Thrombospondin mediates focal adhesion disassembly through interactions with cell surface calreticulin.
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.
Goicoechea
S
S
Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.
Orr
A W
AW
Pallero
M A
MA
Eggleton
P
P
Murphy-Ullrich
J E
JE
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*
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