Neutrophils are critical effector cells in innate immunity as well as in inflammatory settings with pathological consequences, such as sepsis, reperfusion injury, and asthma. It is well established that the neutrophil- expressed, adhesion molecule L-selectin plays an important role in directing these cells to diverse inflammatory settings. The cytoplasmic domain of L-selectin regulates receptor binding activity, cytoskeletal interactions, and endoproteolytic processing (shedding), all of which are known to affect L-selectin adhesion. However, our knowledge of the intracellular factors that interact with the cytoplasmic domain of L-selectin and their mode of function is minimal. Obtaining this knowledge is important, because, it may provide new therapeutic abilities to modulate L- selectin adhesion and manipulate leukocyte recruitment to sites of inflammation. Our long-term goal is to understand how L-selectin adhesion can be modulated for therapeutic purposes. The objective of this application is to determine the means by which the intracellular Ca+2 regulatory protein calmodulin regulates L-selectin adhesion. The central hypothesis of this application is that the co-association of calmodulin with the cytoplasmic domain of L-selectin is important in regulating L-selectin adhesion. This is based on compelling preliminary data that demonstrates a direct and specific interaction between calmodulin and the cytoplasmic domain of L- selectin. Disrupting this interaction profoundly reduces the accumulation of leukocytes on endothelial ligands in an assay system that simulates physiological blood flow conditions. The rationale for the proposed research is that once it is known how the intermolecular interaction between the cytoplasmic domain of L-selectin and calmodulin regulates L- selectin adhesion, this event or specific downstream effects might be manipulated pharmacologically in new and innovate approaches to alter neutrophil accumulation at sites of inflammation. We are particularly well prepared to undertake these studies considering our expertise in the area of adhesion receptor structure/function, our established assay systems, and our novel reagents. This work will be completed in a research environment very conducive to its successful completion. The University of Minnesota contains numerous established and well-funded investigators, for example, as those found in the Center for Immunology and the Cancer Center, which the PI is a member of. We propose to test our hypothesis by performing the following three specific aims. 1. Define the motif in the cytoplasmic domain of L-selectin that is required for calmodulin binding. Based on preliminary findings, our working hypothesis is that the highly basic NH2-terminal region of L-selectin's cytoplasmic domain supports calmodulin binding. Our approach will involve site directed mutagenesis of the human L-selectin cytoplasmic domain, such as truncation and scanning alanine point mutations, to identify critical residues for calmodulin binding. 2. Determine the effects of directly disrupting the co-association of calmodulin with L-selectin on adhesion. Our working hypothesis, again based on preliminary studies, is that calmodulin regulates L-selectin adhesion, in part, through the induction of L-selectin proteolysis. Our approach will involve determining the effects of directly disrupting the co- association of calmodulin with L-selectin on L-selectin adhesion, examined by an in vitro assay that simulates physiologic blood flow conditions. 3. Evaluate the intracellular mechanisms that regulate calmodulin binding to L-selectin. Based on preliminary findings, our working hypothesis is that intracellular Ca+2 concentrations and/or L-selectin phosphorylation regulate calmodulin binding to L-selectin. Our approach will involve determining the effects of L-selectin phosphorylation on calmodulin binding and L-selectin adhesion, and investigating the Ca+2 dependence of calmodulin/L-selectin interactions. The proposed work is innovative, because, it will help elucidate important molecular mechanisms linking inflammatory stimulation to the modulation of leukocyte adhesive functions. In addition, we possess novel reagents to study the transmembrane fragment of L-selectin that remains after endoproteolytic shedding, which to our knowledge is not being performed in any other laboratory. It is our expectation that the proposed study will determine the mode of function of calmodulin in regulating L selectin adhesion. These results will be significant as they are expected to provide new insights into the regulation of L-selectin adhesion, which may result in novel therapeutic interventions for inflammatory diseases.
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