The anti-adhesive activity of thrombospondin is mediated by the N-terminal domain of cell surface calreticulin.
Thrombospondin (TSP) induces reorganization of the actin cytoskeleton and restructuring of focal adhesions through binding of amino acids (aa) 17-35 (hep I peptide) of thrombospondin to a cell surface form of calreticulin (CRT). In this report we provide further evidence for the involvement of calreticulin in thrombospondin signaling and characterize thrombospondin-calreticulin interactions. Wild type but not crt(-/-) cells respond to hep I/TSP. Responsiveness can be restored by incubation of cells with exogenous calreticulin or by transfection with calreticulin. Thrombospondin forms complexes with the CRT-N-domain that are enhanced by physiologic levels of calcium and zinc. Consistent with thrombospondin/CRT-N-domain binding, only the CRT-N-domain blocks hep I- and thrombospondin-stimulated focal adhesion disassembly. A series of glutathione S-transferase-N-domain mutants were used to map the sequence within the N-domain that interacts with TSP/hep I. A construct containing aa 1-43 but not a construct of aa 1-31 supported thrombospondin binding and focal adhesion disassembly. A series of overlapping peptides were used to further map the thrombospondin-binding site. Peptides spanning aa 19-36 (RWIESKHKSDFGKFVLSS) blocked hep I-stimulated focal adhesion disassembly, indicating that the TSP/hep I-binding site is located to this sequence in calreticulin. A mutant fusion protein lacking aa 19-36 (glutathione S-transferase-CRTDeltahep I) failed to restore responsiveness to hep I in crt(-/-) cells, bind thrombospondin, or competitively block focal adhesion disassembly, providing evidence for the role of this calreticulin sequence in mediating thrombospondin signaling.
Goicoechea
Silvia
S
Department of Pathology, Division of Molecular and Cellular Pathology and Cell Adhesion and Matrix Research Center, University of Alabama at Birmingham, Birmingham, Alabama 35294-0019, USA.
Pallero
Manuel Antonio
MA
Eggleton
Paul
P
Michalak
Marek
M
Murphy-Ullrich
Joanne E
JE
D000595:Amino Acid Sequence; D000818:Animals; D001011:Aorta; D001483:Base Sequence; D001792:Blood Platelets / Q000502:physiology; D037282:Calreticulin / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D002448:Cell Adhesion / Q000187:drug effects / Q000502:physiology*; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology; D017931:DNA Primers; D004730:Endothelium, Vascular / Q000187:drug effects / Q000502:physiology; D006801:Humans; D051379:Mice; D018832:Molecular Chaperones / Q000737:chemistry / Q000502:physiology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011817:Rabbits; D019700:Thrombospondin 1 / Q000737:chemistry / Q000494:pharmacology / Q000502:physiology*; D019699:Thrombospondins / Q000502:physiology*
D037282:Calreticulin; D017931:DNA Primers; D018832:Molecular Chaperones; D010446:Peptide Fragments; D019700:Thrombospondin 1; D019699:Thrombospondins