Syndecan-1 Ectodomain Shedding Is Regulated by the Small GTPase Rab5
2008; Elsevier BV; Volume: 283; Issue: 51 Linguagem: Inglês
10.1074/jbc.m804172200
ISSN1083-351X
AutoresKazutaka Hayashida, Philip D. Stahl, Pyong Woo Park,
Tópico(s)Cellular transport and secretion
ResumoThe ectodomain shedding of syndecan-1, a major cell surface heparan sulfate proteoglycan, modulates molecular and cellular processes central to the pathogenesis of inflammatory diseases. Syndecan-1 shedding is a highly regulated process in which outside-in signaling accelerates the proteolytic cleavage of syndecan-1 ectodomains at the cell surface. Several extracellular agonists that induce syndecan-1 shedding and metalloproteinases that cleave syndecan-1 ectodomains have been identified, but the intracellular mechanisms that regulate syndecan-1 shedding are largely unknown. Here we examined the role of the syndecan-1 cytoplasmic domain in the regulation of agonist-induced syndecan-1 shedding. Our results showed that the syndecan-1 cytoplasmic domain is essential because mutation of invariant cytoplasmic Tyr residues abrogates ectodomain shedding, but not because it is Tyr phosphorylated upon shedding stimulation. Instead, our data showed that the syndecan-1 cytoplasmic domain binds to Rab5, a small GTPase that regulates intracellular trafficking and signaling events, and this interaction controls the onset of syndecan-1 shedding. Syndecan-1 cytoplasmic domain bound specifically to Rab5 and preferentially to inactive GDP-Rab5 over active GTP-Rab5, and shedding stimulation induced the dissociation of Rab5 from the syndecan-1 cytoplasmic domain. Moreover, the expression of dominant-negative Rab5, unable to exchange GDP for GTP, interfered with the agonist-induced dissociation of Rab5 from the syndecan-1 cytoplasmic domain and significantly inhibited syndecan-1 shedding induced by several distinct agonists. Based on these data, we propose that Rab5 is a critical regulator of syndecan-1 shedding that serves as an on-off molecular switch through its alternation between the GDP-bound and GTP-bound forms. The ectodomain shedding of syndecan-1, a major cell surface heparan sulfate proteoglycan, modulates molecular and cellular processes central to the pathogenesis of inflammatory diseases. Syndecan-1 shedding is a highly regulated process in which outside-in signaling accelerates the proteolytic cleavage of syndecan-1 ectodomains at the cell surface. Several extracellular agonists that induce syndecan-1 shedding and metalloproteinases that cleave syndecan-1 ectodomains have been identified, but the intracellular mechanisms that regulate syndecan-1 shedding are largely unknown. Here we examined the role of the syndecan-1 cytoplasmic domain in the regulation of agonist-induced syndecan-1 shedding. Our results showed that the syndecan-1 cytoplasmic domain is essential because mutation of invariant cytoplasmic Tyr residues abrogates ectodomain shedding, but not because it is Tyr phosphorylated upon shedding stimulation. Instead, our data showed that the syndecan-1 cytoplasmic domain binds to Rab5, a small GTPase that regulates intracellular trafficking and signaling events, and this interaction controls the onset of syndecan-1 shedding. Syndecan-1 cytoplasmic domain bound specifically to Rab5 and preferentially to inactive GDP-Rab5 over active GTP-Rab5, and shedding stimulation induced the dissociation of Rab5 from the syndecan-1 cytoplasmic domain. Moreover, the expression of dominant-negative Rab5, unable to exchange GDP for GTP, interfered with the agonist-induced dissociation of Rab5 from the syndecan-1 cytoplasmic domain and significantly inhibited syndecan-1 shedding induced by several distinct agonists. Based on these data, we propose that Rab5 is a critical regulator of syndecan-1 shedding that serves as an on-off molecular switch through its alternation between the GDP-bound and GTP-bound forms. Syndecans comprise a major family of cell surface heparan sulfate proteoglycans (1Bernfield M. Götte M. Park P.W. Reizes O. Fitzgerald M.L. Lincecum J. Zako M. Annu. Rev. Biochem. 1999; 68: 729-777Crossref PubMed Scopus (2293) Google Scholar, 2Park P.W. Reizes O. Bernfield M. J. Biol. Chem. 2000; 275: 29923-29926Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar, 3Carey D.J. Biochem. J. 1997; 327: 1-16Crossref PubMed Scopus (600) Google Scholar). There are four syndecans in mammals and all adherent cells express at least one syndecan on their cell surface. Syndecans have been shown or proposed to bind to and regulate various bioactive molecules, such as growth factors, cytokines, chemokines, enzymes, and cell adhesion molecules, in a heparan sulfate (HS) 2The abbreviations used are:HSheparan sulfateBSAbovine serum albuminDNdominant-negativeMMPmatrix metalloproteinaseNMuMGnormal murine mammary glandPDZpost-synaptic density protein (PSD95), Drosophila disc large tumor suppressor, and Zonula occludens-1 proteinPMAphorbol 12-myristate 13-acetatePTKprotein-tyrosine kinaseWTwild typeMAPmitogen-activated protein kinaseTPCKl-1-tosylamido-2-phenylethyl chloromethyl ketoneGFPgreen fluorescent proteinGSTglutathione S-transferaseGTPγSguanosine 5′-3-O-(thio)triphosphatePIPES1,4-piperazinediethanesulfonic acid 2The abbreviations used are:HSheparan sulfateBSAbovine serum albuminDNdominant-negativeMMPmatrix metalloproteinaseNMuMGnormal murine mammary glandPDZpost-synaptic density protein (PSD95), Drosophila disc large tumor suppressor, and Zonula occludens-1 proteinPMAphorbol 12-myristate 13-acetatePTKprotein-tyrosine kinaseWTwild typeMAPmitogen-activated protein kinaseTPCKl-1-tosylamido-2-phenylethyl chloromethyl ketoneGFPgreen fluorescent proteinGSTglutathione S-transferaseGTPγSguanosine 5′-3-O-(thio)triphosphatePIPES1,4-piperazinediethanesulfonic acid-dependent manner (1Bernfield M. Götte M. Park P.W. Reizes O. Fitzgerald M.L. Lincecum J. Zako M. Annu. Rev. Biochem. 1999; 68: 729-777Crossref PubMed Scopus (2293) Google Scholar, 4Fears C.Y. Woods A. Matrix Biol. 2006; 25: 443-456Crossref PubMed Scopus (133) Google Scholar, 5Gotte M. FASEB J. 2003; 17: 575-591Crossref PubMed Scopus (293) Google Scholar). Although all syndecans contain the ligand-binding HS chains, they show distinct temporal and spatial expression patterns and, thus, are likely to function specifically in vivo (1Bernfield M. Götte M. Park P.W. Reizes O. Fitzgerald M.L. Lincecum J. Zako M. Annu. Rev. Biochem. 1999; 68: 729-777Crossref PubMed Scopus (2293) Google Scholar, 6Bernfield M. Kokenyesi R. Kato M. Hinkes M.T. Spring J. Gallo R.L. Lose E.J. Annu. Rev. Cell Biol. 1992; 8: 365-393Crossref PubMed Scopus (960) Google Scholar, 7Kim C.W. Goldberger O.A. Gallo R.L. Bernfield M. Mol. Biol. Cell. 1994; 5: 797-805Crossref PubMed Scopus (346) Google Scholar). For instance, in adult tissues, syndecan-1 is predominantly expressed by epithelial and plasma cells, and to a lesser degree by other cell types (e.g. endothelial cells, fibroblasts). The overall structural design of syndecans is similar: starting at the NH2 terminus, the three major domains are the extracellular ectodomain where HS chains attach distally to the plasma membrane, followed by the highly conserved transmembrane and short cytoplasmic domains.Syndecans function as a coreceptor on the cell surface and also as a soluble heparan sulfate proteoglycan in the extracellular environment because its ectodomain, replete with all its HS chains, can be shed by metalloproteinases (1Bernfield M. Götte M. Park P.W. Reizes O. Fitzgerald M.L. Lincecum J. Zako M. Annu. Rev. Biochem. 1999; 68: 729-777Crossref PubMed Scopus (2293) Google Scholar, 2Park P.W. Reizes O. Bernfield M. J. Biol. Chem. 2000; 275: 29923-29926Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar). Current evidence suggests that the ectodomain shedding of syndecan-1 is an innate host response to tissue injury and inflammation. Syndecan-1 shedding is stimulated in vitro by several inflammatory factors and in vivo under certain pathological conditions. Agonists of syndecan-1 shedding include epidermal growth factor family growth factors, chemokines, stress-related agonists, heparanase, and bacterial virulence factors (8Subramanian S.V. Fitzgerald M.L. Bernfield M. J. Biol. Chem. 1997; 272: 14713-14720Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar, 9Fitzgerald M.L. Wang Z. Park P.W. Murphy G. Bernfield M. J. Cell Biol. 2000; 148: 811-824Crossref PubMed Scopus (343) Google Scholar, 10Park P.W. Foster T.J. Nishi E. Duncan S.J. Klagsbrun M. Chen Y. J. Biol. Chem. 2004; 279: 251-258Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 11Park P.W. Pier G.B. Preston M.J. Goldberger O. Fitzgerald M.L. Bernfield M. J. Biol. 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Liu W. Langford J.K. Pumphrey C.Y. Theus A. Epstein J. Sanderson R.D. Blood. 2002; 100: 610-617Crossref PubMed Scopus (162) Google Scholar). In mouse models of inflammatory diseases, elevated levels of syndecan-1 ectodomains are found in lung and skin homogenates of mice infected with Pseudomonas aeruginosa (23Haynes 3rd, A. Ruda F. Oliver J. Hamood A.N. Griswold J.A. Park P.W. Rumbaugh K.P. Infect. Immun. 2005; 73: 7914-7921Crossref PubMed Scopus (55) Google Scholar, 24Park P.W. Pier G.B. Hinkes M.T. Bernfield M. Nature. 2001; 411: 98-102Crossref PubMed Scopus (205) Google Scholar), in incisional skin wound fluid (20Kato M. Wang H. Kainulainen V. Fitzgerald M.L. Ledbetter S. Ornitz D.M. Bernfield M. Nat. Med. 1998; 4: 691-697Crossref PubMed Scopus (287) Google Scholar, 25Kainulainen V. Nelimarkka L. Jarvelainen H. Laato M. Jalkanen M. Elenius K. J. Biol. Chem. 1996; 271: 18759-18766Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), and in bronchoalveolar lavage fluids of mice challenged with bleomycin (26Li Q. Park P.W. Wilson C.L. Parks W.C. Cell. 2002; 111: 635-646Abstract Full Text Full Text PDF PubMed Scopus (639) Google Scholar) or allergens (27Xu J. Park P.W. Kheradmand F. Corry D.B. J. Immunol. 2005; 174: 5758-5765Crossref PubMed Scopus (89) Google Scholar).Results from animal studies suggest that syndecan-1 shedding modulates the extent and outcome of inflammatory processes. For example, in the mouse model of acute P. aeruginosa pneumonia, syndecan-1 shedding enhanced by LasA, a virulence factor for P. aeruginosa lung infection, promotes bacterial colonization and infectious pneumonia by dysregulating host defense mechanisms in an HS-dependent manner (11Park P.W. Pier G.B. Preston M.J. Goldberger O. Fitzgerald M.L. Bernfield M. J. Biol. 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Immunol. 2005; 174: 5758-5765Crossref PubMed Scopus (89) Google Scholar), whereas syndecan-1 shedding coordinates the generation of a KC (CXCL1) chemokine gradient that guides the transepithelial migration of neutrophils into the airspace in bleomycin-induced acute lung injury (26Li Q. Park P.W. Wilson C.L. Parks W.C. Cell. 2002; 111: 635-646Abstract Full Text Full Text PDF PubMed Scopus (639) Google Scholar). These data highlight the diverse and critical functions of syndecan-1 shedding in modulating inflammatory disorders in vivo.Despite its importance in modulating a variety of key inflammatory processes, the underlying mechanisms of how syndecan-1 shedding is regulated are incompletely understood. Much is known about the identity of extracellular agonists of syndecan-1 shedding, and several syndecan-1 sheddases, such as matrix metalloproteinase-7 (MMP-7, matrilysin) (26Li Q. Park P.W. Wilson C.L. Parks W.C. 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Chemical inhibitor studies have implicated ERK (extracellular signal-regulated kinase) and JNK (c-Jun NH2-terminal kinase) MAP kinases (9Fitzgerald M.L. Wang Z. Park P.W. Murphy G. Bernfield M. J. Cell Biol. 2000; 148: 811-824Crossref PubMed Scopus (343) Google Scholar) and protein-tyrosine kinases (PTKs) in the regulation of syndecan-1 shedding (9Fitzgerald M.L. Wang Z. Park P.W. Murphy G. Bernfield M. J. Cell Biol. 2000; 148: 811-824Crossref PubMed Scopus (343) Google Scholar, 10Park P.W. Foster T.J. Nishi E. Duncan S.J. Klagsbrun M. Chen Y. J. Biol. Chem. 2004; 279: 251-258Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 11Park P.W. Pier G.B. Preston M.J. Goldberger O. Fitzgerald M.L. Bernfield M. J. Biol. Chem. 2000; 275: 3057-3064Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 16Chung M.C. Popova T.G. Millis B.A. Mukherjee D.V. Zhou W. Liotta L.A. Petricoin E.F. Chandhoke V. Bailey C. Popov S.G. J. Biol. Chem. 2006; 281: 31408-31418Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), but precisely how these signaling factors regulate syndecan-1 shedding is not known.The cytoplasmic domain of syndecans is highly conserved and it has been shown to interact with several signaling and scaffolding proteins, including c-Src (30Kinnunen T. Kaksonen M. Saarinen J. Kalkkinen N. Peng H.B. Rauvala H. J. Biol. Chem. 1998; 273: 10702-10708Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar), cortactin (30Kinnunen T. Kaksonen M. Saarinen J. Kalkkinen N. Peng H.B. Rauvala H. J. Biol. Chem. 1998; 273: 10702-10708Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar), protein kinase A (31Hayashida K. Johnston D.R. Goldberger O. Park P.W. J. Biol. Chem. 2006; 281: 24365-24374Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar), protein kinase Cα (32Oh E.S. Woods A. Couchman J.R. J. Biol. Chem. 1997; 272: 8133-8136Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar), syntenin (33Grootjans J.J. Zimmermann P. Reekmans G. Smets A. Degeest G. Durr J. David G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13683-13688Crossref PubMed Scopus (339) Google Scholar), and CASK/LIN2A (34Cohen A.R. Woods D.F. Marfatia S.M. Walther Z. Chishti A.H. Anderson J.M. J. Cell Biol. 1998; 142: 129-138Crossref PubMed Scopus (318) Google Scholar, 35Hsueh Y.P. Yang F.C. Kharazia V. Naisbitt S. Cohen A.R. Weinberg R.J. Sheng M. J. Cell Biol. 1998; 142: 139-151Crossref PubMed Scopus (283) Google Scholar). The syndecan cytoplasmic domain is composed of two invariant regions (C1 and C2), separated by a central variable region (V) that is distinct for each family member (1Bernfield M. Götte M. Park P.W. Reizes O. Fitzgerald M.L. Lincecum J. Zako M. Annu. Rev. Biochem. 1999; 68: 729-777Crossref PubMed Scopus (2293) Google Scholar). Furthermore, the syndecan cytoplasmic domain contains several signaling motifs, including one invariant Ser, four invariant Tyr, and a Glu-Phe-Tyr-Ala PDZ binding domain at the COOH terminus. Collectively, these observations suggest that the cytoplasmic domain plays a vital role in the regulation of syndecan functions in vivo, including syndecan-1 shedding.In this study, we sought to define the regulatory role of syndecan-1 cytoplasmic domain in agonist-induced syndecan-1 ectodomain shedding. Our results indicate that the cytoplasmic domain is essential, but not because it is Tyr phosphorylated upon shedding stimulation. Instead, our results show that the syndecan-1 cytoplasmic domain interacts specifically with the small GTPase Rab5, and the oscillation between the GDP- and GTP-bound forms of Rab5 serves as an intracellular on-off molecular switch of syndecan-1 shedding.EXPERIMENTAL PROCEDURESMaterials—Phorbol 12-myristate 13-acetate (PMA) and C2 ceramide were obtained from Calbiochem (La Jolla, CA). Protein G-agarose beads, enhanced chemiluminescence (ECL) reagents, and Ultralink affinity chromatography resins were purchased from Pierce Chemical. Immobilon Ny+ (cationic nylon) and Immobilon P (polyvinylidene difluoride) membranes were from Millipore (Bedford, MA), and nitrocellulose membrane was from Schleicher & Schuell (Keene, NH). Culture medium, fetal calf serum, and other tissue culture supplements were obtained from Mediatech (Herndon, VA). Reduced glutathione, thrombin cleavage kit, bovine serum albumin (BSA), TPCK-treated trypsin, and soybean trypsin inhibitor were from Sigma. Glutathione-Sepharose 4B and pGEX4T-1 were obtained from GE Healthcare. Purified Staphylococcus aureus β toxin was from Toxin Technology (Sarasota, FL). ViraPower Adenovirus Expression System, AccuPrime Pfx DNA polymerase, pENTER/SD/D-TOPO, pAd/CMV/V5-DEST, LR clonase, and Lipofectamine 2000 were from Invitrogen. PacI was from New England BioLabs (Ipswich, MA). All other materials and reagents were obtained from either Fisher (Pittsburgh, PA) or VWR (Arlington Heights, IL).Cells and Immunochemicals—Normal murine mammary gland (NMuMG) epithelial, human lung adenocarcinoma (A549), and human epidermoid carcinoma (A431) cells were from our collection and cultured as described previously (11Park P.W. Pier G.B. Preston M.J. Goldberger O. Fitzgerald M.L. Bernfield M. J. Biol. Chem. 2000; 275: 3057-3064Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). NMuMG cells were used between passages 18 and 23 for all assays. 293A cells were purchased from Invitrogen. Rat monoclonal anti-mouse syndecan-1 ectodomain antibody (clone 281-2) and mouse monoclonal anti-Rab5 (clone 15), anti-Rab8 (clone 4), anti-β1 integrin (clone 18), anti-β-catenin (clone 14), anti-phosphoinositide-3 kinase (PI3K) (clone 4), anti-early endosome antigen 1 (clone 14), and anti-Stat3 (clone 84) antibodies were from BD Biosciences. Rabbit monoclonal anti-Src antibody (clone 36D10) and rabbit polyclonal anti-Rab5, anti-Akt, and anti-p44/42 MAP kinase antibodies were from Cell Signaling (Danvers, MA). Mouse monoclonal anti-fibroblast growth factor receptor-1 antibody (VBS1) was from Millipore and the mouse monoclonal anti-human syndecan-1 ectodomain antibody (B-B4) was from Serotec (Raleigh, NC). The anti-phosphotyrosine antibodies PY20 and Tyr(P)-100 were from BD Biosciences and Cell Signaling, respectively. Rat monoclonal anti-mouse syndecan-4 ectodomain antibody (Ky8.2) was purified from the conditioned medium of hybridoma cultures by protein G affinity chromatography (11Park P.W. Pier G.B. Preston M.J. Goldberger O. Fitzgerald M.L. Bernfield M. J. Biol. Chem. 2000; 275: 3057-3064Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Affinity purified rabbit polyclonal anti-mouse syndecan-1 cytoplasmic domain antibody directed against the COOH-terminal 16 amino acids was prepared as described previously (31Hayashida K. Johnston D.R. Goldberger O. Park P.W. J. Biol. Chem. 2006; 281: 24365-24374Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Horseradish peroxidase-conjugated donkey anti-rat IgG, goat anti-rabbit IgG, and goat anti-mouse IgG secondary antibodies were from Jackson ImmunoResearch Laboratories (West Grove, PA). Rabbit anti-GFP and Alexa 594-conjugated goat anti-rat IgG antibodies were from Invitrogen.Generation of Recombinant Syndecan-1 Cytoplasmic Domain and Recombinant Human Rab5—PCR-amplified cDNA encoding the entire cytoplasmic domain (Tyr277–Ala311) of mouse syndecan-1 and whole CDS encoding human Rab5a were subcloned in-frame into the GST fusion protein expression vector pGEX 4T-1. The GST-syndecan-1 cytoplasmic domain and GST-Rab5 fusion proteins were expressed and purified according to the manufacturer's instructions. Briefly, Escherichia coli BL21 was transformed with the recombinant constructs, grown to logarithmic growth phase, and induced with 0.5 mm isopropyl 1-thio-β-d-galactopyranoside for 4 h at 37 °C. Cells were lysed by sonication in phosphate-buffered saline with 0.5! (v/v) Triton X-100. Cell lysates were cleared by centrifugation and fractionated by glutathione-Sepharose 4B chromatography, and bound proteins were eluted with 15 mm reduced glutathione. In some experiments, the GST moiety was removed from the recombinant proteins by incubating overnight at 4 °C with 50 μl of thrombin-agarose slurry in cleavage buffer (50 mm Tris, pH 8, 10 mm CaCl2) and removing the cleaved GST by glutathione-Sepharose 4B chromatography. To generate recombinant FLAG-tagged syndecan-1 cytoplasmic domain, the entire mouse syndecan-1 cytoplasmic domain was amplified by PCR using a 5′ primer containing the sequence encoding FLAG tag (DYKDDDDK). The resulting PCR fragment was inserted in-frame into the BamHI site of pET11a. FLAG-tagged syndecan-1 cytoplasmic domain was expressed as described above for GST-tagged syndecan-1 cytoplasmic domain and purified by anti-FLAG M2 affinity chromatography using 100 μg/ml FLAG peptide to elute the bound recombinant protein.Adenoviral Expression of Wild Type and Dominant-negative GFP-Rab5—Adenoviruses harboring GFP-wild type (pAd WT Rab5) or S34N dominant-negative human Rab5 (pAd DN Rab5) were generated using the ViraPower Adenovirus Expression System according to the manufacturer's instructions. Briefly, full-length cDNAs of GFP-WT Rab5 and GFP-DN Rab5 were amplified by PCR with Accuprime using pEGFP human Rab5a (36Li G. Stahl P.D. J. Biol. Chem. 1993; 268: 24475-24480Abstract Full Text PDF PubMed Google Scholar) and pEGFP S34N human Rab5a (36Li G. Stahl P.D. J. Biol. Chem. 1993; 268: 24475-24480Abstract Full Text PDF PubMed Google Scholar) as templates, respectively. Amplified cDNA fragments were subcloned into the pENTER/SD/D-TOPO vector using the pENTER Directional TOPO Cloning kit. After verifying the sequence, the inserts were transferred into the pAd/CMV/V5-DEST vector by the Gateway system using LR clonase II. To obtain virus particles, plasmids were linearized by PacI digestion and transfected into 293A cells with Lipofectamine 2000. When most cells were detached (∼7–10 days), cells were harvested by gentle pipetting in culture medium, lysed by three cycles of freeze/thawing, and centrifuged to collect the supernatant containing crude viral lysates. To amplify virus titers, 100 μl of crude viral lysates were added to fresh 293A cells, and cultured for several days until all cells were detached. Virus-enriched supernatants were collected and viral titers were determined by the plaque-forming assay with 293A cells.Co-sedimentation Assay—Recombinant syndecan-1 cytoplasmic domain free of GST (Sdc1-CPD) was coupled to Ultralink affinity resin according to the manufacturer's instructions. NMuMG total cell lysate was prepared by sonicating cells in lysis buffer A (50 mm HEPES, pH 7.4, 80 mm KCl, 4 mm MgCl2, 2 mm EGTA, 1 mm dithiothreitol, 0.5! Triton X-100) followed by centrifugation at 14,000 × g for 30 min. The cleared total cell lysate (5 mg) was incubated overnight with 10 μg of Sdc1-CPD coupled to Ultralink in the presence of 1 mg of recombinant GST-tagged syndecan-1 cytoplasmic domain or 1 mg of BSA at 4 °C and washed three times with lysis buffer A. Bound proteins were eluted by adding SDS sample buffer and analyzed by SDS-PAGE and immunoblotting.In some experiments, GST-tagged human Rab5 coupled to Ultralink, preloaded with GTPγS or GDP, was used to precipitate syndecan-1 cytoplasmic domain from mildly trypsinized NMuMG total cell lysates. NMuMG cells were treated with 10 μg/ml TPCK-treated trypsin and sonicated in lysis buffer A to obtain cell lysates enriched in syndecan-1 cytoplasmic domain. GST-tagged human Rab5 was coupled to Ultralink and incubated in buffer (20 mm HEPES, pH 7.5, 100 mm NaCl, 1 mm dithiothreitol, 10 mm EDTA, 5 mm MgCl2) containing 1 mm GTPγS or 5 mm GDP at room temperature for 1 h under rotation. The beads were washed and incubated with stabilization buffer (20 mm HEPES, pH 7.5, 100 mm NaCl, 1 mm dithiothreitol, 2 mm MgCl2) containing 100 μm GTPγS or GDP for 10 min at room temperature. NMuMG cell lysate (1 mg) was incubated with either GTPγS or GDP preloaded Rab5-Ultralink beads for 4 h at 4 °C. Beads were washed and bound proteins were eluted with SDS sample buffer.Co-immunoprecipitation—NMuMG cells treated with or without shedding agonists were lysed in lysis buffer B (50 mm HEPES, pH 7.4, 80 mm KCl, 4 mm MgCl2, 2 mm EGTA, 0.5! Triton X-100). The lysate was centrifuged at 14,000 × g for 30 min and the supernatant was incubated overnight with 3 μg of 281-2 anti-syndecan-1 or Ky8.2 anti-syndecan-4 antibody at 4 °C with gentle agitation. Protein G-agarose beads (20 μl) were added and incubated for 2 h at 4 °C. In some experiments, immunoprecipitation was performed with 281-2 antibodies directly coupled to Ultralink beads. Beads were pelleted by centrifugation, washed three times with lysis buffer B, and resuspended in non-reducing SDS-PAGE sample buffer. Immunoprecipitation was also performed in the presence of excess recombinant syndecan-1 cytoplasmic domain devoid of GST (150 μg/ml). Samples were resolved by SDS-PAGE, Western blotted onto nitrocellulose, and probed with rabbit anti-Rab5 or rabbit anti-GFP antibodies. For Western immunoblotting of syndecan-1, samples were resuspended in reducing SDS-PAGE sample buffer, resolved by SDS-PAGE, transferred to Immobilon Ny+ membranes, and developed with the 281-2 antibody.Direct Binding Assay—Direct binding between syndecan-1 cytoplasmic domain and Rab5 was measured by incubating purified FLAG-tagged syndecan-1 cytoplasmic domain (100 ng/ml) with GDP or GTPγS pre-loaded Rab5 coupled to Ultralink in 20 mm HEPES, pH 7.5, 100 mm NaCl, 1 mm dithiothreitol, 2 mm MgCl2, and 1 mg/ml BSA for 4 h at 4 °C. Beads were washed and bound proteins were eluted with SDS sample buffer and analyzed by SDS-PAGE and immunoblotting.Measurement of Cell Surface and Shed Syndecan-1—Syndecan-1 shedding was assessed as described previously (10Park P.W. Foster T.J. Nishi E. Duncan S.J. Klagsbrun M. Chen Y. J. Biol. Chem. 2004; 279: 251-258Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar) with slight modifications. Briefly, confluent cultures of NMuMG, A431, or A549 epithelial cells were transduced with adenovirus harboring LacZ, GFP-WT human Rab5, or GFP-DN human Rab5 at a multiplicity of infection of 10 for NMuMG and A431 cells and 2 for A549 cells. At day 3, cells were incubated with various test samples for the indicated times at 37 °C. To quantify shedding, the conditioned medium was collected, spun down to remove cells, and acidified by addition of NaOAc (pH 4.5), NaCl, and Tween 20 to a final concentration of 50 mm, 150 mm, and 0.1! (v/v), respectively. Various volumes of the acidified samples were dot blotted onto Immobilon Ny+ and developed with specific antibodies and ECL.Quantification of cell surface syndecan-1 was performed by mild trypsinization and measurement of the released syndecan-1 ectodomains by dot immunoblotting as described previously (31Hayashida K. Johnston D.R. Goldberger O. Park P.W. J. Biol. Chem. 2006; 281: 24365-24374Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Briefly, confluent NMuMG cells transduced with or without adenovirus harboring LacZ, WT Rab5, or D
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