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.

Thrombospondin Induces RhoA Inactivation through FAK-dependent Signaling to Stimulate Focal Adhesion Disassembly

“…10). The hep I sequence in TSP1 binds to a receptor co-complex consisting of cell surface CRT and LRP, stimulating the activation of a PTX-sensitive G␣ i signal (39,40,42). This G protein signal induces activation of PI 3-kinase and ERK, both of which are required for TSP1/hep I-induced focal adhesion turnover (41,42).…”

“…The hep I sequence of TSP1 signals through a CRT/LRP receptor co-complex to stimulate focal adhesion disassembly (39,40). Signaling through the hep I sequence of TSP1 is…”

The Calreticulin-Binding Sequence of Thrombospondin 1 Regulates Collagen Expression and Organization During Tissue Remodeling

“…Invading macrophages and fibroblasts perceive the exogenous collagen I as a provisional matrix, phagocytose the GAM, and thus become locally transfected. 33 To express the secreted CRT-binding sequence of TSP1 (aa [17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35] in the GAM, a plasmid encoding the TSP1 signal peptide and the first 35 amino acids of TSP1 with downstream EGFP was constructed [NTD (1-35)-EGFP]. In prior studies, we established that substitution of lysines at positions 24 and 32 renders the sequence inactive.…”

“…66 However, work from our lab and others have demonstrated stimulation of intracellular signaling via TSP1 binding to cell surface CRT, which stimulates engagement of LRP1-mediated signaling. 19,20,63 LRP1 similarly has dual functions in mediating endocytic clearance of molecules, including TSP1 and matrix metalloproteinases, and it also acts as a signaling receptor. 67,68 It is therefore possible that the presence of cell surface CRT dictates whether LRP1 will respond to TSP1 by activating signaling pathways or by internalizing TSP1.…”

“…15 These diverse and sometimes paradoxical activities can be ascribed to its interactions with multiple receptors, including integrins, syndecans, 13,16 CD47, 17 CD36, 18 low-density lipoprotein receptor-related protein 1 (LRP1), 19 and calreticulin (CRT). 20 To date, the role of TSP1 in tissue remodeling has largely been studied through injury models in TSP1-null mice. 9,21,22 These models have been useful for identifying many functions of TSP1 but are limited in their ability to mimic tissue remodeling in a normal organism, in which TSP1 expression, proteolysis, and interactions with multiple receptors will be modulated both temporally and spatially.…”