title: SIRPbeta1 is expressed as a disulfide-linked homodimer in leukocytes and positively regulates neutrophil transepithelial migration.
abstract: Signal regulatory proteins (SIRPs) comprise a family of cell surface signaling receptors differentially expressed in leukocytes and the central nervous system. Although the extracellular domains of SIRPs are highly similar, classical motifs in the cytoplasmic or transmembrane domains distinguish them as either activating (beta) or inhibitory (alpha) isoforms. We reported previously that human neutrophils (polymorphonuclear leukocytes (PMN)) express multiple SIRP isoforms and that SIRPalpha binding to its ligand CD47 regulates PMN transmigration. Here we further characterized the expression of PMN SIRPs, and we reported that the major SIRPalpha and SIRPbeta isoforms expressed in PMN include Bit/PTPNS-1 and SIRPbeta1, respectively. Furthermore, although SIRPalpha (Bit/PTPNS-1) is expressed as a monomer, we showed that SIRPbeta1 is expressed on the cell surface as a disulfide-linked homodimer with bond formation mediated by Cys-320 in the membrane-proximal Ig loop. Subcellular fractionation studies revealed a major pool of SIRPbeta1 within the plasma membrane fractions of PMN. In contrast, the majority of SIRPalpha (Bit/PTPNS-1) is present in fractions enriched in secondary granules and is translocated to the cell surface after chemoattractant (formylmethionylleucylphenylalanine) stimulation. Functional studies revealed that antibody-mediated ligation of SIRPbeta1 enhanced formylmethionylleucylphenylalanine-driven PMN transepithelial migration. Co-immunoprecipitation experiments to identify associated adaptor proteins revealed a 10-12-kDa protein associated with SIRPbeta1 that was tyrosine-phosphorylated after PMN stimulation and is not DAP10/12 or Fc receptor gamma chain. These results provide new insights into the structure and function of SIRPs in leukocytes and their potential role(s) in fine-tuning responses to inflammatory stimuli.
authors.affiliation: Department of Biology, Georgia State University, Atlanta, Georgia 30302, USA. yliu@gsu.edu
authors.forename: Yuan
authors.initials: Y
authors.lastname: Liu
authors.forename: Ileana
authors.initials: I
authors.lastname: Soto
authors.forename: Qiao
authors.initials: Q
authors.lastname: Tong
authors.forename: Alex
authors.initials: A
authors.lastname: Chin
authors.forename: Hans-Jörg
authors.initials: HJ
authors.lastname: Bühring
authors.forename: Tao
authors.initials: T
authors.lastname: Wu
authors.forename: Ke
authors.initials: K
authors.lastname: Zen
authors.forename: Charles A
authors.initials: CA
authors.lastname: Parkos
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D015153:Blotting, Western; D051928:CD47 Antigen / Q000096:biosynthesis; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D002465:Cell Movement; D002522:Chlorocebus aethiops; D003545:Cysteine / Q000737:chemistry; D003593:Cytoplasm / Q000378:metabolism; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D004847:Epithelial Cells / Q000166:cytology; D018922:HL-60 Cells; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D007249:Inflammation; D007962:Leukocytes / Q000166:cytology* / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009154:Mutation; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000502:physiology*; D011961:Receptors, Fc / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions / Q000378:metabolism; D013997:Time Factors; D014443:Tyrosine / Q000737:chemistry
chemical_list: D000911:Antibodies, Monoclonal; D051928:CD47 Antigen; D004220:Disulfides; D020033:Protein Isoforms; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D011961:Receptors, Fc; D011993:Recombinant Fusion Proteins; C501921:SIRPB1 protein, human; D014443:Tyrosine; D009240:N-Formylmethionine Leucyl-Phenylalanine; D003545:Cysteine