P=selectin is a cell adhesion molecule stored in the secretory granules of resting endothelial cells and platelets. Upon stimulation of these cells, P-selectin migrates to the plasma membrane to function as a leukocyte receptor. The phosphorylation of P-selectin after cell stimulation has recently been identified in both platelets and endothelial cells. This proposal aims to further examine phosphorylation events in endothelial cells and isolate protein(s) binding to the cytoplasmic tail of P-selectin. Specifically, the kinetics of phosphorylation will be determined by quantifying the amount of 32P- labeled P-selectin recovered by immunoprecipitation of endothelial cells lysed at selected time points after thrombin stimulation. The resulting phospho-amino acids will be identified through acid and base hydrolysis. Thus, the temporal relationship of P-selectin phosphorylation to events such as P-selectin intracellular transport and cellular activation will be established. The effect of phosphorylation on the affinity of P- selectin for its ligand on HL-60 cells will also be ascertained, as there is evidence or "inside-out" signaling from the integrin family of cell adhesion molecules. Furthermore, isolation of protein(s) which interact(s) with P-selectin will be accomplished through co- immunoprecipitation from cytosol, cytoskeleton, and membrane cytoskeleton fractious of resting vs. activated endothelial cells. The Mr-and subunit structure of the protein will be determined by SDS PAGE. The metal ion and nucleotide tripbosphate dependence of the binding interaction will be characterized. N-terminal sequencing will aid in identifying what may prove to be a kinase or phosphatase a chaperone molecule, or a protein involved in a signal cascade. In defining a role for this post- translational phosphorylation event, the proposed project has broad implications for the understanding of endothelial cell and platelet function in the thrombotic and inflammatory responses. The methodology proposed to accomplish the stated aims will add mastery of protein and phospholipid chemistry as well as immunochemistry techniques to the candidates prior molecular biology experience. expanding her armamentarium of weapons essential to attack fundamental questions in the field of signal transduction and cell-cell interaction.