Novel Structural Determinants on SIRPα that Mediate Binding to CD47

Abstract

Signal regulatory proteins (SIRP-α, -β, and -γ) are important regulators of several innate immune functions that include leukocyte migration. Membrane distal (D1) domains of SIRPα and SIRPγ, but not SIRPβ, mediate binding to a cellular ligand termed CD47. Because the extracellular domains of all SIRPs are highly homologous, we hypothesized that some of the 16 residues unique to SIRPα.D1 mediate binding to CD47. By site-directed mutagenesis, we determined that SIRPα binding to CD47 is independent of N-glycosylation. We also identified three residues critical for CD47 binding by exchanging residues on SIRPα with corresponding residues from SIRPβ. Cumulative substitutions of the critical residues into SIRPβ resulted in de novo binding of the mutant protein to CD47. Homology modeling of SIRPα.D1 revealed topological relationships among critical residues and allowed the identification of critical residues common to SIRPα and SIRPβ. Mapping these critical residues onto the recently reported crystal structure of SIRPα.D1 revealed a novel region that is required for CD47 binding and is distinct and lateral to another putative CD47 binding site described on that crystal structure. The importance of this lateral region in mediating SIRPα.D1 binding to CD47 was confirmed by epitope mapping analyses of anti-SIRP Abs. These observations highlight a complex nature of the ligand binding requirements for SIRPα that appear to be dependent on two distinct but adjacent regions on the membrane distal Ig loop. A better understanding of the structural basis of SIRPα/CD47 interactions may provide insights into therapeutics targeting pathologic inflammation.

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].…”

Surfactant Protein D (Sp-D) Binds to Membrane-proximal Domain (D3) of Signal Regulatory Protein α (SIRPα), a Site Distant from Binding Domain of CD47, while Also Binding to Analogous Region on Signal Regulatory Protein β (SIRPβ)

“…Rabbit polyclonal antiserum against recombinant SIRP␣ ectodomain was produced by Covance Research Products. Monoclonal antibodies against SIRP␣ D1 (SAF17.2) and SIRP␣ D3 (SAF4.2) have been described previously (9). LPS from Salmonella minnesota R595 was obtained from List Biological Laboratories.…”

“…Forward and reverse end primers were designed with HindIII and BamHI sites. DNA constructs encoding various ectodomains of SIRP␣ tagged with His 10 (SIRP␣-His) were cloned into pcDNA3 (Invitrogen) as described previously (9). All constructs were verified by DNA sequencing.…”

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

“…Rabbit polyclonal antiserum against recombinant SIRP␣ ectodomain as well as murine monoclonal antibodies against SIRP␣D1 (SAF17.2) or SIRP␣D3 (SAF4.2) were generated as described previously (28). A hybridoma that secretes a murine monoclonal antibody against Myc (9E10) was obtained from ATCC.…”

“…However, little is known about the contributions of the membrane-proximal immunoglobulin domains of SIRP␣. Previously published analyses of protein sequences indicated that proximal domains contained IgC motifs and are highly homologous among members of the SIRP family (6,(27)(28)(29)(30). Given that many known IgC domains associate with other IgC domains, others have speculated that SIRP␣ may form dimers and higher order structures.…”