Association of GP Ib with actin-binding protein does not require GP IX.

GP IX is necessary for optimal expression of the GP Ib-IX complex on the surface of transfected cells, and presumably also on the surface of the platelet. The authors investigated whether increasing complex association with the cytoskeleton is one mechanism by which GP IX exerts its effect. CHO and L cell lines that express high levels of GP Ib were used to determine whether GP Ib (GPIb alpha and GPIb beta) associated with the cytoskeleton. GP Ib in these cells was found in the insoluble cytoskeletal fraction from Triton X-100 lysates in a proportion similar to that found in cells expressing the full complex. As in platelets and cells expressing the full complex, the association of GP Ib with the cytoskeleton was shown to be mediated by actin-binding protein (ABP). This was demonstrated by the observation that a monoclonal antibody against GPIb alpha precipitated ABP from GP Ib-expressing cells, and polyclonal anti-ABP antibodies specifically coprecipitated GP Ib. In addition, colocalization of the two components in intact cells was demonstrated by confocal microscopy. These data indicate that the association of GP Ib with the cytoskeleton is independent of GP IX, which therefore must increase surface expression of the complex by another mechanism.