The Role of cis Dimerization of Signal Regulatory Protein (SIRP ) in Binding to CD47*

Interaction of SIRP 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 . 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 cishas ledothers tohypothesize that SIRP forms higher order structures on the cell surface. Here we report that SIRP forms noncovalently linked cis homodimers. Treatment of SIRP -expressing cells with amembrane-impermeable cross-linker resulted in the formation of SDS-stable SIRP dimers and oligomers. Biochemical analyses of soluble recombinant extracellular regions of SIRP , including domain truncation mutants, revealed that each of the three extracellular immunoglobulin loops of SIRP formed dimers in solution. Coimmunoprecipitation experiments using cells transfected with different affinity-tagged SIRP molecules revealed that SIRP forms cis dimers. Interestingly, in cells treated with tunicamycin, SIRP dimerization but not CD47 binding was inhibited, suggesting that a SIRP dimer is probably bivalent. Last, we demonstrate robust dimerization of SIRPa in adherent, stimulated human neutrophils. Collectively, these data are consistent with SIRP being expressed on the cell surface as a functional cis-linked dimer.