Tyrosine sulfation of glycoprotein Ib alpha

Glycoprotein Ib alpha (GP Ib alpha), the ligand binding subunit of the plat elet glycoprotein Ib-M-V complex, is sulfated on three tyrosine residues (T yr-276, Tyr-278, and Tyr-279). This posttranslational modification is known to be critical for von Willebrand factor (VWF) binding; yet it remains unc lear whether it provides a specific structure or merely contributes negativ e charges. To investigate this issue, we constructed cell lines expressing GP Ib alpha polypeptides with the three tyrosine residues converted to eith er Glu or Phe and studied the ability of these mutants to bind vWF in the p resence of modulators or shear stress. The mutants were expressed normally on the cell surface as GP m-M complexes, with the conformation of the ligan d-binding domain preserved, as judged by the binding of conformation-sensit ive monoclonal antibodies. In contrast to their normal expression, both mut ants were functionally abnormal. Cells expressing the Phe mutant failed to bind vWF in the presence of either ristocetin or botrocetin. These cells ad hered to and rolled on immobilized vWF only when their surface receptor den sity was increased to twice the level that supported adhesion of cells expr essing the wild-type receptor and even then only 20% as many rolled and rol led significantly faster than wild-type cells. Cells expressing the Glu mut ant, on the other hand, were normal with respect to ristocetin-induced vWF binding and adhesion to immobilized VWF but were markedly defective in botr ocetin-induced vWF binding. These results indicate that GP Ib alpha tyrosin e sulfation influences the interaction of this polypeptide with vWF primari ly by contributing negative charges under physiological conditions and when the interaction is induced by ristocetin but contributes a specific struct ure to the botrocetin-induced interaction.