Cysteinyl leukotrienes activate the cysteinyl leukotriene type 1 receptor (CysLT1R) to regulate numerous cell functions important in inflammatory processes and diseases such as asthma. Despite its physiologic importance, no studies to date have examined the regulation of CysLT1R signaling or trafficking. We have established model systems for analyzing recombinant human CysLT1R and found regulation of internalization and signaling of the CysLT1R to be unique among G protein-coupled receptors. Rapid and profound LTD4-stimulated internalization was observed for the wild type (WT) CysLT1R, whereas a C-terminal truncation mutant exhibited impaired internalization yet signaled robustly, suggesting a region within amino acids 310–321 as critical to internalization. Although overexpression of WT arrestins significantly increased WT CysLT1R internalization, expression of dominant-negative arrestins had minimal effects, and WT CysLT1R internalized in murine embryonic fibroblasts lacking both arrestin-2 and arrestin-3, suggesting that arrestins are not the primary physiologic regulators of CysLT1Rs. Instead, pharmacologic inhibition of protein kinase C (PKC) was shown to profoundly inhibit CysLT1R internalization while greatly increasing both phosphoinositide (PI) production and calcium mobilization stimulated by LTD4 yet had almost no effect on H1 histamine receptor internalization or signaling. Moreover, mutation of putative PKC phosphorylation sites within the CysLT1R C-tail (CysLT1RS(313–316)A) reduced receptor internalization, increased PI production and calcium mobilization by LTD4, and significantly attenuated the effects of PKC inhibition. These findings characterized the CysLT1R as the first G protein-coupled receptor identified to date in which PKC is the principal regulator of both rapid agonist-dependent internalization and rapid agonist-dependent desensitization. Cysteinyl leukotrienes activate the cysteinyl leukotriene type 1 receptor (CysLT1R) to regulate numerous cell functions important in inflammatory processes and diseases such as asthma. Despite its physiologic importance, no studies to date have examined the regulation of CysLT1R signaling or trafficking. We have established model systems for analyzing recombinant human CysLT1R and found regulation of internalization and signaling of the CysLT1R to be unique among G protein-coupled receptors. Rapid and profound LTD4-stimulated internalization was observed for the wild type (WT) CysLT1R, whereas a C-terminal truncation mutant exhibited impaired internalization yet signaled robustly, suggesting a region within amino acids 310–321 as critical to internalization. Although overexpression of WT arrestins significantly increased WT CysLT1R internalization, expression of dominant-negative arrestins had minimal effects, and WT CysLT1R internalized in murine embryonic fibroblasts lacking both arrestin-2 and arrestin-3, suggesting that arrestins are not the primary physiologic regulators of CysLT1Rs. Instead, pharmacologic inhibition of protein kinase C (PKC) was shown to profoundly inhibit CysLT1R internalization while greatly increasing both phosphoinositide (PI) production and calcium mobilization stimulated by LTD4 yet had almost no effect on H1 histamine receptor internalization or signaling. Moreover, mutation of putative PKC phosphorylation sites within the CysLT1R C-tail (CysLT1RS(313–316)A) reduced receptor internalization, increased PI production and calcium mobilization by LTD4, and significantly attenuated the effects of PKC inhibition. These findings characterized the CysLT1R as the first G protein-coupled receptor identified to date in which PKC is the principal regulator of both rapid agonist-dependent internalization and rapid agonist-dependent desensitization. The cysteinyl (Cys) 1The abbreviations used are: Cys, cysteinyl; LT, leukotriene; CysLT1R, CysLT type 1 receptor; GPCR, G protein-coupled receptor; β2AR, β2-adrenergic receptor; GRK, GPCR kinase; PI, phosphoinositide; PKC, protein kinase C; PKA, protein kinase A; MEF, murine embryonic fibroblast; H1 HR, H1 histamine receptor; DMEM, Dulbecco's modified Eagle's medium; PMA, phorbol 12-myristate 13-acetate; Bis, bisindolylmaleimide; TP, thromboxane A2; GFP, green fluorescent protein; ELISA, enzyme-linked immunosorbent assay.1The abbreviations used are: Cys, cysteinyl; LT, leukotriene; CysLT1R, CysLT type 1 receptor; GPCR, G protein-coupled receptor; β2AR, β2-adrenergic receptor; GRK, GPCR kinase; PI, phosphoinositide; PKC, protein kinase C; PKA, protein kinase A; MEF, murine embryonic fibroblast; H1 HR, H1 histamine receptor; DMEM, Dulbecco's modified Eagle's medium; PMA, phorbol 12-myristate 13-acetate; Bis, bisindolylmaleimide; TP, thromboxane A2; GFP, green fluorescent protein; ELISA, enzyme-linked immunosorbent assay. leukotriene (LT) LTC4, and its conversion products LTD4 and LTE4, regulate numerous cell and organ system functions (1Lewis R.A. Austen K.F. Soberman R.J. N. Engl. J. Med. 1990; 323: 645-655Crossref PubMed Scopus (1167) Google Scholar, 2Samuelsson B. Science. 1983; 220: 568-575Crossref PubMed Scopus (2311) Google Scholar). Most notably, CysLTs have been identified as important mediators of asthmatic attacks and asthma pathogenesis; they are potent bronchoconstrictors (3Barnes N.C. Piper P.J. Costello J.F. Thorax. 1984; 39: 500-504Crossref PubMed Scopus (253) Google Scholar), and also seem important in modulating airway inflammation and remodeling (4Holgate S.T. Peters-Golden M. Panettieri R.A. Henderson Jr., W.R. J. Allergy Clin. Immunol. 2003; 111: S18-S36Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar). Despite the detection of specific LTD4 binding to guinea pig lung membranes in 1993 by Metters et al. (5Metters K.M. Zamboni R.J. J. Biol. Chem. 1993; 268: 6487-6495Abstract Full Text PDF PubMed Google Scholar), the cloning of a high affinity CysLT receptor was frustrated for years, until the ultimate reporting of the human CysLT type 1 receptor (CysLT1R) by Lynch et al. (6Lynch K.R. O'Neill G.P. Liu Q. Im D.S. Sawyer N. Metters K.M. Coulombe N. Abramovitz M. Figueroa D.J. Zeng Z. Connolly B.M. Bai C. Austin C.P. Chateauneuf A. Stocco R. Greig G.M. Kargman S. Hooks S.B. Hosfield E. Williams Jr., D.L. Ford-Hutchinson A.W. Caskey C.T. Evans J.F. Nature. 1999; 399: 789-793Crossref PubMed Scopus (882) Google Scholar) and Saura et al. (7Sarau H.M. Ames R.S. Chambers J. Ellis C. Elshourbagy N. Foley J.J. Schmidt D.B. Muccitelli R.M. Jenkins O. Murdock P.R. Herrity N.C. Halsey W. Sathe G. Muir A.I. Nuthulaganti P. Dytko G.M. Buckley P.T. Wilson S. Bergsma D.J. Hay D.W. Mol. Pharmacol. 1999; 56: 657-663Crossref PubMed Scopus (303) Google Scholar) in 1999. The CysLT1R is expressed in spleen, peripheral blood leukocytes, and airway smooth muscle, has nanomolar affinity for LTD4, and couples to the heterotrimeric G protein Gq to promote calcium flux. LTC4 is also a full agonist of the CysLT1R but is 10 times less potent. A second CysLT receptor subtype, CysLT type 2 receptor, has recently been cloned (8Heise C.E. O'Dowd B.F. Figueroa D.J. Sawyer N. Nguyen T. Im D.S. Stocco R. Bellefeuille J.N. Abramovitz M. Cheng R. Williams Jr., D.L. Zeng Z. Liu Q. Ma L. Clements M.K. Coulombe N. Liu Y. Austin C.P. George S.R. O'Neill G.P. Metters K.M. Lynch K.R. Evans J.F. J. Biol. Chem. 2000; 275: 30531-30536Abstract Full Text Full Text PDF PubMed Scopus (578) Google Scholar, 9Nothacker H.P. Wang Z. Zhu Y. Reinscheid R.K. Lin S.H. Civelli O. Mol. Pharmacol. 2000; 58: 1601-1608Crossref PubMed Scopus (163) Google Scholar). CysLT type 2 receptor is expressed in leukocytes, heart, and brain, and binds LTD4 and LTC4 with equal affinity. Although no specific CysLT type 2 receptor antagonists currently exist, CysLT1R antagonists have been established as effective anti-asthma drugs (10Peters S.P. J. Allergy Clin. Immunol. 2003; 111: S62-S70Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 11Drazen J.M. Proc. Assoc. Am. Physicians. 1999; 111: 547-559Crossref PubMed Scopus (30) Google Scholar).Since the initial characterization of the CysLT1R only a handful of studies, focused primarily on pharmacologic properties, have been published examining this receptor. This lack of studies is due primarily to difficulties in expressing recombinant CysLT1R in mammalian cells (6Lynch K.R. O'Neill G.P. Liu Q. Im D.S. Sawyer N. Metters K.M. Coulombe N. Abramovitz M. Figueroa D.J. Zeng Z. Connolly B.M. Bai C. Austin C.P. Chateauneuf A. Stocco R. Greig G.M. Kargman S. Hooks S.B. Hosfield E. Williams Jr., D.L. Ford-Hutchinson A.W. Caskey C.T. Evans J.F. Nature. 1999; 399: 789-793Crossref PubMed Scopus (882) Google Scholar, 12Figueroa D.J. Breyer R.M. Defoe S.K. Kargman S. Daugherty B.L. Waldburger K. Liu Q. Clements M. Zeng Z. O'Neill G.P. Jones T.R. Lynch K.R. Austin C.P. Evans J.F. Am. J. Respir. Crit. Care Med. 2001; 163: 226-233Crossref PubMed Google Scholar). Although expression of recombinant CysLT1R in HEK 293T cells appears useful for high-throughput screening of potential CysLT1R ligands using an automated assay of intracellular calcium mobilization (7Sarau H.M. Ames R.S. Chambers J. Ellis C. Elshourbagy N. Foley J.J. Schmidt D.B. Muccitelli R.M. Jenkins O. Murdock P.R. Herrity N.C. Halsey W. Sathe G. Muir A.I. Nuthulaganti P. Dytko G.M. Buckley P.T. Wilson S. Bergsma D.J. Hay D.W. Mol. Pharmacol. 1999; 56: 657-663Crossref PubMed Scopus (303) Google Scholar, 9Nothacker H.P. Wang Z. Zhu Y. Reinscheid R.K. Lin S.H. Civelli O. Mol. Pharmacol. 2000; 58: 1601-1608Crossref PubMed Scopus (163) Google Scholar), features of the CysLT1R beyond basic pharmacologic receptor-ligand interactions remain uncharacterized.Understanding the mechanisms by which the responsiveness of a given G protein-coupled receptor (GPCR) is regulated not only can provide insight into the functional impact of the receptor in physiologic and disease states but also can identify regulatory molecules as potential therapeutic targets (13Koch W.J. J. Card Fail. 2002; 8: S526-S531Abstract Full Text PDF PubMed Scopus (4) Google Scholar, 14Lefkowitz R.J. Nat. Biotechnol. 1996; 14: 283-286Crossref PubMed Scopus (40) Google Scholar, 15Penn R.B. Pronin A.P. Benovic J.L. Trends Cardiovasc. Med. 2000; 10: 81-89Crossref PubMed Scopus (185) Google Scholar). For example, the responsiveness of the β2-adrenergic receptor (β2AR) is tightly regulated by phosphorylation of the receptor by GPCR kinases (GRKs) and the subsequent binding of arrestin proteins. GRK-mediated phosphorylation partially uncouples the β2AR from Gs and promotes the binding of arrestins to the receptor, which in turn sterically inhibits β2AR-Gs interaction while promoting receptor internalization into endocytotic vesicles. Sensitivity of the β2AR to this mode of regulation may explain a differential efficacy of β-agonists among airway cells that influences airway function and response to therapy (16McGraw D.W. Liggett S.B. J. Biol. Chem. 1997; 272: 7338-7344Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar) and may contribute to the pathology of chronic heart failure, in which β2AR hyporesponsiveness is associated with elevated GRK levels in cardiac myocytes (17Koch W.J. Lefkowitz R.J. Rockman H.A. Annu. Rev. Physiol. 2000; 62: 237-260Crossref PubMed Scopus (135) Google Scholar). Importantly, cardiac expression of a GRK2 "minigene" that effectively inhibits GRK2 activity can reverse β2AR hyporesponsiveness and the pathologic phenotype in animal models of heart failure (18White D.C. Hata J.A. Shah A.S. Glower D.D. Lefkowitz R.J. Koch W.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5428-5433Crossref PubMed Scopus (196) Google Scholar, 19Rockman H.A. Chien K.R. Choi D.J. Iaccarino G. Hunter J.J. Ross Jr., J. Lefkowitz R.J. Koch W.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7000-7005Crossref PubMed Scopus (431) Google Scholar, 20Koch W.J. Rockman H.A. Samama P. Hamilton R.A. Bond R.A. Milano C.A. Lefkowitz R.J. Science. 1995; 268: 1350-1353Crossref PubMed Scopus (635) Google Scholar), thereby establishing the utility of targeting GRK2, and possibly other GPCR regulatory molecules, in disease therapy. Moreover, differential sensitivity to GRK/arrestin-mediated regulation seems to explain, in part, differences in the signaling capacity and functional effects among GPCRs in a given cell type. The prostaglandin E2 EP2 receptor, which is resistant to GRK-mediated phosphorylation and arrestin binding, is much more efficacious than the β2AR in stimulating cAMP production in analyses of both recombinant and endogenous receptors (21Penn R.B. Pascual R.M. Kim Y.-M. Mundell S.J. Krymskaya V.P. Panettieri Jr., R.A. Benovic J.L. J. Biol. Chem. 2001; 276: 32648-32656Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). The enhanced signaling capacity of EP2 receptors in human airway smooth muscle likely contributes to the significantly greater effect of prostaglandin E2 (relative to β-agonists) in modulating growth, migration, and contraction of human airway smooth muscle (22Goncharova E.A. Billington C.K. Irani C. Vorotnikov A.V. Tkachuk V.A. Penn R.B. Krymskaya V.P. Panettieri Jr., R.A. Am. J. Respir. Cell Mol. Biol. 2003; 29: 19-27Crossref PubMed Scopus (93) Google Scholar, 23Billington C.K. Penn R.B. Respir. Res. 2003; 4: 2Crossref PubMed Google Scholar, 24Tilley S.L. Hartney J.M. Erikson C.J. Jania C. Nguyen M. Stock J. McNeisch J. Valancius C. Panettieri Jr., R.A. Penn R.B. Koller B.H. Am. J. Physiol. 2003; 284: L599-L606Crossref PubMed Scopus (108) Google Scholar).In the current study, we have provided mechanistic insight into the regulatory features of the signaling and trafficking of the human CysLT1R. Results demonstrated that the CysLT1R undergoes rapid agonist-dependent internalization, yet, unlike most GPCRs characterized to date, this effect appeared largely GRK- and arrestin-independent. Instead, internalization and desensitization were most dramatically affected by PKC activity, characterizing the CysLT1R as the only GPCR examined to date in which both agonist-dependent internalization and desensitization are primarily PKC-dependent phenomena.EXPERIMENTAL PROCEDURESMaterials—FuGENE 6 transfection reagent and all reagents used for cloning were purchased from Roche Applied Science. Anti-CysLT1R antibody and LTD4 were purchased from Cayman Chemical Company (Ann Arbor, MI). [3H]LTD4 (183 Ci/mmol) and myo-[2-3H]-inositol (10–25 Ci/mmol) were purchased from PerkinElmer Life Sciences. All bisindolylmaleimide (Bis) compounds and MK-571 were purchased from Calbiochem. Anti-FLAG M1 and M2 antibodies were purchased from Sigma. Anti-PKCα antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-GFP antibody was from Covance (Princeton, NJ). A construct encoding PKCβ-GFP was purchased from Clontech. The HG55 cDNA (6Lynch K.R. O'Neill G.P. Liu Q. Im D.S. Sawyer N. Metters K.M. Coulombe N. Abramovitz M. Figueroa D.J. Zeng Z. Connolly B.M. Bai C. Austin C.P. Chateauneuf A. Stocco R. Greig G.M. Kargman S. Hooks S.B. Hosfield E. Williams Jr., D.L. Ford-Hutchinson A.W. Caskey C.T. Evans J.F. Nature. 1999; 399: 789-793Crossref PubMed Scopus (882) Google Scholar) encoding human CysLT1R was provided by Jilly Evans of Merck (West Point, PA). Murine embryonic fibroblast (MEF) cultures, derived from transgenic mice in which arrestin-2 and arrestin-3 expression were ablated (arr2/3–/–) and from paired nontransgenic controls, were provided by Robert Lefkowitz (Duke University, Durham, NC). Adenovirus shuttle plasmids and corresponding viral vectors pAdEGI and pAdVgRXR (25Johns D.C. Marx R. Mains R.E. O'Rourke B. Marban E. J Neurosci. 1999; 19: 1691-1697Crossref PubMed Google Scholar) were provided by David Johns (Johns Hopkins University, Baltimore, MD).Sources of other reagents either are identified below or are from previously identified sources (21Penn R.B. Pascual R.M. Kim Y.-M. Mundell S.J. Krymskaya V.P. Panettieri Jr., R.A. Benovic J.L. J. Biol. Chem. 2001; 276: 32648-32656Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar).Generation of Receptor Constructs—More detailed descriptions of construct generation are provided in supplemental material. A signal sequence (26Guan X.M. Kobilka T.S. Kobilka B.K. J. Biol. Chem. 1992; 267: 21995-21998Abstract Full Text PDF PubMed Google Scholar) was inserted upstream of the 3-FLAG cassette in pcDNA3–3FLAG, and this plasmid was used to generate all WT and mutant CysLT1R clones. The open reading frame encoding the human CysLT1R in HG55 was amplified by PCR and inserted in-frame immediately downstream of the 3-FLAG cassette. C-terminal truncation mutants CysLT1R321stop, CysLT1R309stop, and CysLT1R300stop, as well as CysLT1RS(313–316)A, in which serines 313, 315, and 316 were mutated to alanines, were similarly generated by PCR cloning with specific antisense primers. Generation of pcDNA3 plasmid encoding FLAG-tagged human H1 histamine receptor (H1 HR) in pcDNA3 was described previously (27Iwata K. Luo J. Penn R.B. Benovic J.L. J. Biol. Chem. 2005; 280: 2197-2204Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar).The sequence encoding the CysLT1R open reading frame, the signal sequence motif, and the 3-FLAG epitope were PCR-amplified and cloned into the shuttle vector pAdEGI to make pAdEGI-CysLT1R. Recombinant adenovirus was generated by co-transfection of pAdEGI-CysLT1R DNA with ψ5 viral DNA as described previously (25Johns D.C. Marx R. Mains R.E. O'Rourke B. Marban E. J Neurosci. 1999; 19: 1691-1697Crossref PubMed Google Scholar). The resulting virus, AdEGI-CysLT1R, was plaque-purified, expanded, and purified by two rounds of cesium chloride density centrifugation followed by exhaustive dialysis against 10 mm Tris, pH 8.0, 140 mm NaCl, 1 mm MgCl2, and 5% w/v sucrose. Viral particle numbers were determined by dilution in 0.1% SDS followed by measuring absorbance at 260 nm, and viral titers were determined by plaque analysis on 911 cells. The particle to infective/particle ratio was between 25 and 45 for all of the viruses used. Sequence fidelity of all of the clones was verified by dideoxynucleotide sequencing.Cell Culture, Transfection, Infection—HEK 293 cells and COS-1 cells were maintained in Dulbecco's modified Eagles medium (DMEM) supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. Cells were plated into 60-mm dishes 1 day prior to transfection so that they were 65–75% confluent immediately prior to transfection. All transfections were carried out using 10 μl of FuGENE 6 transfection reagent and 3 μg of total DNA. Eighteen h after transfection, cells were harvested and plated on 6- or 24-well plates for subsequent immunoblot analysis, receptor sequestration ELISA, analysis of phosphoinositide (PI) production, or onto poly-l-lysine-coated coverslips for immunocytochemical analysis of receptor subcellular distribution. For transfection of MEFs with pcDNA3-FLAGβ2AR, 20 μl of Lipofectamine 2000 (Invitrogen) in 500 μl of OptiMEM (Invitrogen) were mixed with 8 μg of pcDNA3-FLAGβ2AR, and the mixture was added dropwise to MEFs seeded in 60-mm dishes; the cells were passaged 24 h later into 24-well plates. For infection of MEFs using AdEGI-CysLT1R, MEFs were harvested with trypsin/EDTA, washed in phosphate-buffered saline, and resuspended in DMEM/10% fetal bovine serum medium lacking antibiotics. 1 × 1010 particles of CysLT1R adenovirus plus 5 × 109 particles of receptor plasmid VgRXR adenovirus, or 5 × 109 particles of VgRXR alone, were combined with 500,000 cells and were plated into 60-mm dishes. Fifteen h later, cells were again harvested, washed, and plated into 24-well plates in the presence of 10 μm (final concentration) ponasterone to induce expression. Twenty-four h later, receptor internalization was assessed by ELISA as described below.Receptor Internalization—Agonist-induced changes in cell surface receptor distribution were measured by ELISA. Forty-eight h after transfection, cells in 24-well plates were washed once and then incubated at 37 °C for 30 min in plain DMEM with various inhibitors or vehicle (typically 0.03% Me2SO). After pretreatment, cells were stimulated with LTD4, histamine, isoproterenol, or vehicle (typically 0.05% EtOH) for 0–30 min. The medium was aspirated, and cells were fixed with 3.7% formaldehyde for 10 min at room temperature, washed three times with Tris-buffered saline, and then blocked for 45 min with 1% bovine serum albumin in Tris-buffered saline. Cells were then incubated for 1 h with a 1:1200 dilution of an anti-Flag M2 antibody conjugated to alkaline phosphatase, washed three times with Tris-buffered saline, and then incubated with alkaline phosphatase substrate at 37 °C until adequate color development was visible. Reactions were stopped by adding 0.1 ml from each sample well into 0.1 ml of 0.4 m NaOH, and the sample absorbances were read at 405 nm using a Bio-Rad microplate reader and Microplate Manager software.Treatment of cells with 10 mm l-cysteine throughout the course of cell treatment with LTD4, in experiments assessing either receptor internalization or PI production, had no effect on results, suggesting that the metabolism of LTD4 in culture media was not of consequence. Experimental results were also qualitatively similar whether cells were maintained in serum-containing or serum-free medium for 18 h prior to acute pretreatment and treatment. Whenever possible, the effect of experimental variables (e.g. receptor mutations, pharmacologic inhibition) was tested in assays performed under optimally matched conditions (e.g. simultaneously performed using same passage or transfected cells, same prepared reagents) to minimize intra- and inter-experimental variability.Radioligand Binding—Binding of [3H]LTD4 (183 Ci/mmol) to membrane fractions prepared from COS-1 cells expressing WT CysLT1R was performed by incubating membranes in 10 mm HEPES, pH 7.4, containing 20 mm CaCl2, protease inhibitor mixture (Sigma), 20 mm l-penicillamine, and 0–1.5 nm [3H]LTD4, for 60 min at room temperature, followed by filtration on Whatman GF/C filters using a Brandel Cell Harvester and five 5-ml washes with 10 mm HEPES, 0.01% bovine serum albumin. Nonspecific binding was determined using 10 μm MK-571.Assay of Phosphoinositide Generation—Cells passaged into 24-well plates as described above were loaded with 2 μCi/ml myo-[3H]inositol for 18 h. Cells were then washed briefly with phosphate-buffered saline, incubated in DMEM containing 5 mm LiCl plus various inhibitors or vehicle for 15 min, and then stimulated with LTD4, histamine, or vehicle for 30 min. Reactions were terminated by replacing medium with cold 20 mm formic acid. The inositol and PI fractions were separated by anion exchange chromatography, mixed with Ultima Gold and Ultima Flo AF scintillation fluid (Packard), respectively, and counted. To normalize for loading variability among wells, PI production was calculated as PI/(PI + inositol), and values were reported as fold basal (vehicle-stimulated) production, as described previously (28Sterne-Marr R. Tesmer J.J. Day P.W. Stracquatanio R.P. Cilente J.A. O'Connor K.E. Pronin A.N. Benovic J.L. Wedegaertner P.B. J. Biol. Chem. 2003; 278: 6050-6058Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar).Analysis of Agonist-induced Ca2+Flux—COS-1 cells grown on glass coverslips and transfected with WT CysLT1R, CysLT1RS(313–316)A, or H1 HR constructs were loaded with 10 μm fura-2/AM, a ratiometric calcium indicator dye. All of the calcium measurements were carried out at 37 °C in Hanks' balanced salt solution containing 10 mm HEPES, 11 mm glucose, 2.5 mm CaCl2, and 1.2 mm MgCl2 (pH 7.4) using a dual excitation fluorescence photomultiplier system (IonOptix, Milton, MA). The ratio of intensities of fura-2 emissions at excitation wavelengths 340 and 380 nm was calculated every second, and the intracellular calcium concentration (nm) was calculated by extrapolation from a calibration curve, as described previously (29Deshpande D.A. Walseth T.F. Panettieri R.A. Kannan M.S. FASEB J. 2003; 17: 452-454Crossref PubMed Scopus (145) Google Scholar). Cells were pretreated for 15 min with either vehicle (0.03% Me2SO) or 10 μm Bis I and then were stimulated with agonist (1 μm LTD4 or 1 μm histamine), and changes in the intracellular calcium concentrations were recorded for 3–4 min. Calcium flux upon agonist stimulation was calculated by subtracting the mean basal intracellular calcium concentration from that of the peak intracellular calcium concentration elicited by agonist. Comparable expression of WT CysLT1R and CysLT1RS(313–316)A was confirmed by the immunoblotting of lysates from cells plated in parallel in 6-well plates.Analysis of PKC Translocation—COS-1 cells were transiently transfected with pcDNA3wtCysLT1R or vector control or were co-transfected with a construct encoding PKCβ-GFP plus either pcDNA3wtCysLT1R or pcDNA3 vector and subsequently passaged into 60-mm dishes. Cells were serum-starved and then stimulated for 20 min with vehicle, 100 nm LTD4, or 100 nm PMA. Cells were harvested by scraping them into 500 μl of homogenization buffer (20 mm Tris, pH 7.5, 0.25 m sucrose, 10 mm EDTA, 2 mm EGTA, 10 μm phenylmethylsulfonyl fluoride, 10 μg/ml benzamidine, 10 μg/ml aprotinin, and 10 μg/ml leupeptin). Lysates were passed through a 26-gauge needle three times and were centrifuged at 100,000 × g for 60 min. The resultant supernatant was saved as the cytosolic fraction, and the pellet (crude membrane fraction) was resuspended in 125 μl of homogenization buffer containing 0.1% Triton. 25 μl of each fraction were resolved by SDS-PAGE and immunoblots were probed with anti-PKCα or anti-GFP antibody.Immunofluorescence Microscopy and Immunoblotting—Visualization of the agonist-induced changes in CysLT1R distribution was performed on cells plated on poly-l-lysine-coated coverslips using a Nikon Eclipse E800 fluorescence microscope, as described previously for EP receptors (21Penn R.B. Pascual R.M. Kim Y.-M. Mundell S.J. Krymskaya V.P. Panettieri Jr., R.A. Benovic J.L. J. Biol. Chem. 2001; 276: 32648-32656Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Immunoblot analysis of WT and mutant CysLT1R expression was performed by harvesting cells and scraping them into Laemmli buffer (0.75 m Tris, pH 6.5, 5% β-mercaptoethanol, 10% glycerol, and 4% SDS). For samples generated for radioligand binding studies and analysis of membrane/cytosolic distribution of CysLT1R, cells were scraped into homogenization buffer (as described above) and homogenized with a Dounce homogenizer. To generate membrane and cytosolic fractions, whole cell lysates were centrifuged at 30,000 × g for 30 min, and the supernatant was saved and the pellet resuspended in homogenization buffer. For radioligand binding studies, resuspended pellets of the 30,000 × g fraction were centrifuged a second time and resuspended in binding buffer. For immunoblot analysis, protein samples were resolved by SDS-PAGE, transferred to nitrocellulose membrane, and then probed with the appropriate antibodies. Bands were visualized by enhanced chemiluminescence (Pierce).RESULTS AND DISCUSSIONInitial attempts to generate a useful recombinant CysLT1R produced constructs that either expressed poorly or, when expressed, resulted in proteins that migrated at too small a size (∼35 kDa), suggesting either folding problems or difficulties in processing through the endoplasmic reticulum and the Golgi (data not shown). Ultimately a construct encoding an N-terminal 3-FLAG epitope and an upstream signal sequence to facilitate endoplasmic reticulum processing (26Guan X.M. Kobilka T.S. Kobilka B.K. J. Biol. Chem. 1992; 267: 21995-21998Abstract Full Text PDF PubMed Google Scholar) was generated that expressed well in both COS-1 and HEK 293 cells and migrated at the predicted size (Fig. 1A). Multiple bands ranging from ∼45 to 52 kDa in size were detected (as well as a possible dimer at ∼90 kDa), suggesting the existence of multiple glycosylation sites. Importantly, antibodies against the N-terminal FLAG epitope and the C terminus of the human CysLT1R identified the same size bands (see below). Radioligand binding analysis of this construct expressed in COS-1 revealed saturable binding of [3H]LTD4 (Bmax = 450 fmol/mg protein) at levels ∼10-fold higher than that reported for the original HG55 clone (6Lynch K.R. O'Neill G.P. Liu Q. Im D.S. Sawyer N. Metters K.M. Coulombe N. Abramovitz M. Figueroa D.J. Zeng Z. Connolly B.M. Bai C. Austin C.P. Chateauneuf A. Stocco R. Greig G.M. Kargman S. Hooks S.B. Hosfield E. Williams Jr., D.L. Ford-Hutchinson A.W. Caskey C.T. Evans J.F. Nature. 1999; 399: 789-793Crossref PubMed Scopus (882) Google Scholar), yet with a similar Kd value (0.4 nm) (Fig. 1B). Comparable levels of CysLT1R were expressed among groups in which additional constructs were co-expressed (see supplemental materials). In assays of agonist-stimulated PI production (Fig. 1C), signaling capacity of wt CysLT1R was robust (∼5-fold of basal (vehicle-stimulated)), albeit less than that mediated by the H1 histamine receptor (H1 HR).Exposure of COS-1 cells expressing wt CysLT1R to LTD4 caused a rapid internalization of receptor as demonstrated by the loss of cell surface binding of anti-FLAG antibody in ELISA experiments (Fig. 2A) and as suggested by punctate aggregation of receptor, visualized by immunocytochemistry, following the addition of LTD4 (Fig. 2B). In unstimulated cells, CysLT1R was visualized primarily at the plasma membrane, although some puncta were frequently observed, suggesting that the tendency of receptors to internalize in the absence of agonist. Co-expression of CysLT1R with either arrestin-2 or arrestin-3 increased LTD4-stimulated internalization (Fig. 2C), suggesting a role for arrestins in mediating agonist-dependent internalization similar to th
Chronic obstructive pulmonary disease (COPD) is the most prevalent lung disease, and macrophages play a central role in the inflammatory response in COPD. We here report a comprehensive characterization of circulating short non-coding RNAs (sncRNAs) in plasma from patients with COPD. While circulating sncRNAs are increasingly recognized for their regulatory roles and biomarker potential in various diseases, the conventional RNA-seq method cannot fully capture these circulating sncRNAs due to their heterogeneous terminal structures. By pre-treating the plasma RNAs with T4 polynucleotide kinase, which converts all RNAs to those with RNA-seq susceptible ends (5'-phosphate and 3'-hydroxyl), we comprehensively sequenced a wide variety of non-microRNA sncRNAs, such as 5'-tRNA halves containing a 2',3'-cyclic phosphate. We discovered a remarkable accumulation of the 5'-half derived from tRNA
Chronic obstructive pulmonary disease (COPD) is the most prevalent lung disease, and macrophages play a central role in the inflammatory response in COPD. We here report a comprehensive characterization of circulating short non-coding RNAs (sncRNAs) in plasma from patients with COPD. While circulating sncRNAs are increasingly recognized for their regulatory roles and biomarker potential in various diseases, the conventional RNA sequencing (RNA-seq) method cannot fully capture these circulating sncRNAs due to their heterogeneous terminal structures. By pre-treating the plasma RNAs with T4 polynucleotide kinase, which converts all RNAs to those with RNA-seq susceptible ends (5'-phosphate and 3'-hydroxyl), we comprehensively sequenced a wide variety of non-microRNA sncRNAs, such as 5'-tRNA halves containing a 2',3'-cyclic phosphate. We discovered a remarkable accumulation of the 5'-half derived from tRNA
The transmembrane glycoprotein CD38 catalyzes the synthesis of the calcium mobilizing molecule cyclic ADP-ribose from NAD. In human airway smooth muscle (HASM) cells, the expression and function of CD38 are augmented by the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha), leading to increased intracellular calcium response to agonists. A glucocorticoid response element in the CD38 gene has been computationally described, providing evidence for transcriptional regulation of its expression. In the present study, we investigated the effects of dexamethasone, a glucocorticoid, on CD38 expression and ADP-ribosyl cyclase activity in HASM cells stimulated with TNF-alpha. In HASM cells, TNF-alpha augmented CD38 expression and ADP-ribosyl cyclase activity, which were attenuated by dexamethasone. TNF-alpha increased NF-kappaB expression and its activation, and dexamethasone partially reversed these effects. TNF-alpha increased the expression of IkappaBalpha, and dexamethasone increased it further. An inhibitor of NF-kappaB activation or transfection of cells with IkappaB mutants decreased TNF-alpha-induced CD38 expression. The results indicate that TNF-alpha-induced CD38 expression involves NF-kappaB expression and its activation and dexamethasone inhibits CD38 expression through NF-kappaB-dependent and -independent mechanisms.
Second generation antiandrogens have improved overall survival for men with metastatic castrate resistant prostate cancer; however, the antiandrogens result in suppression of androgen receptor (AR) activity in all tissues resulting in dose limiting toxicity. We sought to overcome this limitation through encapsulation in a prostate specific membrane antigen (PSMA)–conjugated nanoparticle. We designed and characterized a novel nanoparticle containing an antiandrogen, enzalutamide. Selectivity and enhanced efficacy was achieved through coating the particle with PSMA. The PSMA-conjugated nanoparticle was internalized selectively in AR expressing prostate cancer cells. It did not elicit an inflammatory effect. The efficacy of enzalutamide was not compromised through insertion into the nanoparticle; in fact, lower systemic drug concentrations of enzalutamide resulted in comparable clinical activity. Normal muscle cells were not impacted by the PSMA-conjugated containing antiandrogen. This approach represents a novel strategy to increase the specificity and effectiveness of antiandrogen treatment for men with castrate resistant prostate cancer. The ability to deliver higher drug concentrations in prostate cancer cells may translate into improved clinical end points including overall survival.
The proton-sensing receptor, ovarian cancer G protein-coupled receptor (OGR1), has been shown to be expressed in airway smooth muscle (ASM) cells and is capable of promoting ASM contraction in response to decreased extracellular pH. OGR1 knockout (OGR1KO) mice are reported to be resistant to the asthma features induced by inhaled allergen. We recently described certain benzodiazepines as OGR1 activators capable of mediating both procontractile and prorelaxant signaling in ASM cells. Here we assess the effect of treatment with the benzodiazepines lorazepam or sulazepam on the asthma phenotype in wild-type (WT) and OGR1KO mice subjected to inhaled house dust mite (HDM;
