Normal ligand binding and signaling by CD47 (integrin-associated protein) requires a long range disulfide bond between the extracellular and membrane-spanning domains.
CD47 is a unique member of the Ig superfamily with a single extracellular Ig domain followed by a multiply membrane-spanning (MMS) domain with five transmembrane segments, implicated in both integrin-dependent and -independent signaling cascades. Essentially all functions of CD47 require both the Ig and MMS domains, raising the possibility that interaction between the two domains is required for normal function. Conservation of Cys residues among CD47 homologues suggested the existence of a disulfide bond between the Ig and MMS domains that was confirmed by chemical digestion and mapped to Cys(33) and Cys(263). Subtle changes in CD47 conformation in the absence of the disulfide were suggested by decreased binding of two anti-Ig domain monoclonal antibodies, decreased SIRPalpha1 binding, and reduced CD47/SIRPalpha1-mediated cell adhesion. Mutagenesis to prevent formation of this disulfide completely disrupted CD47 signaling independent of effects on ligand binding, as assessed by T cell interleukin-2 secretion and Ca(2+) responses. Loss of the disulfide did not affect membrane raft localization of CD47 or its association with alpha(v)beta(3) integrin. Thus, a disulfide bond between the Ig and MMS domains of CD47 is required for normal ligand binding and signal transduction.
Rebres
R A
RA
Program in Microbial Pathogenesis and Host Defense and Department of Medicine, University of California, San Francisco, California 94143, USA.
Vaz
L E
LE
Green
J M
JM
Brown
E J
EJ
D000595:Amino Acid Sequence; D015703:Antigens, CD / Q000737:chemistry* / Q000502:physiology; D000943:Antigens, Differentiation; D051928:CD47 Antigen; D002118:Calcium / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry* / Q000502:physiology; D002462:Cell Membrane / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry*; D006801:Humans; D019169:Jurkat Cells; D008024:Ligands; D008562:Membrane Glycoproteins / Q000378:metabolism; D021962:Membrane Microdomains / Q000737:chemistry; D008969:Molecular Sequence Data; D039842:Neural Cell Adhesion Molecule L1; D019006:Neural Cell Adhesion Molecules / Q000378:metabolism; D011948:Receptors, Antigen, T-Cell / Q000502:physiology; D011971:Receptors, Immunologic; D014357:Trypsin / Q000494:pharmacology
D015703:Antigens, CD; D000943:Antigens, Differentiation; D051928:CD47 Antigen; C497894:CD47 protein, human; D002352:Carrier Proteins; D004220:Disulfides; D008024:Ligands; D008562:Membrane Glycoproteins; D039842:Neural Cell Adhesion Molecule L1; D019006:Neural Cell Adhesion Molecules; D011948:Receptors, Antigen, T-Cell; D011971:Receptors, Immunologic; D014357:Trypsin; D002118:Calcium