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Novel Structural Determinants on SIRP that Mediate Binding to CD47¹

Winston Y. Lee^*, Dominique A. Weber^*, Oskar Laur, Eric A. Severson^*, Ingrid McCall^*, Rita P. Jen^*, Alex C. Chin^*, Tao Wu^*, Kim M. Gernet and Charles A. Parkos^2,*

^* Department of Pathology and Laboratory Medicine, Emory University, Atlanta, GA 30322; Emory Vaccine Center, Department of Microbiology and Immunology, Emory University, Atlanta, GA 30329; and BioMolecular Computing Resource, Emory University, Atlanta, GA 30322

Signal regulatory proteins (SIRP-, -β, and -) are important regulators of several innate immune functions that include leukocyte migration. Membrane distal (D1) domains of SIRP and SIRP, 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 SIRP.D1 mediate binding to CD47. By site-directed mutagenesis, we determined that SIRP binding to CD47 is independent of N-glycosylation. We also identified three residues critical for CD47 binding by exchanging residues on SIRP 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 SIRP.D1 revealed topological relationships among critical residues and allowed the identification of critical residues common to SIRP and SIRPβ. Mapping these critical residues onto the recently reported crystal structure of SIRP.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 SIRP.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 SIRP 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 SIRP/CD47 interactions may provide insights into therapeutics targeting pathologic inflammation.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by National Institutes of Health Grant R01 DK79392. The work at BimCore was supported by Emory School of Medicine, Graduate Division of Biological and Biomedical Sciences and Emory Woodruff Fund.

² Address correspondence and reprint requests to Dr. Charles A. Parkos, Division of Gastrointestinal Pathology, Emory University, Whitehead Biomedical Building, Room 105B, 615 Michael Street, Atlanta, GA 30322. E-mail address: cparkos{at}emory.edu

³ Abbreviations used in this paper: PMN, polymorphonuclear neutrophil; SIRP, signal regulatory proteins; RT, room temperature; RPS-BLAST, Reverse Position Specific Basic Local Alignment Search Tool.