P-selectin, a protein of platelets and endothelials cells, plays a role in inflammation and thrombosis following vascular injury. P-selectin is a cell adhesion molecule that resides in the alpha granules of resting platelets and the Weibel-Palade bodies of endothelial cells. Upon stimulation of these cells, the protein is translocated to the plasma membrane where it functions as a leukocyte receptor for neutrophils and monocytes. Since our discovery of this protein 12 years ago, our laboratory, in tandem with others, has characterized this protein, defined its function and determined the features of the counterreceptor that it recognizes on leukocytes. The current application represents a continuation of studies that address the structure of P-selectin, the structure and biology of the P-selectin ligand on leukocytes, and cell effector function and signal transduction stimulated by the interaction of P-selectin with the P-selectin ligand. To determine the three dimensional structure of the lectin and the lectin-EGF domains of P- selectin, these domains will be expressed in a bacterial expression system to obtain suitable quantities of biologically active peptide. These domains will be functionally characterized in terms of sialylated Lewis x binding, calcium ion binding and inhibitor of cell adhesion. Amino acid residues on P-selectin that define these binding functions will be identified by site-specific mutagenesis. Crystallization and determination of the three dimensional structure of the lectin domain and the lectin-EGF domain in the presence and absence of sialylated Lewis x will be performed in collaboration with Dr. William Weis. Signal transduction and effector function induced by cell activation of P- selectin binding to the P-selectin ligand will be studied in platelets and endothelial cells, with special attention to phosphorylation of P- selectin during platelet activation and endothelial cell stimulation. The kinases and phosphatases that act on P-selectin will be identified. Finally, effector functions induced in P-selectin ligand-expressing cells, including neutrophils and monocytes, during P-selectin binding will be analyzed, such as phosphorylation of the P-selectin ligand. The biology of PSGL-1, the P-selectin ligand, will be evaluated by site- specific mutagenesis to determine the function of the putative propeptide, the 14 dodecameric repeats and the cytoplasmic tail. The complete P-selectin ligand will be defined by identifying either additional proteins that form a complex with PSGL-1 or enzymes that posttranslationally modify PSGL-1 to yield the fully functional P- selectin ligand. Furthermore, we propose to clone the E-selectin ligand to allow direct comparison to the P-selectin ligand and the L-selectin ligand. These studies should contribute to our understanding of platelet and vascular biology, with specific emphasis on the role of selectins in thrombosis, hemostasis and the inflammatory response. The grant proposal is officially a new grant, but it is actually the competitive renewal of Project V of Program Project Grant HL42443 (Membrane Proteins in Blood Coagulation).
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