Phosphorylation of NG2 Proteoglycan by Protein Kinase C-α Regulates Polarized Membrane Distribution and Cell Motility

Protein kinase C (PKC)-α phosphorylation of recombinant NG2 cytoplasmic domain and phorbol ester-induced PKC-dependent phosphorylation of full-length NG2 expressed in U251 cells are both blocked by mutation of Thr2256, identifying this residue as a primary phosphorylation site. In untreated U251/NG2 cells, NG2 is present along with ezrin and α3β1 integrin in apical cell surface protrusions. Phorbol ester treatment causes redistribution of all three components to lamellipodia, accompanied by increased cell motility. U251 cells expressing NG2 with a valine substitution at position 2256 are resistant to phorbol ester treatment: NG2 remains in membrane protrusions and cell motility is unchanged. In contrast, NG2 with a glutamic acid substitution at position 2256 redistributes to lamellipodia even without phorbol ester treatment, rendering transfected U251 cells spontaneously motile. PKC-α-mediated NG2 phosphorylation at Thr2256 is therefore a key step for initiating cell polarization and motility. Protein kinase C (PKC)-α phosphorylation of recombinant NG2 cytoplasmic domain and phorbol ester-induced PKC-dependent phosphorylation of full-length NG2 expressed in U251 cells are both blocked by mutation of Thr2256, identifying this residue as a primary phosphorylation site. In untreated U251/NG2 cells, NG2 is present along with ezrin and α3β1 integrin in apical cell surface protrusions. Phorbol ester treatment causes redistribution of all three components to lamellipodia, accompanied by increased cell motility. U251 cells expressing NG2 with a valine substitution at position 2256 are resistant to phorbol ester treatment: NG2 remains in membrane protrusions and cell motility is unchanged. In contrast, NG2 with a glutamic acid substitution at position 2256 redistributes to lamellipodia even without phorbol ester treatment, rendering transfected U251 cells spontaneously motile. PKC-α-mediated NG2 phosphorylation at Thr2256 is therefore a key step for initiating cell polarization and motility. Transmembrane proteoglycans such as CD44 and syndecans make important contributions to communication between the exterior and interior of the cell (1Thorne R.F. Legg J.W. Isacke C.M. J. Cell Sci. 2004; 117: 373-380Crossref PubMed Scopus (192) Google Scholar, 2Couchman J.R. Nat. Rev. Mol. Cell. Biol. 2003; 4: 926-937Crossref PubMed Scopus (344) Google Scholar). We are elucidating specific signaling functions for NG2, a membrane-spanning chondroitin sulfate proteoglycan found on several types of immature progenitor cells and on a variety of tumor types (3Stallcup W.B. J. Neurocytol. 2002; 31: 423-435Crossref PubMed Scopus (243) Google Scholar). Two hallmarks of both progenitor and tumor cells are increased motility and proliferation, both of which are influenced by NG2 (4Burg M.A. Grako K.A. Stallcup W.B. J. Cell. Physiol. 1998; 177: 299-312Crossref PubMed Scopus (102) Google Scholar, 5Grako K.A. Ochiya T. Barritt D. Nishiyama A. Stallcup W.B. J. Cell Sci. 1999; 112: 905-915PubMed Google Scholar, 6Nishiyama A. Lin X.H. Giese N. Heldin C.H. Stallcup W.B. J. Neurosci. Res. 1996; 43: 315-330Crossref PubMed Scopus (206) Google Scholar, 7Ozerdem U. Stallcup W.B. Angiogenesis. 2004; (in press)Google Scholar). Several mechanisms have been suggested to account for the contribution of NG2 to these processes. These include sequestration of growth factors (5Grako K.A. Ochiya T. Barritt D. Nishiyama A. Stallcup W.B. J. Cell Sci. 1999; 112: 905-915PubMed Google Scholar, 8Goretzki L. Burg M.A. Grako K.A. Stallcup W.B. J. Biol. Chem. 1999; 274: 16831-16837Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar), modulation of the activity of kringle domain proteins (9Goretzki L. Lombardo C.R. Stallcup W.B. J. Biol. Chem. 2000; 275: 28625-28633Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 10Chekenya M. Hjelstuen M. Enger P.O. Thorsen F. Jacob A.L. Probst B. Haraldseth O. Pilkington G. Butt A. Levine J.M. Bjerkvig R. FASEB J. 2002; 16: 586-588Crossref PubMed Scopus (83) Google Scholar) and matrix metalloproteinases (11Iida J. Pei D. Kang T. Simpson M.A. Herlyn M. Furcht L.T. McCarthy J.B. J. Biol. Chem. 2001; 276: 18786-18794Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), and interaction with other cell surface molecules or with extracellular matrix components that regulate signaling pathways involved in cell proliferation and motility (12Iida J. Meijne A.M. Spiro R.C. Roos E. Furcht L.T. McCarthy J.B. Cancer Res. 1995; 55: 2177-2185PubMed Google Scholar, 13Fang X. Burg M.A. Barritt D. Dahlin-Huppe K. Nishiyama A. Stallcup W.B. Mol. Biol. Cell. 1999; 10: 3373-3387Crossref PubMed Scopus (74) Google Scholar, 14Tillet E. Gential B. Garrone R. Stallcup W.B. J. Cell. Biochem. 2002; 86: 726-736Crossref PubMed Scopus (81) Google Scholar, 15Majumdar M. Vuori K. Stallcup W.B. Cell. Signal. 2003; 15: 79-84Crossref PubMed Scopus (38) Google Scholar). NG2 engagement has been shown to result in activation of the small GTPases cdc42 and rac (15Majumdar M. Vuori K. Stallcup W.B. Cell. Signal. 2003; 15: 79-84Crossref PubMed Scopus (38) Google Scholar, 16Eisenmann K.M. McCarthy J.B. Simpson M.A. Keely P.J. Guan J.L. Tachibana K. Lim L. Manser E. Furcht L.T. Iida J. Nat. Cell Biol. 1999; 1: 507-513Crossref PubMed Scopus (170) Google Scholar) and in β1 integrin-dependent activation of focal adhesion kinase and ERK-1/2 1The abbreviations used are: ERK, extracellular signal-regulated kinase; GF109203X, bisindolylmaleimide I; GRIP-1, glutamate receptor interacting protein; MUPP1, multi-PDZ domain protein; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; GST, glutathione S-transferase. (17Yang J. Price M.A. Neudauer C.L. Wilson C. Ferrone S. Xia H. Iida J. Simpson M.A. McCarthy J.B. J. Cell Biol. 2004; 165: 881-891Crossref PubMed Scopus (128) Google Scholar, 18Fukushi J. Makagiansar I.T. Stallcup W.B. Mol. Biol. Cell. 2004; 15: 3580-3590Crossref PubMed Scopus (263) Google Scholar). The fundamental importance of these pathways in cell physiology underscores the potential significance of elucidating the specific contributions of NG2 to transmembrane communication. Protein phosphorylation and dephosphorylation are recognized as critical aspects of intracellular signaling. By altering protein conformation and by creating docking sites for protein-protein interaction, phosphorylation and dephosphorylation of cytoplasmic tyrosine, serine, and threonine residues provide an extremely versatile means of regulating signaling pathways (19Manning G. Whyte D.B. Martinez R. Hunter T. Sudarsanam S. Science. 2002; 298: 1912-1934Crossref PubMed Scopus (6315) Google Scholar, 20Alonso A. Sasin J. Bottini N. Friedberg I. Osterman A. Godzik A. Hunter T. Dixon J. Mustelin T. Cell. 2004; 117: 699-711Abstract Full Text Full Text PDF PubMed Scopus (1536) Google Scholar). Although the NG2 cytoplasmic domain contains several threonine residues that might serve as phosphorylation sites (21Nishiyama A. Dahlin K.J. Prince J.T. Johnstone S.R. Stallcup W.B. J. Cell Biol. 1991; 114: 359-371Crossref PubMed Scopus (233) Google Scholar), to date we have had no experimental verification of this type of modification in NG2 and no information about potential functional consequences. The current work sheds initial light on these questions by identifying Thr2256 as the primary site for NG2 modification by PKC-α. In addition, we show that phosphorylation at this site changes the distribution of NG2 on the cell surface. Whereas non-phosphorylated NG2 is localized to small membrane protrusions distributed over most of the cell surface, Thr2256-phosphorylated NG2 is largely associated with extensive lamellipodia at the cell periphery. This redistribution of NG2 and the resulting polarization of cells are accompanied by significant increases in cell motility. Reagents—PMA (phorbol 12-myristate 13-acetate), GF109203X, and Gö6976 were purchased from Calbiochem. Calyculin A was purchased from Cell Signaling Technology (Beverly, MA). G418 (Geneticin) and Lipofectamine were purchased from Invitrogen. Antibodies—Affinity-purified rabbit and guinea pig antibodies against rat NG2 were prepared in our laboratory (22Ozerdem U. Grako K.A. Dahlin-Huppe K. Monosov E. Stallcup W.B. Dev. Dyn. 2001; 222: 218-227Crossref PubMed Scopus (476) Google Scholar). Rabbit antibodies specific for the XTpX(K/R) phosphothreonine motif and for phosphorylated ezrin (Thr567) were obtained from Cell Signaling Technology. Monoclonal antibody against PKC-α was from BD Biosciences. Monoclonal antibody against the human α3 integrin subunit was obtained from Chemicon (Temecula, CA). Rabbit antibodies against ezrin and glutathione S-transferase (GST) were generous gifts from Dr. Heinz Furthmayr (Stanford University) and Dr. Elena Pasquale (The Burnham Institute), respectively. Fluorochome-coupled second antibodies were purchased from BIOSOURCE International (Camarillo, CA). Goat anti-rabbit immunoglobulin coupled to 6-nm colloidal gold was obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). Rhodamine-labeled phalloidin was obtained from Molecular Probes (Eugene, OR). Cell Culture and Stable Transfections—U251MG human astrocytoma cells (23Ponten J. Westermark B. Med. Biol. 1978; 56: 184-193PubMed Google Scholar) transfected with cDNA for rat NG2 have been previously described (24Nishiyama A. Lin X.H. Stallcup W.B. Mol. Biol. Cell. 1995; 6: 1819-1832Crossref PubMed Scopus (94) Google Scholar). Cells were grown at 37 °C in 5% CO2 in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (Tissue Culture Biologicals, Tulare, CA). Mutant NG2 constructs (in pcDNA/ampI, Invitrogen) in which specific threonine residues were replaced by either valine or glutamic acid were made using Stratagene's QuikChange mutagenesis kit. For this purpose, overlapping pairs of oligonucleotide primers containing appropriately altered codons were purchased from FisherOligos. The resulting mutations were confirmed by DNA sequencing. Stable transfection of U251MG cells with these mutant NG2 cDNAs was accomplished as described by Stallcup and Dahlin-Huppe (25Stallcup W.B. Dahlin-Huppe K. J. Cell Sci. 2001; 114: 2315-2325PubMed Google Scholar). Unless otherwise specified, cell populations used for our experiments contained >85% NG2-positive cells. Immunoprecipitation and Western Blotting—Immunoprecipitation and treatment of NG2 were to Stallcup and Dahlin-Huppe (25Stallcup W.B. Dahlin-Huppe K. J. Cell Sci. 2001; 114: 2315-2325PubMed Google Scholar). were by on and to MA). The were with antibodies against NG2, GST, or the XTpX(K/R) phosphothreonine were with or anti-rabbit and proteins were with a NG2 cytoplasmic of NG2 was by from a rat NG2 cDNA the and sites of the and used to The resulting protein was from using Mutant of the NG2 cytoplasmic in which were replaced by glutamic were using the described for full-length NG2 In of NG2 by of type or mutant protein to was with of recombinant PKC-α for using the described by T. M. J. D. A. M. S. P.J. P.J. J. 2001; PubMed Scopus Google Scholar). this the protein were with and was to on were to and components were by using and were in Dulbecco's modified Eagle's medium for at 37 °C and for with of in Cells were with or without A or the of NG2 were by on to localized by and for acid of NG2 was as described previously Hunter T. PubMed Scopus Google Scholar). The were by and on the of with was to the of K. Google Scholar). a cells were at °C to the of NG2 on the cell surface. In most cells were with for at to was as described by Stallcup and Dahlin-Huppe (25Stallcup W.B. Dahlin-Huppe K. J. Cell Sci. 2001; 114: 2315-2325PubMed Google Scholar). were using a for and A was used for all were using and on were prepared and as described for that second antibodies were replaced by anti-rabbit immunoglobulin to 6-nm colloidal the cells were as described by S. I. J. Cell Biol. PubMed Scopus Google Scholar) and in CA). were on an and with and were at using a Cell cell were in and with a of using a T. M. J. D. A. M. S. P.J. P.J. J. 2001; PubMed Scopus Google Scholar). medium and cells were and replaced with either with or without a were with and for NG2 and was by NG2-positive cells that had the as by from the of cells in the The of cells in a was by the of the to a for cells of in of three was for cell type experimental NG2 on cytoplasmic domain of rat NG2 contains and threonine residues (21Nishiyama A. Dahlin K.J. Prince J.T. Johnstone S.R. Stallcup W.B. J. Cell Biol. 1991; 114: 359-371Crossref PubMed Scopus (233) Google Scholar). of these residues as phosphorylation acid was on NG2 from transfected U251 cells in the of the kinase A these NG2 contains phosphothreonine of the cytoplasmic of rat NG2 using a phosphorylation A. N. S. Res. 1999; PubMed Scopus Google Thr2256, and as potential phosphorylation on the of the for involved in protein-protein interaction the as a potential site for This Thr2256 is in the human G. M. A. T. Sci. U. S. A. 1996; PubMed Scopus Google Scholar), S. F. D. H. K. A. T. M. J. J. Neurosci. 2001; PubMed Google Scholar), and of the with the that the motif have functional The Thr2256 site therefore to a for cytoplasmic phosphorylation of Protein C a of NG2 threonine phosphorylation of NG2, the proteoglycan was from U251/NG2 cells and using a phosphothreonine antibody specific for the of cells with increased the phosphorylation of NG2 over the that for NG2 G. C. F. P.J. Biochem. J. 1999; PubMed Scopus Google Scholar) and NG2, an was in the of the A A. S. A. H. T. N. M. K. 2002; 10: Full Text Full Text PDF PubMed Scopus Google Scholar). This treatment in threonine phosphorylation of NG2 an of with important in cell proliferation, and S. J. Biochem. 2002; PubMed Scopus Google Scholar, I. Cell. Mol. Sci. 2003; PubMed Scopus Google Scholar). We to specific in NG2 is an of and Gö6976 is a of the and K.M. H. C. N. 1996; PubMed Scopus Google Scholar). and of NG2 threonine phosphorylation were or U251/NG2 cells were with these of NG2 phosphorylation by a PKC-α is the of the expressed in U251 cells A. Mol. Cell. Biol. 2000; PubMed Scopus Google Scholar). In of the that PKC-α is for NG2 we used to the of an the treatment of U251/NG2 cells with In of the NG2 by that PKC-α NG2 a protein containing the cytoplasmic of NG2 was used as the for in kinase modification by recombinant PKC-α. of phosphorylated from this a to In contrast, is a for PKC-α phosphorylation In addition, of with the antibody phosphorylated components at and The to that threonine is phosphorylated by PKC-α in the in The is PKC-α at the kinase domain J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). with the on NG2 phosphorylation in U251/NG2 cells, these in to the that PKC-α threonine phosphorylation in the NG2 cytoplasmic The of phosphorylated PKC-α and from our that PKC-α to Thr2256 the in the site of NG2 threonine phosphorylation by we prepared three proteins containing NG2 cytoplasmic in which Thr2256, and were to glutamic acid These mutant proteins were used along with the type protein as for in phosphorylation by recombinant PKC-α. that phosphorylation of the mutant from the other three The the present in the other of a that the cytoplasmic domain a PKC-α phosphorylation site. the of phosphorylation in the mutant that Thr2256 is the site for PKC-α phosphorylation of an means of Thr2256 as the site of NG2 we of full-length NG2 and treatment of U251/NG2 cells with in the of These are obtained with the NG2 cytoplasmic domain This in to of the full-length to the of containing the phosphorylated For the of the cytoplasmic to the transmembrane domain might at these sites J.R. Nat. 2003; PubMed Scopus Google Scholar). In the of treatment several are on the of type treatment to the of and of phosphorylation at a site in the In contrast, treatment of U251 cells expressing an NG2 in which the threonine at position 2256 is replaced by valine to of these with the that Thr2256 is the key site of threonine phosphorylation in of Cell and of untreated U251/NG2 cells, NG2 is associated with a of membrane protrusions distributed over the apical cell surface NG2 is in these protrusions with the protein ezrin and with α3β1 integrin The of this of to the cell surface is confirmed by to NG2 antibody is at °C on cells The of NG2 on cell surface membrane protrusions in of U251/NG2 cells of that these cells with NG2-positive membrane protrusions are and to the In cells NG2 is to lamellipodia cells are to for lamellipodia and NG2-positive membrane protrusions the of the In lamellipodia is of NG2 with ezrin and α3β1 integrin with a antibody T. M. J. D. A. M. S. P.J. P.J. J. 2001; PubMed Scopus Google Scholar) that phosphorylated ezrin (Thr567) is present in the lamellipodia of cells, as as in the membrane protrusions of untreated cells in lamellipodia and of in cells with Cells in the transfected U251 NG2 over a of several in These cells are to lamellipodia in to treatment, of these and A of lamellipodia was made using a in which of the cells had NG2 lamellipodia were in of the NG2-positive cells. In contrast, of the cells lamellipodia, a functional for NG2 lamellipodia in to redistribution of NG2 to lamellipodia is blocked by the and The of this to phosphorylation of NG2 that phosphorylation of the proteoglycan a in redistribution to This was U251 cells expressing mutant of NG2 in which Thr2256, and were replaced by valine the of these three NG2 to cell surface protrusions is from that with type NG2 and The of the and to treatment that of type U251/NG2 cells and in NG2 is to In contrast, are resistant to the of Although of NG2-positive lamellipodia NG2 remains associated with apical protrusions in most cells. These are in by the of cells in that NG2 in lamellipodia as to apical membrane protrusions. treatment NG2 from membrane protrusions to lamellipodia in for the with the that phosphorylation of Thr2256 is for this type NG2, the is with ezrin and α3β1 integrin in membrane protrusions. of ezrin to in these as by with a substitution of a acid the of phosphorylation C. A.L. M. M. J.W. E. Pasquale 2000; PubMed Scopus Google Scholar), we transfected U251 cells with NG2 constructs in which glutamic acid residues replaced and The and and to from the valine and the type These mutant NG2 were localized to apical protrusions in untreated cells and were to lamellipodia In contrast, NG2 in lamellipodia both in the and of and of Thr2256 with glutamic acid the of NG2 and α3β1 integrin are in lamellipodia along with the in Cell and of in U251/NG2 cells are of by cells. We used to changes in cell motility. of a and the of U251/NG2 cells to the and Although cell motility is the motility by treatment is these are for NG2 and the of lamellipodia in the cells is the contains both NG2-positive in and cells in the cells are cells the and are in the a U251/NG2 in which of the cells had NG2 of that of the cells expressed were with the mutant NG2 U251/NG2 cells a and the motility of U251 cells expressing the and is by treatment and In contrast, treatment has on the motility of cells expressing the mutant and are spontaneously in the with and of this NG2 to lamellipodia and the and from type or the and and These are in We that these are by the of PKC-α to NG2 in the mutant and by the of glutamic acid substitution to phosphorylation at In we have been to that PKC-α the NG2 cytoplasmic In cell work we have used as a means of PKC-α-mediated phosphorylation of to to both and A. Mol. Cell. Biol. 2000; PubMed Scopus Google Scholar), as as other intracellular Mol. 2000; PubMed Scopus Google Scholar), on protein phosphorylation and on signaling processes. In work therefore important to NG2 phosphorylation growth and engagement of extracellular matrix in which intracellular signaling pathways are in a even against the of our provide that the phosphorylation of NG2 is a significant of NG2 at Thr2256 is accompanied by redistribution of the proteoglycan on the cell surface, by polarization of the cell and several key and by significant increases in cell motility. NG2 phosphorylation is a an of these is by the of the and These mutations are of the of NG2 and molecules that serve as of the The of the to and polarization increases in cell in to treatment that phosphorylation at Thr2256 is at a in for these to This is by the that and along with increased spontaneously in the glutamic acid residue threonine The of NG2 to cell surface of NG2 as a potential of membrane a to of the J.R. Nat. Rev. Mol. Cell. Biol. 2003; 4: 926-937Crossref PubMed Scopus (344) Google Scholar). In cells NG2 is localized to membrane protrusions from the cell surface, of found on the surface of human cells S. I. J. Cell Biol. PubMed Scopus Google Scholar). and PKC-α are in cells T. M. J. D. A. M. S. P.J. P.J. J. 2001; PubMed Scopus Google Scholar), with our that NG2, and α3β1 integrin all to present in membrane protrusions on the surface of U251/NG2 cells. has been that membrane protrusions of this serve as for of molecules that and S. I. J. Cell Biol. PubMed Scopus Google Scholar). These protrusions to from type of that contains NG2 (25Stallcup W.B. Dahlin-Huppe K. J. Cell Sci. 2001; 114: 2315-2325PubMed Google Scholar). are associated with the of cells and apical membrane protrusions. at Thr2256 in of NG2 to type of membrane the The or mechanisms redistribution of phosphorylated NG2 are We previously that treatment of U251/NG2 cells in of NG2 (24Nishiyama A. Lin X.H. Stallcup W.B. Mol. Biol. Cell. 1995; 6: 1819-1832Crossref PubMed Scopus (94) Google Scholar), the of a between NG2 phosphorylation and NG2 these of on phosphorylation of NG2 and redistribution of NG2 from membrane protrusions to lamellipodia In contrast, of significant of NG2 is for of The most for of NG2 remains that PKC-dependent protein is to the for NG2 (24Nishiyama A. Lin X.H. Stallcup W.B. Mol. Biol. Cell. 1995; 6: 1819-1832Crossref PubMed Scopus (94) Google Scholar). for the of NG2 phosphorylation on membrane of a that the interaction of the proteoglycan with a cytoplasmic or extracellular resulting in a in site of membrane on of NG2 with ezrin and α3β1 in with the of an interaction between and β1 T. M. J. D. A. M. S. P.J. P.J. J. 2001; PubMed Scopus Google Scholar), the of a between NG2, and PKC-α. We have the of NG2 to with α3β1 J. Makagiansar I.T. Stallcup W.B. Mol. Biol. Cell. 2004; 15: 3580-3590Crossref PubMed Scopus (263) Google Scholar). Although an interaction between NG2 and ezrin has been previously the of such an interaction the of NG2 with the X.H. Dahlin-Huppe K. Stallcup W.B. J. Cell. Biochem. 1996; PubMed Scopus Google Scholar). are no the of NG2 phosphorylation on these NG2 is with ezrin and α3β1 in both membrane protrusions and lamellipodia, is that phosphorylation interaction with either phosphorylation and dephosphorylation of NG2 interaction with In this of to the between NG2 and and Pasquale Stallcup W.B. J. Cell Biochem. 2000; PubMed Scopus (83) Google Scholar, J. H. K.A. J. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar) and This of and antibodies for with both the NG2 cytoplasmic domain and the full-length proteoglycan a interaction between NG2 and PKC-α. The proteoglycan therefore in the kinase to key sites activity signaling pathways that cell and motility. a has been previously for the transmembrane proteoglycan A. J.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). in to in NG2, PKC-α is for the phosphorylation of ezrin at T. M. J. D. A. M. S. P.J. P.J. J. 2001; PubMed Scopus Google Scholar). 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Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) in regulating changes in cell In the of U251 cells, is of polarization and motility in the cells. In the of NG2 these on U251 cells are by other such as A. Wilson J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). current that of NG2 in U251 cells has the of the of on cell and cell motility a that is on PKC-α-mediated phosphorylation of NG2 and redistribution of this proteoglycan on the cell surface. work at elucidating of the mechanisms that these We are to Monosov for and to Elena Pasquale for and