Selectins and their ligands (P-selectin glycoprotein ligand-1, PSGL-1, in particular) are important adhesion molecules with demonstrated roles both in normal inflammatory responses and in diseases. They are clearly defined biological systems and have been well characterized biochemically. Therefore, they represent an ideal system for studying kinetic properties using biophysical approaches. Kinetic properties are essential determinants of cell adhesion, as these parameters govern how likely, how rapidly and how strongly cells bind, as well as how long they remain bound in the presence and absence of external forces. Our overall hypothesis is that kinetic rates and binding affinity (including their force dependence) are intrinsic properties that are determined by the structure of the interacting molecules as well as their cell surface organizations. These properties govern the cell adhesion functions through a complex interplay with extrinsic biophysical/biochemical factors. As intrinsic properties, kinetic rates and binding affinity themselves are independent of any particular experimental technique used to measure their values and relationships, so long as the same properties are measured under comparable conditions. We have recently developed the micropipet and centrifugation techniques to quantify kinetic rates and binding affinity as well as the mathematical models to relate these properties to adhesion functions. Applying these new methods and using a variety of constructs of selectins and PSGL-1, we propose to: I. determine and compare the kinetic rates and binding affinity in the absence of force and their dependence on force of three selectins to their respective and common ligands on prototype leukocytes and carcinoma cells; II. delineate the extracellular contributions to the structure-function relationship by measuring the influences of the length and orientation of selectins as well as cell surface organization on the kinetic rates; and III. elucidate the cytoplasmic contributions to the structure-function relationship by quantifying the effects of P-selectin clustering and its interactions with other cell surface or cytoskeletal proteins on the kinetic rates and on the probability of uprooting the molecule from the cell surface. Not only will the information generated from this research advance our understanding of the workings of the selectin family of adhesion molecules, but it may also provide guidance to the development of selectin-based anti-inflammatory drugs which is currently being actively pursued by many biotechnological and pharmaceutical companies.
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