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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 K(d) approximately 8 microM at 37 degrees C with a k(off )>/=2.1 s(-1). The membrane-distal SIRP V-like domain was sufficient for binding to CD47.

Original publication

DOI

10.1002/1521-4141(2000)30:8<2130::AID-IMMU2130>3.0.CO;2-8

Type

Journal article

Journal

Eur J Immunol

Publication Date

08/2000

Volume

30

Pages

2130 - 2137

Keywords

Animals, Antibodies, Monoclonal, Antigens, CD, Antigens, CD47, Antigens, Differentiation, Binding Sites, COS Cells, Carrier Proteins, Humans, Membrane Glycoproteins, Mice, Neural Cell Adhesion Molecule L1, Neural Cell Adhesion Molecules, Rats, Receptors, Immunologic, Signal Transduction