Thrombospondin mediates focal adhesion disassembly through interactions with cell surface calreticulin

Thrombospondin induces reorganization of the actin cytoskeleton and restruc turing of focal adhesions. This activity is localized to amino acids 17-35 in the N-terminal heparin-binding domain of thrombospondin and can be repli cated by a peptide (hep I) with this sequence. Thrombospondin/hep I stimula te focal adhesion disassembly through a mechanism involving phosphoinositid e 3-kinase, activation. However, the receptor for this thrombospondin seque nce is unknown. We now report that calreticulin on the cell surface mediate s focal adhesion disassembly by thrombospondin/hep I. A 60-kDa protein from endothelial cell detergent extracts has homology and-immunoreactivity to c alreticulin, binds a hep I affinity column, and neutralizes thrombospondin/ hep I-mediated focal adhesion disassembly, Calreticulin on the cell Surface was confirmed by biotinylation, confocal microscopy, and by fluorescence-a ctivated cell sorting analyses, Thrombospondin and calreticulin potentially bind through the hep I sequence, since thrombospondin-calreticulin complex formation can be blocked specifically by hep I peptide. Antibodies to calr eticulin and preincubation of thrombospondin/hep I with glutathione S-trans ferase-calreticulin block thrombospondin/hep I-mediated focal adhesion disa ssembly and :phosphoinositide 3-kinase activation, suggesting that calretic ulin is a component of the thrombospondin-induced signaling cascade that re gulates cytoskeletal organization. These data identify both a novel recepto r for the N terminus of thrombospondin and a distinct role for cell surface calreticulin in cell adhesion.