Membrane-cytoskeleton linkages occur in all eukaryotic cells where they play important roles in cell surface modulation, signal transduction, adhesion, and motility. Our long term goal is to understand the molecular architecture, regulation and functions of such membrane-cytoskeleton attachments in platelets. Platelets are fantastic cells that exhibit exquisitely regulated adhesive properties. When resting platelets encounter a discontinuity in a blood vessel wall, they become activated. Upon activation, the platelet fibrinogen receptor, alpha-IIb beta-3 integrin, develops ligand-binding capacity and is linked to the actin cytoskeleton. The membrane-cytoskeleton linkages generated during the platelet activation response are required for transmembrane signalling events that govern the regulated participation of the platelets in aggregation and clot retraction. Hemostasis thus depends on the cycles of transmembrane signalling that occur in activated platelets. Although the requirement for transmembrane signalling in the regulated adhesive response of blood platelets is well-documented, the detailed molecular machinery involved in the signalling events remains to be elucidated. Because integrin engagement and cytoskeletal association appear to be necessary for much of the transmembrane signalling, we have suggested that at least some of the downstream effectors are likely to be localized proximal to the site of integrin-cytoskeleton linkage. Sites of integrin- cytoskeleton linkage may provide a scaffold for the assembly of signalling machineries. One candidate for participation in transmembrane signalling events that are regulated by integrin occupancy is zyxin. Zyxin exhibits an unusual proline-rich domain as well as three copies of a zinc- finger motif (the LIM motif) that we postulate are involved in protein-protein interactions. The LIM motif is present in a variety of proteins that have well-established roles in cellular signalling events. We have recently identified zyxin in human blood platelets. To learn more about the structure and function of this protein we propose to define its subcellular distribution in platelets by immunoelectron microscopy, to examine the post-translational modification of zyxin in response to platelet activation, to isolate and characterize cDNAs that encode the human platelet protein, and to use biochemical and molecular approaches to identify zyxin-binding proteins in the platelet. Knowledge of the structure, subcellular location, and interactive partners of human platelet zyxin should significantly enhance our understanding of its physiological function and its involvement in the transmembrane signalling events required for hemostasis.
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