SIRPβ1 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 (␤) or inhibitory (␣) isoforms. We reported previously that human neutrophils (polymorphonuclear leukocytes (PMN)) express multiple SIRP isoforms and that SIRP␣ binding to its ligand CD47 regulates PMN transmigration. Here we further characterized the expression of PMN SIRPs, and we reported that the major SIRP␣ and SIRP␤ isoforms expressed in PMN include Bit/PTPNS-1 and SIRP␤1, respectively. Furthermore, although SIRP␣ (Bit/PTPNS-1) is expressed as a monomer, we showed that SIRP␤1 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 SIRP␤1 within the plasma membrane fractions of PMN. In contrast, the majority of SIRP␣ (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 SIRP␤1 enhanced formylmethionylleucylphenylalanine-driven PMN transepithelial migration. Co-immunoprecipitation experiments to identify associated adaptor proteins revealed a 10 -12-kDa protein associated with SIRP␤1 that was tyrosine-phosphorylated after PMN stimulation and is not DAP10/12 or Fc receptor ␥ 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. Signal regulatory proteins (SIRPs)3 are a family of transmembrane receptor-like signaling proteins that are abundantly expressed in hematopoietic cells, including granulocytes, monocytes, dendritic cells, and lymphocytes (1-3). In addition, SIRPs are expressed in neuronal cells (4 -6) and certain types of cancer cells (7-10). SIRPs can be divided into two subfamilies, SIRP␣ and SIRP␤, based on the putative structures of their C-terminal intracellular domains (11). SIRPs share typical immunoglobulin superfamily structures with an N-terminal extracellular domain containing three cysteine-bound Ig-like loops, a single membrane-spanning transmembrane domain, and a C-terminal intracellular domain (11). The C-terminal intracellular domains of the SIRP␣ subfamily contain a relatively long amino acid sequence (110 amino acids for SIRP␣1) that includes four tyrosine residues to form two immunoreceptor tyrosine-based inhibition motifs (ITIM). Conversely, SIRP␤ subfamily members have a short intracellular domain containing only a few amino acids (4 amino acids for SIRP␤1). Despite a short cytoplasmic tail, SIRP␤1 contains a positively charged lysine in the transmembrane domain that can mediate interactions with an immunoreceptor tyrosi...

Signal Regulatory Proteins in the Immune System

“…The positively charged lysine residues of the transmembrane regions of the SIRP␤ members, which mediate or are proposed to mediate, an association with DAP12 are indicated with a "ϩ". In case of SIRP␤1, mSIRP␤1, and mSIRP␤2, an interaction with DAP12 is supported by direct evidence (16,17,38,40) protein DAP12. Consistent with this, SIRP␤2 is expressed by cells of the monocyte-macrophage lineage (as determined by PCR and analysis of EST data), and there is alternative splicing of the extracellular IgSF domains (E. M. Van Beek et al, unpublished observations).…”

Transepithelial Migration of Neutrophils

“…Furthermore, antagonism of SIRPa results in inhibition of neutrophil transmigration rather than the delay in transmigration attributable to CD47 (105). In addition, the related protein SIRPb1, which is not a CD47 ligand, also regulates neutrophil transmigration, probably in an inhibitory fashion via signaling through an unidentified adaptor protein (107). This evidence further suggests that SIRP family members control neutrophil transmigration via CD47-independent mechanisms.…”

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

“…Dimers on the Cell Surface-We have previously reported that SIRP␤ but not SIRP␣ forms a covalent homodimer in cells through a disulfide bond at the membrane-proximal Ig domain (D3) (31). Because the ectodomains of SIRP␣ and SIRP␤ exhibit a very high degree of homology in protein sequences (Fig.…”

“…1A) and the general belief that monomeric partners of a dimer must pair noncovalently in a thermodynamically favorable fashion in order to facilitate the formation of an intermolecular disulfide bond (35,36), we hypothesized that SIRP␣ may form a noncovalently linked dimer on the cell surface. To address this possibility, we analyzed cell lysates of HL-60, a human promyelocytic leukemia cell line that is known to express SIRP␣ and SIRP␤ (31,37), by immunoblot for the presence of SIRP␣ dimers by SDS-PAGE. Under non-reducing conditions, covalent SIRP␤ dimers but not SIRP␣ dimers were detected.…”

“…Given that many known IgC domains associate with other IgC domains, others have speculated that SIRP␣ may form dimers and higher order structures. Interestingly, we have previously shown that SIRP␤ is present on the cell surface as a cis homodimer that is covalently linked through a disulfide bond on the most membrane-proximal domain (D3) (31). Although SIRP␣ lacks this particular cysteine, the marked similarities in protein sequences as well as the propensity for IgC domains to dimerize led us to hypothesize that SIPR␣ may form noncovalently linked cis homodimers on the cell surface.…”