The aim is to further our understanding of the way in which actin filaments associate with platelet plasma membranes and of the role that such interactions have in platelet function. We have developed the hypothesis that actin filaments are attached to the membrane glycoprotein complex GP Ib-IX, with actin-binding protein serving as a linkage protein. We suggest that these filaments serve as a membrane skeleton, regulating the shape of the platelet and the function of the membrane glycoproteins to which they are attached. Experiments are designed (i) to characterize the interaction of actin-binding protein with GP Ib-IX, (ii) to test the hypothesis that actin-binding protein links actin filaments to membrane glycoproteins in other cells as well, and (iii) to identify platelet functions affected by this interaction. Actin-binding protein and the GP Ib-IX complex will be purified, and protocols are described for reconstituting the interaction between actin-binding protein and the glycoprotein complex. The reconstituted glycoprotein-actin-binding protein complex will be visualized by rotary shadowing. Experiments will be performed to determine to which glycoprotein actin-binding protein binds and to characterize the binding domains involved in this interaction. Preliminary experiments show that endothelial cells contain a polypeptide that reacts with GP Ib antibodies on Western blots. This protein will be purified, characterized, and studied to determine whether it is a membrane glycoprotein that binds to actin-binding protein. Antibodies against the cytoplasmic domain of the glycoprotein to which actin-binding protein binds in platelets will be raised and used to determine whether an antigenic determinant with a similar function exists in other nucleated cells. Experiments will be performed to determine whether the membrane-associated actin filaments affect the stability and the shape of the platelet and whether they regulate the mobility or function of the GP Ib-IX complex to which they are linked. Since GP Ib is the receptor for von Willebrand factor on platelets and is essential for adhesion of platelets to subendothelium, these experiments may identify potential sites at which platelet function can be regulated. By increasing our knowledge of the function of actin-membrane interactions in other cells, these studies may provide insight into processes such as ligand-induced receptor redistribution and regulation of receptor function.
| O'Toole, Timothy E; Bialkowska, Katarzyna; Li, Xiaohong et al. (2011) Tiam1 is recruited to ?1-integrin complexes by 14-3-3? where it mediates integrin-induced Rac1 activation and motility. J Cell Physiol 226:2965-78 |
