THE ASSOCIATION OF CD47 WITH INTEGRIN Mac-1 REGULATES MACROPHAGE RESPONSES BY STABILIZING THE EXTENDED INTEGRIN CONFORMATION

Abstract

CD47 is a ubiquitously expressed cell surface integrin-associated protein. Recently, we have demonstrated that integrin Mac-1 (M{beta}2, CD11b/CD18, CR3), the major adhesion receptor on the surface of myeloid cells, can be coprecipitated with CD47. However, the molecular basis for the CD47-Mac-1 interaction and its functional consequences remain unclear. Here, we demonstrated that CD47 regulates macrophage functions directly interacting with Mac-1. In particular, adhesion, spreading, migration, phagocytosis, and fusion of CD47-deficient macrophages were significantly decreased. The functional link between CD47 and Mac-1 was validated by co-immunoprecipitation analysis using various Mac-1-expressing cells. In HEK293 cells expressing individual M and {beta}2 integrin subunits, CD47 has been found to bind both subunits. Interestingly, the amount of CD47 recovered with the free {beta}2 subunit was higher than in the complex with the whole integrin. Furthermore, activating Mac-1-expressing HEK293 cells with PMA, Mn2+, and activating antibody increased CD47 in complex with Mac-1, suggesting greater stability of the complex with integrin in the extended conformation. Notably, on the surface of cells lacking CD47, fewer Mac-1 molecules could convert into an extended conformation in response to activation. The binding site in CD47 for Mac-1 was identified in its constituent IgV domain. The complementary binding sites for CD47 in Mac-1 were localized in integrin epidermal growth factor-like domains 3 and 4 of the {beta}2 and calf-1 and calf-2 domains of the subunits. These results indicate that Mac-1 forms a lateral complex with CD47, which regulates essential macrophage functions by stabilizing the extended integrin conformation.