Novel structural determinants on SIRP alpha that mediate binding to CD47.
Signal regulatory proteins (SIRP-alpha, -beta, and -gamma) are important regulators of several innate immune functions that include leukocyte migration. Membrane distal (D1) domains of SIRPalpha and SIRPgamma, but not SIRPbeta, 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 SIRPalpha.D1 mediate binding to CD47. By site-directed mutagenesis, we determined that SIRPalpha binding to CD47 is independent of N-glycosylation. We also identified three residues critical for CD47 binding by exchanging residues on SIRPalpha with corresponding residues from SIRPbeta. Cumulative substitutions of the critical residues into SIRPbeta resulted in de novo binding of the mutant protein to CD47. Homology modeling of SIRPalpha.D1 revealed topological relationships among critical residues and allowed the identification of critical residues common to SIRPalpha and SIRPbeta. Mapping these critical residues onto the recently reported crystal structure of SIRPalpha.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 SIRPalpha.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 SIRPalpha 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 SIRPalpha/CD47 interactions may provide insights into therapeutics targeting pathologic inflammation.
Lee
Winston Y
WY
Department of Pathology and Laboratory Medicine, Emory University, Atlanta, GA 30322, USA.
Weber
Dominique A
DA
Laur
Oskar
O
Severson
Eric A
EA
McCall
Ingrid
I
Jen
Rita P
RP
Chin
Alex C
AC
Wu
Tao
T
Gernert
Kim M
KM
Gernet
Kim M
KM
Parkos
Charles A
CA
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000937:Antigen-Antibody Reactions; D000943:Antigens, Differentiation / Q000737:chemistry / Q000276:immunology*; D051928:CD47 Antigen / Q000276:immunology*; D019556:COS Cells; D002462:Cell Membrane / Q000276:immunology; D002522:Chlorocebus aethiops; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000276:immunology; D011971:Receptors, Immunologic / Q000737:chemistry / Q000276:immunology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology
D000911:Antibodies, Monoclonal; D000943:Antigens, Differentiation; D051928:CD47 Antigen; D007074:Immunoglobulin G; D011956:Receptors, Cell Surface; D011971:Receptors, Immunologic; D011994:Recombinant Proteins; C501920:SIRPA protein, human; C501921:SIRPB1 protein, human