The Cytoplasmic Domain of Glycoprotein (GP) Ibα Constrains the Lateral Diffusion of the GP Ib−IX Complex and Modulates von Willebrand Factor Binding

Abstract

To study the role of the glycoprotein (GP) Ibα cytoplasmic domain in the mobility of the GP Ib−IX complex within the plasma membrane and in its ability to bind vWf, we established eight cell lines expressing GP Ib−IX complexes (these complexes lack GP V but function normally as receptors for vWf) that contain either wild-type GP Ibα or one of a series of GP Ibα truncation mutants missing different lengths of the cytoplasmic domain. To test the mobility of these complexes within the plasma membrane, we used the technique of fluorescence recovery after photobleaching after labeling them with a fluorescein-conjugated anti-GP Ibα monoclonal antibody. Fluorescence recovery within a bleached area on the cell surface was evaluated by scanning the cell surface with a low-intensity laser for 3 min after bleaching and then extrapolating the recovery values to infinite time. Fluorescence recovery in cells expressing wild-type GP Ibα was negligible. However, when only six amino acids were removed from the GP Ibα carboxyl terminus (t604 mutant, polypeptide length of 604 vs 610 residues for wild-type GP Ibα), complex mobility increased greatly, as judged by a more rapid recovery of fluorescence in the bleached area (48% recovery). The mobility increased further in the t594 mutant and remained approximately the same through the t534 mutant (55−67% recovery). A further increase in mobility was observed with the t518 mutant (>80% recovery), which lacks almost all of the GP Ibα cytoplasmic domain. The ristocetin-dependent binding of the mutant cell lines was also evaluated. Binding of vWf to cells expressing any of the mutant complexes was markedly lower than that to cells expressing the wild-type complex. These studies demonstrate that the cytoplasmic domain of GP Ibα fixes the position of the GP Ib−IX complex on the platelet surface and that this orientation is an important determinant of the complex's ability to bind vWf.