035917312 hasConcept C170493617 @default.
• W2035917312 hasConcept C181199279 @default.
• W2035917312 hasConcept C185592680 @default.
• W2035917312 hasConcept C20352681 @default.
• W2035917312 hasConcept C2776370428 @default.
• W2035917312 hasConcept C2776885963 @default.
• W2035917312 hasConcept C29688787 @default.
• W2035917312 hasConcept C55493867 @default.
• W2035917312 hasConcept C86803240 @default.
• W2035917312 hasConcept C90474776 @default.
• W2035917312 hasConcept C95444343 @default.
• W2035917312 hasConceptScore W2035917312C134018914 @default.
• W2035917312 hasConceptScore W2035917312C170493617 @default.
• W2035917312 hasConceptScore W2035917312C181199279 @default.
• W2035917312 hasConceptScore W2035917312C185592680 @default.
• W2035917312 hasConceptScore W2035917312C20352681 @default.
• W2035917312 hasConceptScore W2035917312C2776370428 @default.
• W2035917312 hasConceptScore W2035917312C2776885963 @default.
• W2035917312 hasConceptScore W2035917312C29688787 @default.
• W2035917312 hasConceptScore W2035917312C55493867 @default.
• W2035917312 hasConceptScore W2035917312C86803240 @default.
• W2035917312 hasConceptScore W2035917312C90474776 @default.
• W2035917312 hasConceptScore W2035917312C95444343 @default.
• W2035917312 hasIssue "45" @default.
• W2035917312 hasLocation W20359173121 @default.
• W2035917312 hasOpenAccess W2035917312 @default.
• W2035917312 hasPrimaryLocation W20359173121 @default.
• W2035917312 hasRelatedWork W1973811817 @default.
• W2035917312 hasRelatedWork W1993414773 @default.
• W2035917312 hasRelatedWork W1996846474 @default.
• W2035917312 hasRelatedWork W2007102871 @default.
• W2035917312 hasRelatedWork W2030077661 @default.
• W2035917312 hasRelatedWork W2033116575 @default.
• W2035917312 hasRelatedWork W2144939206 @default.
• W2035917312 hasRelatedWork W2397094813 @default.
• W2035917312 hasRelatedWork W2406023723 @default.
• W2035917312 hasRelatedWork W2414761485 @default.
• W2035917312 hasVolume "279" @default.

• next