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

Abstract

ABSTRACTCD47 is a ubiquitously expressed cell surface integrin-associated protein. Recently, we have demonstrated that integrin Mac-1 (αMβ2, 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 αM and β2 integrin subunits, CD47 has been found to bind both subunits. Interestingly, the amount of CD47 recovered with the free β2 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 β2 and calf-1 and calf-2 domains of the α subunits. These results indicate that Mac-1 forms a lateral complex with CD47, which regulates essential macrophage functions by stabilizing the extended integrin conformation.