title: Thrombospondin mediates focal adhesion disassembly through interactions with cell surface calreticulin.
abstract: Thrombospondin induces reorganization of the actin cytoskeleton and restructuring of focal adhesions. This activity is localized to amino acids 17-35 in the N-terminal heparin-binding domain of thrombospondin and can be replicated by a peptide (hep I) with this sequence. Thrombospondin/hep I stimulate focal adhesion disassembly through a mechanism involving phosphoinositide 3-kinase activation. However, the receptor for this thrombospondin sequence is unknown. We now report that calreticulin on the cell surface mediates focal adhesion disassembly by thrombospondin/hep I. A 60-kDa protein from endothelial cell detergent extracts has homology and immunoreactivity to calreticulin, 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-activated 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 calreticulin and preincubation of thrombospondin/hep I with glutathione S-transferase-calreticulin block thrombospondin/hep I-mediated focal adhesion disassembly and phosphoinositide 3-kinase activation, suggesting that calreticulin is a component of the thrombospondin-induced signaling cascade that regulates cytoskeletal organization. These data identify both a novel receptor for the N terminus of thrombospondin and a distinct role for cell surface calreticulin in cell adhesion.
authors.affiliation: Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.
authors.forename: S
authors.initials: S
authors.lastname: Goicoechea
authors.forename: A W
authors.initials: AW
authors.lastname: Orr
authors.forename: M A
authors.initials: MA
authors.lastname: Pallero
authors.forename: P
authors.initials: P
authors.lastname: Eggleton
authors.forename: J E
authors.initials: JE
authors.lastname: Murphy-Ullrich
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000494:pharmacology; D019921:Biotinylation; D018955:CD36 Antigens / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D037282:Calreticulin; D002417:Cattle; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism; D004730:Endothelium, Vascular / Q000737:chemistry / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D022001:Focal Adhesions / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology / Q000378:metabolism / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D012261:Ribonucleoproteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D019699:Thrombospondins / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D000906:Antibodies; D018955:CD36 Antigens; D002135:Calcium-Binding Proteins; D037282:Calreticulin; D046911:Macromolecular Substances; D008565:Membrane Proteins; D010446:Peptide Fragments; D012261:Ribonucleoproteins; D019699:Thrombospondins; D019869:Phosphatidylinositol 3-Kinases