Prominent 85‐kDa Oligomannosidic Glycoproteins of Rat Brain Are Signal Regulatory Proteins and Include the SHP Substrate‐1 for Tyrosine Phosphatases

Bartoszewicz, Zbigniew; Jaffe, Howard; Sasaki, Mika; Möller, Johanna R.; Stebbins, J. W.; Gebrekristos, H.; Quarles, Richard H.

doi:10.1046/j.1471-4159.1999.721688.x

Cited by 11 publications

(7 citation statements)

References 28 publications

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“…Similarly, KARAP/DAP-12 is also expressed in non-hematopoietic cells [8]. In particular, SIRP molecules are present in brain and neurons [28,37], and KARAP/DAP-12 transcripts have been detected by RT-PCR in cells of neural origin such as in the N2A cell line [8], as well as in vitro primary neural cell cultures (data not shown). The developmentally regulated expression of ITAM-bearing polypeptides in neural cells has also been reported for CD3´ [38].…”

Section: Discussionmentioning

confidence: 96%

Association of signal-regulatory proteins β with KARAP/DAP-12

et al. 2000

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The signal‐regulatory proteins (SIRP) are Ig‐like cell surface receptors detected in hematopoietic and non‐hematopoietic cells. SIRP are classified as SIRPα molecules, containing a 110‐ to 113‐amino acid long, or SIRPβ molecules, with a 5‐amino acid long intracytoplasmic domain. SIRPα molecules belong to inhibitory immunoreceptor tyrosine‐based inhibition motif (ITIM)‐bearing molecules. The majority of ITIM‐bearing receptors are paired with activating isoforms, which share highly related extracytoplasmic domains but harbor a shorter cytoplasmic domain devoid of ITIM and contain a charged amino acid residue in their transmembrane domain. Activating receptors are associated with immunoreceptor tyrosine‐based activation motif (ITAM)‐bearing proteins, such as KARAP/DAP‐12 and FcRγ. In this report, we show that human SIRPβ1 is included in an oligomeric complex with KARAP/DAP‐12 in hematopoietic and non‐hematopoietic transfectant cells as well as in human monocytes. The physical association between SIRPβ1 and KARAP/DAP‐12 results in the functional coupling of SIRPβ1 engagement to the recruitment of the protein tyrosine kinase Syk and to serotonin release in RBL cell transfectants. Therefore our results show that SIRPβ1 acts as an activating isoform of SIRPα molecules, confirming the co‐existence of inhibitory ITIM‐bearing molecules, recruiting SHP‐1 and SHP‐2 protein tyrosine phosphatases, and activating counterparts, whose engagement couples to protein tyrosine kinases via ITAM‐bearing molecules.

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Section: Discussionmentioning

confidence: 96%

Association of signal-regulatory proteins β with KARAP/DAP-12

et al. 2000

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“…As the recombinant forms of SIRP and CD47 produced in heterologous cells bind each other it is likely that it is a protein-protein interaction in spite of the demonstration of high carbohydrate content of SIRP in rat brain [22]. Furthermore, there is a remarkable difference between the number of potential N-linked glycosylation sites with 15 in the rodent and bovine SIRP and only 4 in human SIRP (Fig.…”

Section: Discussionmentioning

confidence: 99%

CD47 is a ligand for rat macrophage membrane signal regulatory protein SIRP (OX41) and human SIRPα 1

et al. 2000

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The rat OX41 antigen is a cell surface protein containing three immunoglobulin superfamily domains and intracellular immunoreceptor tyrosine‐based inhibitory motifs (ITIM). It is a homologue of the human signal‐regulatory protein (SIRP) also known as SHPS‐1, BIT or MFR. Cell activation‐induced phosphorylation of the intracellular ITIM motifs induces association with the tyrosine phosphatases SHP‐1 and SHP‐2. To identify the physiological OX41 ligand, recombinant OX41‐CD4d3+4 fusion protein was coupled to fluorescent beads to produce a multivalent cell binding reagent. The OX41‐CD4d3+4 beads bound to thymocytes and concanavalin A‐stimulated splenocytes. This interaction was blocked by the monoclonal antibody (mAb) OX101. Affinity chromatography with OX101 mAb and peptide sequencing revealed the rat SIRP ligand to be CD47 (integrin‐associated protein). A direct interaction between human SIRP and human CD47 was demonstrated using purified recombinant proteins and surface plasmon resonance ruling out the involvement of other proteins known to be associated with CD47. The affinity of the SIRP/CD47 interaction was Kd ≈ 8 μM at 37°C with a koff ≥2.1 s–1. The membrane‐distal SIRP V‐like domain was sufficient for binding to CD47.

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“…In the mouse retina, the expression of CD47 and SIRP␣ is colocalized, and CD47-deficient mice show an altered pattern of SIRP␣ expression, especially in the cellular and plexiform layers of the retina, suggesting a functional association between the two molecules (12,16). In rats, SIRP␣ expressed in neuronal cells demonstrates reduced glycosylation compared with SIRP␣ from myeloid cells, resulting in altered binding affinity to tissue sections or plant lectins (13,22,23), and SIRP␣ on neuronal cells exhibits differential glycosylation during embryonic development (15). Evidence of differential glycosylation is also seen in the MM5/C1 mouse mammary carcinoma and the A431 human epidermal carcinoma cell lines (4), and rat SIRP␣ expressed on peritoneal macrophages exhibits reduced glycosylation compared with SIRP␣ on alveolar macrophages (24).…”

Section: Introductionmentioning

confidence: 99%

Expression and Activation of Signal Regulatory Protein α on Astrocytomas

Chen

Brown²,

Huang³

et al. 2004

Cancer Research

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High-grade astrocytomas and glioblastomas are usually unresectable because they extensively invade surrounding brain tissue. Here, we report the expression and function of a receptor on many astrocytomas that may alter both the proliferative and invasive potential of these tumors. Signal regulatory protein (SIRP) ␣1 is an immunoglobulin superfamily transmembrane glycoprotein that is normally expressed in subsets of myeloid and neuronal cells. Transfection of many cell types with SIRP␣1, including glioblastomas, has been shown to inhibit their proliferation in response to a range of growth factors. Furthermore, the expression of a murine SIRP␣1 mutant has been shown to enhance cell adhesion and initial cell spreading but to inhibit cell extension and movement. The extracellular portion of SIRP␣1 binds CD47 (integrin-associated protein), although this interaction is not required for integrin-mediated activation of SIRP␣1. On phosphorylation, SIRP␣1 recruits the tyrosine phosphatases SHP-1 and SHP-2, which are important in its functions. Although SHP-1 is uniquely expressed on hematopoietic cells, SHP-2 is ubiquitously expressed, so that SIRP␣1 has the potential to function in many cell types, including astrocytomas. Because SIRP␣1 regulates cell functions that may contribute to the malignancy of these tumors, we examined the expression of SIRPs in astrocytoma cell lines by flow cytometry using a monoclonal antibody against all SIRPs. Screening of nine cell lines revealed clear cell surface expression of SIRPs on five cell lines, whereas Northern blotting for SIRP␣ transcripts showed mRNA present in eight of nine cell lines. All nine cell lines expressed the ligand for SIRP␣1, CD47. To further examine the expression and function of SIRPs, we studied the SF126 and U373MG astrocytoma cell lines, both of which express SIRPs, in greater detail. SIRP transcripts in these cells are identical in sequence to SIRP␣1. The expressed deglycosylated protein is the same size as SIRP␣1, but in the astrocytoma cells, it is underglycosylated compared with SIRP␣1 produced in transfected Chinese hamster ovary cells. It is nonetheless still capable of binding soluble CD47. Moreover, SIRP␣1 in each of the two cell lines recruited SHP-2 on phosphorylation, and SIRP␣1 phosphorylation in cultured cells is CD47 dependent. Finally, examination of frozen sections from 10 primary brain tumor biopsies by immunohistochemistry revealed expression of SIRPs on seven of the specimens, some of which expressed high levels of SIRPs. Most of the tumors also expressed CD47. This is the first demonstration that astrocytomas can express SIRP␣. Given the known role of SIRP␣ in regulating cell adhesion and responses to mitogenic growth factors, the expression of SIRP␣1 on astrocytomas may be of considerable importance in brain tumor biology, and it offers the potential of a new avenue for therapeutic intervention.

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Prominent 85‐kDa Oligomannosidic Glycoproteins of Rat Brain Are Signal Regulatory Proteins and Include the SHP Substrate‐1 for Tyrosine Phosphatases

Cited by 11 publications

References 28 publications

Association of signal-regulatory proteins β with KARAP/DAP-12

Association of signal-regulatory proteins β with KARAP/DAP-12

CD47 is a ligand for rat macrophage membrane signal regulatory protein SIRP (OX41) and human SIRPα 1

Expression and Activation of Signal Regulatory Protein α on Astrocytomas

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