Abstract
SIRPα, an ITIMs-containing signaling receptor, negatively regulates leukocyte responses through extracellular interactions with CD47. However, the dynamics of SIRPα-CD47 interactions on the cell surface and the governing mechanisms remain unclear. Here we report that while the purified SIRPα binds to CD47 and that SIRPα is expressed on monocytes and monocytic THP-1 or U937, these SIRPα 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 SIRPα to be diffused on naïve monocytes but highly clustered on transmigrated monocytes and macrophages. Protein crosslink and equilibrium centrifugation confirm that SIRPα in the latter cells forms oligomerized complexes resulting in increased avidity for CD47 binding. Furthermore, formation of SIRPα complexes/clusters requires the plasma membrane ‘lipid rafts’ and the activity of Src family kinase during macrophage differentiation. These results together suggest that ‘clustering’ SIRPα into plasma membrane microdomains is essential for activated monocytes and macrophages to effectively interact with CD47 and initiate intracellular signaling.
Highlights
Signal regulatory protein α (SIRPα) is a cell surface receptor-like signaling protein predominantly expressed in myeloid leukocytes including granulocytes, monocytes, macrophages and dendritic cells [1,2]
It has been suggested that the extracellular binding of SIRPα to CD47 triggers tyrosine phosphorylation in its cytoplasmic immunoreceptor tyrosinebased inhibitory motifs (ITIMs), resulting in the association of the SH2 domain-containing protein tyrosine phosphatase (SHP-1 or 2), which initiates negative signaling events leading to the inhibition of cell function [11,12,13,14,15,16]
Cell surface immunofluorescence labeling showed that in peripheral blood mononuclear cells (PBMC), SIRPα is selectively expressed on CD14+ monocytes but not on lymphocyte-specific protein tyrosine kinase (Lck)+ T cells or B220+ B lymphocytes (Figure 1B), confirming previous results [25]
Summary
Signal regulatory protein α (SIRPα) ( known as SHPS1, CD172A, or P84) is a cell surface receptor-like signaling protein predominantly expressed in myeloid leukocytes including granulocytes, monocytes, macrophages and dendritic cells [1,2]. SIRPα has been reported to be crucial for leukocyte functions, including monocyte and neutrophil (PMN) chemotactic transmigration [6,7], macrophage phagocytosis [8], macrophage fusion [9], dendritic cell antigen presentation [10], and other possible cellular functions such as neuronal axonal growth and synapse formation [3] and kidney filtration [4]. The regulation of all these cellular functions by SIRPα has been attributed to its extracellular binding interactions with the ligand CD47, another important cell surface protein broadly expressed on most cells, and to the SIRPα ITIMs-mediated intracellular signaling. It has been suggested that increased CD47 expression on cancer cells serves as a crucial mechanism for the evasion of immune clearance [23,24]
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