Tyrosine Sulfation of Glycoprotein Ibα (2001)

Glycoprotein Ibα (GP Ibα), the ligand binding subunit of the platelet glycoprotein Ib-IX-V complex, is sulfated on three tyrosine residues (Tyr-276, Tyr-278, and Tyr-279). This posttranslational modification is known to be critical for von Willebrand factor (vWF) binding; yet it remains unclear whether it provides a specific structure or merely contributes negative charges. To investigate this issue, we constructed cell lines expressing GP Ibα polypeptides with the three tyrosine residues converted to either Glu or Phe and studied the ability of these mutants to bind vWF in the presence of modulators or shear stress. The mutants were expressed normally on the cell surface as GP Ib-IX complexes, with the conformation of the ligand-binding domain preserved, as judged by the binding of conformation-sensitive monoclonal antibodies. In contrast to their normal expression, both mutants were functionally abnormal. Cells expressing the Phe mutant failed to bind vWF in the presence of either ristocetin or botrocetin. These cells adhered to and rolled on immobilized vWF only when their surface receptor density was increased to twice the level that supported adhesion of cells expressing the wild-type receptor and even then only 20% as many rolled and rolled significantly faster than wild-type cells. Cells expressing the Glu mutant, on the other hand, were normal with respect to ristocetin-induced vWF binding and adhesion to immobilized vWF but were markedly defective in botrocetin-induced vWF binding. These results indicate that GP Ibα tyrosine sulfation influences the interaction of this polypeptide with vWF primarily by contributing negative charges under physiological conditions and when the interaction is induced by ristocetin but contributes a specific structure to the botrocetin-induced interaction. Glycoprotein Ibα (GP Ibα), the ligand binding subunit of the platelet glycoprotein Ib-IX-V complex, is sulfated on three tyrosine residues (Tyr-276, Tyr-278, and Tyr-279). This posttranslational modification is known to be critical for von Willebrand factor (vWF) binding; yet it remains unclear whether it provides a specific structure or merely contributes negative charges. To investigate this issue, we constructed cell lines expressing GP Ibα polypeptides with the three tyrosine residues converted to either Glu or Phe and studied the ability of these mutants to bind vWF in the presence of modulators or shear stress. The mutants were expressed normally on the cell surface as GP Ib-IX complexes, with the conformation of the ligand-binding domain preserved, as judged by the binding of conformation-sensitive monoclonal antibodies. In contrast to their normal expression, both mutants were functionally abnormal. Cells expressing the Phe mutant failed to bind vWF in the presence of either ristocetin or botrocetin. These cells adhered to and rolled on immobilized vWF only when their surface receptor density was increased to twice the level that supported adhesion of cells expressing the wild-type receptor and even then only 20% as many rolled and rolled significantly faster than wild-type cells. Cells expressing the Glu mutant, on the other hand, were normal with respect to ristocetin-induced vWF binding and adhesion to immobilized vWF but were markedly defective in botrocetin-induced vWF binding. These results indicate that GP Ibα tyrosine sulfation influences the interaction of this polypeptide with vWF primarily by contributing negative charges under physiological conditions and when the interaction is induced by ristocetin but contributes a specific structure to the botrocetin-induced interaction. von Willebrand factor Chinese hamster ovary α-minimal essential medium phosphate-buffered saline glycoprotein The platelet receptor for von Willebrand factor (vWF),1 the glycoprotein (GP) Ib-IX-V complex, is composed of four polypeptide subunits (GP Ibα, GP Ibβ, GP IX, and GP V (1Berndt M.C. Gregory C. Kabral A. Zola H. Fournier D. Castaldi P.A. Eur. J. Biochem. 1985; 151: 637-649Crossref PubMed Scopus (150) Google Scholar, 2López J.A. Dong J.F. Curr. Opin. Hematol. 1997; 4: 323-329Crossref PubMed Scopus (93) Google Scholar)) encoded by four separate genes (3López J.A. Chung D.W. Fujikawa K. Hagen F.S. Papayannopoulou T. Roth G.J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5615-5619Crossref PubMed Scopus (281) Google Scholar, 4López J.A. Chung D.W. Fujikawa K. Hagen F.S. Davie E.W. Roth G.J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2135-2139Crossref PubMed Scopus (193) Google Scholar, 5Hickey M.J. Williams S.A. Roth G.J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6773-6777Crossref PubMed Scopus (124) Google Scholar, 6Lanza F. Morales M. de La Salle C. Cazenave J.-P. Clemetson K.J. Shimomura T. Phillips D.R. J. Biol. Chem. 1993; 268: 20801-20807Abstract Full Text PDF PubMed Google Scholar). GP Ibα is the largest subunit within the complex and so far the only subunit implicated in ligand binding, being capable of binding vWF (7López J.A. Blood Coagul. Fibrinolysis. 1994; 5: 97-119Crossref PubMed Scopus (291) Google Scholar), α-thrombin (7López J.A. Blood Coagul. Fibrinolysis. 1994; 5: 97-119Crossref PubMed Scopus (291) Google Scholar), P-selectin (8Romo G.M. Dong J.F. Schade A.J. Gardiner E.E. Li C. Kansas G.S. McIntire L.V. Berndt M.C. López J.A. J. Exp. Med. 1999; 190: 803-831Crossref PubMed Scopus (288) Google Scholar), and leukocyte Mac-1 (9Simon D.I. Chen Z. Xu H. Li C.Q. Dong J.F. McIntire L.V. Ballantyne C.M. Zhang L. Furman M.I. Berndt M.C. López J.A. J. Exp. Med. 2000; 192: 193-204Crossref PubMed Scopus (498) Google Scholar). The ligand-binding region resides within ∼300 amino acids at the GP Ibα N terminus and is held high above the cell membrane by a stiff, highly O-glycosylated mucin-like domain (Fig.1) (3López J.A. Chung D.W. Fujikawa K. Hagen F.S. Papayannopoulou T. Roth G.J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5615-5619Crossref PubMed Scopus (281) Google Scholar, 10Titani K. Takio K. Handa M. Ruggeri Z.M. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5610-5614Crossref PubMed Scopus (102) Google Scholar). The ligand-binding region can be divided into three distinct structural subdomains that are all implicated in vWF binding (7López J.A. Blood Coagul. Fibrinolysis. 1994; 5: 97-119Crossref PubMed Scopus (291) Google Scholar): seven tandem leucine-rich repeats, disulfide loops flanking the leucine-rich repeats, and a highly negatively charged sequence spanning residues Asp-269 to Asp-287 (3López J.A. Chung D.W. Fujikawa K. Hagen F.S. Papayannopoulou T. Roth G.J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5615-5619Crossref PubMed Scopus (281) Google Scholar,10Titani K. Takio K. Handa M. Ruggeri Z.M. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5610-5614Crossref PubMed Scopus (102) Google Scholar). Three tyrosine residues (Tyr-276, Tyr-278, and Tyr-279) are embedded in this negatively charged sequence (2López J.A. Dong J.F. Curr. Opin. Hematol. 1997; 4: 323-329Crossref PubMed Scopus (93) Google Scholar, 7López J.A. Blood Coagul. Fibrinolysis. 1994; 5: 97-119Crossref PubMed Scopus (291) Google Scholar) and each is fully sulfated, a modification critical for the binding of vWF and α-thrombin (11Dong J.-F. Li C.Q. López J.A. Biochemistry. 1994; 33: 13946-13953Crossref PubMed Scopus (107) Google Scholar, 12Marchese P. Murata M. Mazzucato M. Pradella P. De Marco L. Ware J. Ruggeri Z.M. J. Biol. Chem. 1995; 270: 9571-9578Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 13Ward C.M. Andrews R.K. Smith A.I. Berndt M.C. Biochemistry. 1996; 35: 4929-4938Crossref PubMed Scopus (180) Google Scholar).Tyrosine sulfation is a widespread posttranslational modification of proteins (14Carew J.A. Browning P.J. Lynch D.C. Blood. 1990; 76: 2530-2539Crossref PubMed Google Scholar, 15Huttner W.B. Nature. 1982; 299: 273-276Crossref PubMed Scopus (180) Google Scholar, 16Hortin G.L. Blood. 1990; 76: 946-952Crossref PubMed Google Scholar, 17Sako D. Comess K.M. Barone K.M. Camphausen R.T. Cumming D.A. Shaw G.D. Cell. 1995; 83: 323-331Abstract Full Text PDF PubMed Scopus (392) Google Scholar, 18Li F. Wilkins P.P. Crawley S. Weinstein J. Cummings R.D. McEver R.P. J. Biol. Chem. 1996; 271: 3255-3264Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar, 19Leyte A. van Schijndel H.B. Niehrs C. Huttner W.B. Verbeet M.P. Mertens K. van Mourik J.A. J. Biol. Chem. 1991; 266: 740-746Abstract Full Text PDF PubMed Google Scholar, 20Hortin G.L. Farries T.C. Graham J.P. Atkinson J.P. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1338-1342Crossref PubMed Scopus (66) Google Scholar), but few studies on sulfation in at the as of negative charges for or a specific structure for or To the of tyrosine sulfation in the interaction vWF and GP Ibα, we cell lines that GP Ibα mutants in the three sulfated tyrosine residues were by either Phe or Glu In the Phe mutant, the negative charges by sulfation are in the Glu mutant, the is but the is we on the of the on cell surface of the GP Ib-IX complex, vWF binding, and on cell adhesion to immobilized vWF under shear sulfation is a widespread posttranslational modification that in the it is by the W.B. Nature. 1982; 299: 273-276Crossref PubMed Scopus (180) Google W.B. Biochem. Sci. 1987; Full Text PDF Scopus Google Scholar, Biochem. PubMed Scopus Google Scholar, A. J. Biochem. PubMed Scopus Google Scholar). of proteins to this but only in a few A. van Schijndel H.B. Niehrs C. Huttner W.B. Verbeet M.P. Mertens K. van Mourik J.A. J. Biol. Chem. 1991; 266: 740-746Abstract Full Text PDF PubMed Google Scholar, H. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J.A. D. J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar), GP Ibα being of (11Dong J.-F. Li C.Q. López J.A. Biochemistry. 1994; 33: 13946-13953Crossref PubMed Scopus (107) Google Scholar, 12Marchese P. Murata M. Mazzucato M. Pradella P. De Marco L. Ware J. Ruggeri Z.M. J. Biol. Chem. 1995; 270: 9571-9578Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 13Ward C.M. Andrews R.K. Smith A.I. Berndt M.C. Biochemistry. 1996; 35: 4929-4938Crossref PubMed Scopus (180) Google Scholar). with the of sulfation for GP Ibα and only in other to J. Shaw G.D. Camphausen R.T. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). The by tyrosine sulfation in is by either or contributing to a specific structure or by contributing negative charges. by the of J. Shaw G.D. Camphausen R.T. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar) that the sulfated of glycoprotein in when this In the we whether is for GP Ibα or whether for the vWF binding this by cell lines expressing either wild-type GP Ibα or mutant the three with either or The Phe the of the but the The Glu on the other hand, the but their mutants were expressed on the cell surface normally and the normal conformation of the GP Ibα N as by the binding of conformation-sensitive antibodies. The monoclonal failed to bind both mutants the sulfated region binding C.M. Andrews R.K. Smith A.I. Berndt M.C. Biochemistry. 1996; 35: 4929-4938Crossref PubMed Scopus (180) Google the vWF binding on that binding was vWF binding was induced by a known to bind vWF and both mutants were to be markedly defective This is with that the sulfated of GP Ibα is in botrocetin-induced vWF binding and that even of this can binding (7López J.A. Blood Coagul. Fibrinolysis. 1994; 5: 97-119Crossref PubMed Scopus (291) Google Scholar, 12Marchese P. Murata M. Mazzucato M. Pradella P. De Marco L. Ware J. Ruggeri Z.M. J. Biol. Chem. 1995; 270: 9571-9578Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, Dong J. Schade A. McIntire L. D. Berndt M.C. López J.A. Andrews R.K. Blood. 2000; PubMed Google Scholar). In only the Phe mutant was defective when vWF binding was induced by ristocetin a that we to physiological vWF binding, binding induced by high shear or by vWF a surface J.F. Berndt M.C. Schade A. McIntire L.V. Andrews R.K. López J. Blood. PubMed Scopus Google Scholar). This in ristocetin-induced vWF binding was by a in of the cells to immobilized vWF in a surface of the Phe mutant to on wild-type cells that and rolled on the cells expressing the mutant failed to to the surface the cells and a few but of the surface by This is in contrast to the Glu and rolled on the surface in a the wild-type cells. with to the physiological of GP vWF binding of the sulfated with residues to fully results that tyrosine sulfation influences the interaction GP Ibα and vWF either by or by both of can be by the of the the of the on vWF binding and on ristocetin-induced vWF binding is with that ristocetin is a of the interaction than is J.F. Berndt M.C. Schade A. McIntire L.V. Andrews R.K. López J. Blood. PubMed Scopus Google Scholar). The specific sequence is as critical for ristocetin-induced binding as for botrocetin-induced binding, is markedly by both the Phe and the Glu The that the sulfated region of GP Ibα at a for botrocetin-induced vWF binding, with the being for the interaction. the results that the three negative charges by the sulfated are in the of the GP interaction. The Phe these negative with immobilized vWF under conditions of by the presence of cells and by the that cells that to the surface by than by This was by the surface of the mutant we that sulfation of tyrosine residues within the sulfated region of platelet GP Ibα contributes negative charges that are critical for the interaction of GP Ibα with vWF under and in the presence of than a specific sequence is for these the is for the interaction induced by botrocetin. results the that is than binding on GP Ibα for vWF and that the of that on the by the interaction is The platelet receptor for von Willebrand factor (vWF),1 the glycoprotein (GP) Ib-IX-V complex, is composed of four polypeptide subunits (GP Ibα, GP Ibβ, GP IX, and GP V (1Berndt M.C. Gregory C. Kabral A. Zola H. Fournier D. Castaldi P.A. Eur. J. Biochem. 1985; 151: 637-649Crossref PubMed Scopus (150) Google Scholar, 2López J.A. Dong J.F. Curr. Opin. Hematol. 1997; 4: 323-329Crossref PubMed Scopus (93) Google Scholar)) encoded by four separate genes (3López J.A. Chung D.W. Fujikawa K. Hagen F.S. Papayannopoulou T. Roth G.J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5615-5619Crossref PubMed Scopus (281) Google Scholar, 4López J.A. Chung D.W. Fujikawa K. Hagen F.S. Davie E.W. Roth G.J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2135-2139Crossref PubMed Scopus (193) Google Scholar, 5Hickey M.J. Williams S.A. Roth G.J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6773-6777Crossref PubMed Scopus (124) Google Scholar, 6Lanza F. Morales M. de La Salle C. Cazenave J.-P. Clemetson K.J. Shimomura T. Phillips D.R. J. Biol. Chem. 1993; 268: 20801-20807Abstract Full Text PDF PubMed Google Scholar). GP Ibα is the largest subunit within the complex and so far the only subunit implicated in ligand binding, being capable of binding vWF (7López J.A. Blood Coagul. Fibrinolysis. 1994; 5: 97-119Crossref PubMed Scopus (291) Google Scholar), α-thrombin (7López J.A. Blood Coagul. Fibrinolysis. 1994; 5: 97-119Crossref PubMed Scopus (291) Google Scholar), P-selectin (8Romo G.M. Dong J.F. Schade A.J. Gardiner E.E. Li C. Kansas G.S. McIntire L.V. Berndt M.C. López J.A. J. Exp. Med. 1999; 190: 803-831Crossref PubMed Scopus (288) Google Scholar), and leukocyte Mac-1 (9Simon D.I. Chen Z. Xu H. Li C.Q. Dong J.F. McIntire L.V. Ballantyne C.M. Zhang L. Furman M.I. Berndt M.C. López J.A. J. Exp. Med. 2000; 192: 193-204Crossref PubMed Scopus (498) Google Scholar). The ligand-binding region resides within ∼300 amino acids at the GP Ibα N terminus and is held high above the cell membrane by a stiff, highly O-glycosylated mucin-like domain (Fig.1) (3López J.A. Chung D.W. Fujikawa K. Hagen F.S. Papayannopoulou T. Roth G.J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5615-5619Crossref PubMed Scopus (281) Google Scholar, 10Titani K. Takio K. Handa M. Ruggeri Z.M. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5610-5614Crossref PubMed Scopus (102) Google Scholar). The ligand-binding region can be divided into three distinct structural subdomains that are all implicated in vWF binding (7López J.A. Blood Coagul. Fibrinolysis. 1994; 5: 97-119Crossref PubMed Scopus (291) Google Scholar): seven tandem leucine-rich repeats, disulfide loops flanking the leucine-rich repeats, and a highly negatively charged sequence spanning residues Asp-269 to Asp-287 (3López J.A. Chung D.W. Fujikawa K. Hagen F.S. Papayannopoulou T. Roth G.J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5615-5619Crossref PubMed Scopus (281) Google Scholar,10Titani K. Takio K. Handa M. Ruggeri Z.M. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5610-5614Crossref PubMed Scopus (102) Google Scholar). Three tyrosine residues (Tyr-276, Tyr-278, and Tyr-279) are embedded in this negatively charged sequence (2López J.A. Dong J.F. Curr. Opin. Hematol. 1997; 4: 323-329Crossref PubMed Scopus (93) Google Scholar, 7López J.A. Blood Coagul. Fibrinolysis. 1994; 5: 97-119Crossref PubMed Scopus (291) Google Scholar) and each is fully sulfated, a modification critical for the binding of vWF and α-thrombin (11Dong J.-F. Li C.Q. López J.A. Biochemistry. 1994; 33: 13946-13953Crossref PubMed Scopus (107) Google Scholar, 12Marchese P. Murata M. Mazzucato M. Pradella P. De Marco L. Ware J. Ruggeri Z.M. J. Biol. Chem. 1995; 270: 9571-9578Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 13Ward C.M. Andrews R.K. Smith A.I. Berndt M.C. Biochemistry. 1996; 35: 4929-4938Crossref PubMed Scopus (180) Google Scholar). sulfation is a widespread posttranslational modification of proteins (14Carew J.A. Browning P.J. Lynch D.C. Blood. 1990; 76: 2530-2539Crossref PubMed Google Scholar, 15Huttner W.B. Nature. 1982; 299: 273-276Crossref PubMed Scopus (180) Google Scholar, 16Hortin G.L. Blood. 1990; 76: 946-952Crossref PubMed Google Scholar, 17Sako D. Comess K.M. Barone K.M. Camphausen R.T. Cumming D.A. Shaw G.D. Cell. 1995; 83: 323-331Abstract Full Text PDF PubMed Scopus (392) Google Scholar, 18Li F. Wilkins P.P. Crawley S. Weinstein J. Cummings R.D. McEver R.P. J. Biol. Chem. 1996; 271: 3255-3264Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar, 19Leyte A. van Schijndel H.B. Niehrs C. Huttner W.B. Verbeet M.P. Mertens K. van Mourik J.A. J. Biol. Chem. 1991; 266: 740-746Abstract Full Text PDF PubMed Google Scholar, 20Hortin G.L. Farries T.C. Graham J.P. Atkinson J.P. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1338-1342Crossref PubMed Scopus (66) Google Scholar), but few studies on sulfation in at the as of negative charges for or a specific structure for or To the of tyrosine sulfation in the interaction vWF and GP Ibα, we cell lines that GP Ibα mutants in the three sulfated tyrosine residues were by either Phe or Glu In the Phe mutant, the negative charges by sulfation are in the Glu mutant, the is but the is we on the of the on cell surface of the GP Ib-IX complex, vWF binding, and on cell adhesion to immobilized vWF under shear stress. sulfation is a widespread posttranslational modification that in the it is by the W.B. Nature. 1982; 299: 273-276Crossref PubMed Scopus (180) Google W.B. Biochem. Sci. 1987; Full Text PDF Scopus Google Scholar, Biochem. PubMed Scopus Google Scholar, A. J. Biochem. PubMed Scopus Google Scholar). of proteins to this but only in a few A. van Schijndel H.B. Niehrs C. Huttner W.B. Verbeet M.P. Mertens K. van Mourik J.A. J. Biol. Chem. 1991; 266: 740-746Abstract Full Text PDF PubMed Google Scholar, H. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J.A. D. J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar), GP Ibα being of (11Dong J.-F. Li C.Q. López J.A. Biochemistry. 1994; 33: 13946-13953Crossref PubMed Scopus (107) Google Scholar, 12Marchese P. Murata M. Mazzucato M. Pradella P. De Marco L. Ware J. Ruggeri Z.M. J. Biol. Chem. 1995; 270: 9571-9578Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 13Ward C.M. Andrews R.K. Smith A.I. Berndt M.C. Biochemistry. 1996; 35: 4929-4938Crossref PubMed Scopus (180) Google Scholar). with the of sulfation for GP Ibα and only in other to J. Shaw G.D. Camphausen R.T. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). The by tyrosine sulfation in is by either or contributing to a specific structure or by contributing negative charges. by the of J. Shaw G.D. Camphausen R.T. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar) that the sulfated of glycoprotein in when this In the we whether is for GP Ibα or whether for the vWF binding this by cell lines expressing either wild-type GP Ibα or mutant the three with either or The Phe the of the but the The Glu on the other hand, the but their mutants were expressed on the cell surface normally and the normal conformation of the GP Ibα N as by the binding of conformation-sensitive antibodies. The monoclonal failed to bind both mutants the sulfated region binding C.M. Andrews R.K. Smith A.I. Berndt M.C. Biochemistry. 1996; 35: 4929-4938Crossref PubMed Scopus (180) Google the vWF binding on that binding was vWF binding was induced by a known to bind vWF and both mutants were to be markedly defective This is with that the sulfated of GP Ibα is in botrocetin-induced vWF binding and that even of this can binding (7López J.A. Blood Coagul. Fibrinolysis. 1994; 5: 97-119Crossref PubMed Scopus (291) Google Scholar, 12Marchese P. Murata M. Mazzucato M. Pradella P. De Marco L. Ware J. Ruggeri Z.M. J. Biol. Chem. 1995; 270: 9571-9578Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, Dong J. Schade A. McIntire L. D. Berndt M.C. López J.A. Andrews R.K. Blood. 2000; PubMed Google Scholar). In only the Phe mutant was defective when vWF binding was induced by ristocetin a that we to physiological vWF binding, binding induced by high shear or by vWF a surface J.F. Berndt M.C. Schade A. McIntire L.V. Andrews R.K. López J. Blood. PubMed Scopus Google Scholar). This in ristocetin-induced vWF binding was by a in of the cells to immobilized vWF in a surface of the Phe mutant to on wild-type cells that and rolled on the cells expressing the mutant failed to to the surface the cells and a few but of the surface by This is in contrast to the Glu and rolled on the surface in a the wild-type cells. with to the physiological of GP vWF binding of the sulfated with residues to fully results that tyrosine sulfation influences the interaction GP Ibα and vWF either by or by both of can be by the of the the of the on vWF binding and on ristocetin-induced vWF binding is with that ristocetin is a of the interaction than is J.F. Berndt M.C. Schade A. McIntire L.V. Andrews R.K. López J. Blood. PubMed Scopus Google Scholar). The specific sequence is as critical for ristocetin-induced binding as for botrocetin-induced binding, is markedly by both the Phe and the Glu The that the sulfated region of GP Ibα at a for botrocetin-induced vWF binding, with the being for the interaction. the results that the three negative charges by the sulfated are in the of the GP interaction. The Phe these negative with immobilized vWF under conditions of by the presence of cells and by the that cells that to the surface by than by This was by the surface of the mutant we that sulfation of tyrosine residues within the sulfated region of platelet GP Ibα contributes negative charges that are critical for the interaction of GP Ibα with vWF under and in the presence of than a specific sequence is for these the is for the interaction induced by botrocetin. results the that is than binding on GP Ibα for vWF and that the of that on the by the interaction is sulfation is a widespread posttranslational modification that in the it is by the W.B. Nature. 1982; 299: 273-276Crossref PubMed Scopus (180) Google W.B. Biochem. Sci. 1987; Full Text PDF Scopus Google Scholar, Biochem. PubMed Scopus Google Scholar, A. J. Biochem. PubMed Scopus Google Scholar). of proteins to this but only in a few A. van Schijndel H.B. Niehrs C. Huttner W.B. Verbeet M.P. Mertens K. van Mourik J.A. J. Biol. Chem. 1991; 266: 740-746Abstract Full Text PDF PubMed Google Scholar, H. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J.A. D. J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar), GP Ibα being of (11Dong J.-F. Li C.Q. López J.A. Biochemistry. 1994; 33: 13946-13953Crossref PubMed Scopus (107) Google Scholar, 12Marchese P. Murata M. Mazzucato M. Pradella P. De Marco L. Ware J. Ruggeri Z.M. J. Biol. Chem. 1995; 270: 9571-9578Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 13Ward C.M. Andrews R.K. Smith A.I. Berndt M.C. Biochemistry. 1996; 35: 4929-4938Crossref PubMed Scopus (180) Google Scholar). with the of sulfation for GP Ibα and only in other to J. Shaw G.D. Camphausen R.T. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). The by tyrosine sulfation in is by either or contributing to a specific structure or by contributing negative charges. by the of J. Shaw G.D. Camphausen R.T. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar) that the sulfated of glycoprotein in when this In the we whether is for GP Ibα or whether for the vWF binding this by cell lines expressing either wild-type GP Ibα or mutant the three with either or The Phe the of the but the The Glu on the other hand, the but their mutants were expressed on the cell surface normally and the normal conformation of the GP Ibα N as by the binding of conformation-sensitive antibodies. The monoclonal failed to bind both mutants the sulfated region binding C.M. Andrews R.K. Smith A.I. Berndt M.C. Biochemistry. 1996; 35: 4929-4938Crossref PubMed Scopus (180) Google Scholar). the vWF binding on that binding was vWF binding was induced by a known to bind vWF and both mutants were to be markedly defective This is with that the sulfated of GP Ibα is in botrocetin-induced vWF binding and that even of this can binding (7López J.A. Blood Coagul. Fibrinolysis. 1994; 5: 97-119Crossref PubMed Scopus (291) Google Scholar, 12Marchese P. Murata M. Mazzucato M. Pradella P. De Marco L. Ware J. Ruggeri Z.M. J. Biol. Chem. 1995; 270: 9571-9578Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, Dong J. Schade A. McIntire L. D. Berndt M.C. López J.A. Andrews R.K. Blood. 2000; PubMed Google Scholar). In only the Phe mutant was defective when vWF binding was induced by ristocetin a that we to physiological vWF binding, binding induced by high shear or by vWF a surface J.F. Berndt M.C. Schade A. McIntire L.V. Andrews R.K. López J. Blood. PubMed Scopus Google Scholar). This in ristocetin-induced vWF binding was by a in of the cells to immobilized vWF in a surface of the Phe mutant to on wild-type cells that and rolled on the cells expressing the mutant failed to to the surface the cells and a few but of the surface by This is in contrast to the Glu and rolled on the surface in a the wild-type cells. with to the physiological of GP vWF binding of the sulfated with residues to fully These results that tyrosine sulfation influences the interaction GP Ibα and vWF either by or by both of can be by the of the the of the on vWF binding and on ristocetin-induced vWF binding is with that ristocetin is a of the interaction than is J.F. Berndt M.C. Schade A. McIntire L.V. Andrews R.K. López J. Blood. PubMed Scopus Google Scholar). The specific sequence is as critical for ristocetin-induced binding as for botrocetin-induced binding, is markedly by both the Phe and the Glu The that the sulfated region of GP Ibα at a for botrocetin-induced vWF binding, with the being for the interaction. the results that the three negative charges by the sulfated are in the of the GP interaction. The Phe these negative with immobilized vWF under conditions of by the presence of cells and by the that cells that to the surface by than by This was by the surface of the mutant In we that sulfation of tyrosine residues within the sulfated region of platelet GP Ibα contributes negative charges that are critical for the interaction of GP Ibα with vWF under and in the presence of than a specific sequence is for these the is for the interaction induced by botrocetin. results the that is than binding on GP Ibα for vWF and that the of that on the by the interaction is H. for in von Willebrand C. Berndt for and for of the