Functional Elements on SIRPα IgV Domain Mediate Cell Surface Binding to CD47

Abstract

SIRPalpha and SIRPbeta1, the two major isoforms of the signal regulatory protein (SIRP) family, are co-expressed in human leukocytes but mediate distinct extracellular binding interactions and divergent cell signaling responses. Previous studies have demonstrated that binding of SIRPalpha with CD47, another important cell surface molecule, through the extracellular IgV domain regulates important leukocyte functions including macrophage recognition, leukocyte adhesion and transmigration. Although SIRPbeta1 shares highly homologous extracellular IgV structure with SIRPalpha, it does not bind to CD47. Here, we defined key amino acid residues exclusively expressing in the IgV domain of SIRPalpha, but not SIRPbeta1, which determine the extracellular binding interaction of SIRPalpha to CD47. These key residues include Gln67, a small hydrophobic amino acid (Ala or Val) at the 57th position and Met102. We found that Gln67 and Ala/Val57 are critical. Mutation of either of these residues abates SIRPalpha directly binding to CD47. Functional cell adhesion and leukocyte transmigration assays further demonstrated central roles of Gln67 and Ala/Val57 in SIRPalpha extracellular binding mediated cell interactions and cell migration. Another SIRPalpha-specific residue, Met102, appears to assist SIRPalpha IgV binding through Gln67 and Ala/Val57. An essential role of these amino acid residues in SIRPalpha binding to CD47 was further confirmed by introducing these residues into the SIRPbeta1 IgV domain, which dramatically converts SIRPbeta1 into a CD47-binding molecule. Our results thus revealed the molecular basis by which SIRPalpha binds to CD47 and shed new light into the structural mechanisms of SIRP isoform mediated distinctive extracellular interactions and cellular responses.

The Structure of the Macrophage Signal Regulatory Protein α (SIRPα) Inhibitory Receptor Reveals a Binding Face Reminiscent of That Used by T Cell Receptors

“…In this respect SIRP␣ d1 is fairly typical; however, the structure shows a number of distinctive features, and the mutagenesis analysis shows that the binding face is unusual in that it involves the loops that are the equivalent of the hypervariable loops of antibodies and TCRs. This is in line with a very recent study (45) using mutational analysis of SIRP␤ and SIRP␣ to find residues crucial in binding CD47. Residues Val-27 and Gln-37 had effects on binding in agreement with the involvement of the BC loop.…”

Signal regulatory protein alpha (SIRPα)/CD47 interaction and function

“…As expected from the sequence similarity of the SIRPα and SIRPβ there is little difference in the overall structures of the domains but subtle differences in the loops were found with evidence for considerable mobility in these [17]. Sequence analysis and mutagenesis have failed to provide a simple explanation for the failure of SIRPβ to bind to CD47 [14,15,[17][18][19] but the structures suggest that this failure is due to subtle differences in the loops and involving indirect changes and not solely contact residues [17].…”

CD47 functions as a molecular switch for erythrocyte phagocytosis

“…In addition, the V56M point mutation within the N-terminal V-set Ig domain that abolishes CD47 binding, as shown by us and others, 33,34 was tested for its ability to bind old erythrocytes through CD47 recognition. These mutants were expressed at a similar level as the SIRP␣ construct from which they were derived ( Figure 2B).…”