Integrin-associated protein (IAP, CD47) is a ubiquitously expressed receptor for thrombospondins (TSPs) and for Signal Inhibitory Regulatory Protein a (SlRPa). CD47 can associate with, 81, 32 and 03 integrins and augment their functions. Thus the biological consequence of CD47 signaling depends on the particular integrin expressed in a given cell type and can lead to enhanced cell motility, spreading, platelet activation, phagocytosis, oxidiative burst and even apoptosis. Many of the effects of CD47 are blocked by pertussis toxin treatment of cells indicating a common role for heterotrimeric Gi family proteins in connecting CD47 to different integrins and their signaling pathways. We have isolated a detergent soluble complex containing integrin, CD47 and Gi and find that stability of this complex requires CD47 and cholesterol, providing a rationale for localization of the complex in cholesterol-rich lipid domains. We have now established experimental systems with which to determine the sites and mechanisms of interaction of the components of this signaling complex. The primary goals of the current proposal are to elucidate the molecular interactions among CD47, the integrins with which it associates and signaling components including heterotrimeric Gi and downstream signaling effectors. The specif~c aims are: (1) To define the molecular interactions responsible for assembly of and signaling by the CD47-integrin-Gi complex using molecular biological and chemical crosslinking approaches. (2) To identify the components of the CD47/integrin signaling complexes, rapidly isolated on mAb- or ligand-coated magnetic beads, employing a panel of cells expressing the integrin of interest and CD47 singly and together. (3) To test the roles of candidate proteins identified in Aim 2 in CD47 ar~d integrin signaling using co-localization studies, antibody, peptide and chemical inhibitors as available, expression of dominant negative and other mutant forms of the proteins, and antisense and genetic deletion strategies.
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