beta2 integrins are the major integrins expressed on leukocytes. They mediate the divalent-cation dependent adhesion functions of these cells including leukocyte homing, firm adhesion, migration, and clearance of pathogens through phagocytosis and cell-mediated killing. beta2 integrins also contribute to injury in many noninfectious diseases where the receptors are pathologically activated; these include heart attacks renal failure, allograft rejection, strokes and diabetic complications. beta2 integrins have thus become very important therapeutic targets in inflammation, autoimmunity and transplantation. The divalent cation-dependent interaction of b2 integrins with their physiologic ligands is tightly regulated by an """"""""inside-out"""""""" activation process triggered intracellularly. It results through poorly understood extracellular conformational changes, in making these receptors ligand-competent. We have traced a component of this conformational change to a major ligand-binding A-domain present in one-half of al integrin alpha subunits, including all beta integrins. We first isolated this domain in a functionally active state determined the crystal structure of its two conformations and showed that these correspond to the low and high affinity states of this domain. More recently, stable and soluble low and high affinity forms of the domain were produced and shown to interact differentially with novel ligands: interaction of the domain with some ligands required activation but that with others did not. The structural basis of these interactions is unknown. We have also solved the crystal structure of a smaller integrin (lacking the aA-domain). We now propose to structurally characterize the interaction of the low and high affinity states of the aA-domain ir complex with activation-dependent and independent ligands and begin to put this information in the wide structural context of a whole integrin by deriving the crystal structure of an integrin containing the A-domain. The beta2 integrin CD11b/CD18, the most abundant integrin of phagocytes, will continue to be the focus of our studies. Results from these studies will have profound impact on understanding the inner workings of A domain containing integrins, and consequently on the regulation of cell adhesion-dependent functions. It should also facilitate rational drug design for this class of therapeutic targets.
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