title: The role of cis dimerization of signal regulatory protein alpha (SIRPalpha) in binding to CD47.
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.
authors.affiliation: Epithelial Pathobiology Unit, Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
authors.forename: Winston Y
authors.initials: WY
authors.lastname: Lee
authors.forename: Dominique A
authors.initials: DA
authors.lastname: Weber
authors.forename: Oskar
authors.initials: O
authors.lastname: Laur
authors.forename: Sean R
authors.initials: SR
authors.lastname: Stowell
authors.forename: Ingrid
authors.initials: I
authors.lastname: McCall
authors.forename: Rakieb
authors.initials: R
authors.lastname: Andargachew
authors.forename: Richard D
authors.initials: RD
authors.lastname: Cummings
authors.forename: Charles A
authors.initials: CA
authors.lastname: Parkos
mesh_terms: D000818:Animals; D000943:Antigens, Differentiation / Q000235:genetics / Q000378:metabolism*; D051928:CD47 Antigen / Q000235:genetics / Q000378:metabolism*; D016466:CHO Cells; D002448:Cell Adhesion / Q000502:physiology; D006224:Cricetinae; D003412:Cricetulus; D057809:HEK293 Cells; D018922:HL-60 Cells; D006801:Humans; D009154:Mutation; D018375:Neutrophil Activation / Q000502:physiology; D009504:Neutrophils / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D055503:Protein Multimerization / Q000502:physiology*; D017434:Protein Structure, Tertiary; D011971:Receptors, Immunologic / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D000943:Antigens, Differentiation; D051928:CD47 Antigen; C497894:CD47 protein, human; D011971:Receptors, Immunologic; D011994:Recombinant Proteins; C501920:SIRPA protein, human
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