'Clustering' SIRPα into the Plasma Membrane Lipid Microdomains Is Required for Activated Monocytes and Macrophages to Mediate Effective Cell Surface Interactions with CD47

SIRP alpha, an ITIMs-containing signaling receptor, negatively regulates leukocyte responses through extracellular interactions with CD47. However, the dynamics of SIRP alpha-CD47 interactions on the cell surface and the governing mechanisms remain unclear. Here we report that while the purified SIRPa binds to CD47 and that SIRPa is expressed on monocytes and monocytic THP-1 or U937, these SIRPa are ineffective to mediate cell binding to immobilized CD47. However, cell binding to CD47 is significantly enhanced when monocytes transmigrating across endothelia, or being differentiated into macrophages. Cell surface labeling reveals SIRP alpha to be diffused on naive monocytes but highly clustered on transmigrated monocytes and macrophages. Protein crosslink and equilibrium centrifugation confirm that SIRP alpha in the latter cells forms oligomerized complexes resulting in increased avidity for CD47 binding. Furthermore, formation of SIRPa complexes/clusters requires the plasma membrane 'lipid rafts' and the activity of Src family kinase during macrophage differentiation. These results together suggest that 'clustering' SIRP alpha into plasma membrane microdomains is essential for activated monocytes and macrophages to effectively interact with CD47 and initiate intracellular signaling.