How the integrin family of receptors for the extracellular matrix associates with the cytoskeleton remains a largely unanswered question. This proposal is aimed at identifying and characterizing proteins that bind to platelet integrin cytoplasmic domains. These proteins may function as links between these receptors and the cytoskeleton. Synthetic peptides that correspond to platelet integrin cytoplasmic domains will be immobilized and used for affinity chromatography to isolate proteins from resting, activated or aggregated platelets that bind to these integrins. Initially we will use peptides that are identical to the cytoplasmic domains of glycoproteins IIb/IIIa, the most abundant platelet integrin. Later we will use peptides that correspond to other platelet integrin cytoplasmic sequences. In addition, we will covalently link the two cytoplasmic domain peptides of glycoproteins Ilb/IIIa at the site where they would emerge from the membrane. Using these joined peptides, we will look for proteins which interact with the complex rather than the individual sequences. Proteins binding to these peptide columns will be purified and characterized with special attention paid to their interactions with intact integrins and cytoskeletal proteins, such as actin, talin and vinculin. These studies will be modeled after our recent experiments in which we have demonstrated an association between alpha-actinin and the cytoplasmic domain of the integrin beta l subunit. The localization of integrin-binding proteins will be determined in normal resting, activated and aggregated platelets by immunoelectron microscopy, and will be compared with their distribution in thrombasthenic platelets, lacking glycoproteins IIb/IIIa. Many of the same integrins are found in endothelial cells, permitting us to use the same peptides to look for related endothelial cell proteins that bind these integrins. To probe the function of these proteins, antibodies will be raised and introduced into endothelial cells by microinjection. We will look for effects on the organization of the actin cytoskeleton and for altered cell adhesion. Finally, using the peptide affinity columns, we will assay for regulatory proteins, particularly protein kinases, that bind to platelet integrin cytoplasmic domains. If kinases are discovered, we will examine their substrates to determine whether phosphorylation modifies their interaction with integrins or cytoskeletal proteins.
