Abstract
Interaction of SIRPα 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 SIRPα. 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 SIRPα forms higher order structures on the cell surface. Here we report that SIRPα forms noncovalently linked cis homodimers. Treatment of SIRPα-expressing cells with a membrane-impermeable cross-linker resulted in the formation of SDS-stable SIRPα dimers and oligomers. Biochemical analyses of soluble recombinant extracellular regions of SIRPα, including domain truncation mutants, revealed that each of the three extracellular immunoglobulin loops of SIRPα formed dimers in solution. Co-immunoprecipitation experiments using cells transfected with different affinity-tagged SIRPα molecules revealed that SIRPα forms cis dimers. Interestingly, in cells treated with tunicamycin, SIRPα dimerization but not CD47 binding was inhibited, suggesting that a SIRPα dimer is probably bivalent. Last, we demonstrate robust dimerization of SIRPa in adherent, stimulated human neutrophils. Collectively, these data are consistent with SIRPα being expressed on the cell surface as a functional cis-linked dimer.
Highlights
It was reported that the inhibition of phagocytosis by tissue-resident macrophages through SIRP␣-CD47 interactions is essential for successful engraftment of hematopoietic stem cells [16, 25]
The general belief that monomeric partners of a dimer must pair noncovalently in a thermodynamically favorable fashion in order to facilitate the formation of an intermolecular disulfide bond [35, 36], we hypothesized that SIRP␣ may form a noncovalently linked dimer on the cell surface
Despite these advances, little is known about the structural role of the proximal Ig constant-like (IgC) domains
Summary
Antibodies—Rabbit polyclonal antibodies against the cytoplasmic tail of SIRP␣ (Prosci) and against green fluorescent protein (GFP) (Abcam) are commercially available. Cell culture and transfections of CHO and HEK293T cells using Lipofectamine (Invitrogen) were performed as described previously [28]. The following wild type (WT) or truncated SIRP␣ constructs containing an intact ctyoplasmic tail and transmembrane domain were produced: SIRP␣D1-Myc, SIRP␣D1D2, and SIRP␣D2D3. MALDI-TOF analyses of purified proteins were performed as described previously [32]. Binding Analysis of Soluble CD47 to SIRP␣ on the Cell Surface—Production and purification of soluble CD47 ectodomain fused with alkaline phosphatase (CD47AP) was as described previously [28, 33]. To stimulate PMNs in suspension, 4 ϫ 106 PMNs in 1 ml of HBSSϩ were activated with 1 g/ml LPS or 10Ϫ6 M fMLP for 20 min at 37 °C, followed by cross-linking and analysis as described above
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