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 I g domain followed by a multiply membrane-spanning (AIMS) domain with five t ransmembrane segments, implicated in both integrin-dependent and -independe nt signaling cascades. Essentially all functions of CD47 require both the I g and AMS domains, raising the possibility that interaction between the two domains is required for normal function. Conservation of Cys residues amon g CD47 homologues suggested the existence of a disulfide bond between the I g and MMS domains that was confirmed by chemical digestion and mapped to Cy s(33) and Cys(263). Subtle changes in CD47 conformation in the absence of t he disulfide were suggested by decreased binding of two anti-Ig domain mono clonal antibodies, decreased SIRP alpha1 binding, and reduced CD47/SIRP alp ha1-mediated cell adhesion. Mutagenesis to prevent formation of this disulf ide completely disrupted CD47 signaling independent of effects on ligand bi nding, as assessed by T cell interleukin-2 secretion and Ca2+ responses. Lo ss of the disulfide did not affect membrane raft localization of CD47 or it s association with alpha (v)beta (3) integrin. Thus, a disulfide bond betwe en the Ig and MMS domains of CD47 is required for normal ligand binding and signal transduction.