The Role of cis Dimerization of Signal Regulatory Protein α (SIRPα) in Binding to CD47

Interaction of SIRP alpha with its ligand, CD47, regulates leukocyte functions, including transmigration, phagocytosis, oxidative burst, and cytokine secretion. Recent progress has provided significant insights into the structural details of the distal IgV domain (D1) of SIRP alpha. However, the structural roles of proximal IgC domains (D2 and D3) have been largely unstudied. The high degree of conservation of D2 and D3 among members of the SIRP family as well as the propensity of known IgC domains to assemble in cis has led others to hypothesize that SIRP alpha forms higher order structures on the cell surface. Here we report that SIRP alpha forms noncovalently linked cis homodimers. Treatment of SIRP alpha-expressing cells with a membrane-impermeable cross-linker resulted in the formation of SDS-stable SIRP alpha dimers and oligomers. Biochemical analyses of soluble recombinant extracellular regions of SIRP alpha, including domain truncation mutants, revealed that each of the three extracellular immunoglobulin loops of SIRP alpha formed dimers in solution. Co-immunoprecipitation experiments using cells transfected with different affinity-tagged SIRP alpha molecules revealed that SIRP alpha forms cis dimers. Interestingly, in cells treated with tunicamycin, SIRP alpha dimerization but not CD47 binding was inhibited, suggesting that a SIRP alpha dimer is probably bivalent. Last, we demonstrate robust dimerization of SIRPa in adherent, stimulated human neutrophils. Collectively, these data are consistent with SIRP alpha being expressed on the cell surface as a functional cis-linked dimer.