Extracellular matrix retention of thrombospondin 1 is controlled by its conserved C-terminal region

Abstract

domain or type 1 repeats. Using a novel mRFP-tagged TSP1 C-terminal trimer, we demonstrate that ECM retention involves the RGD site and a novel site in the L-lectin domain with structural similarity to the ligand-binding site of cargo transport proteins. CD47 and ␤1 integrins are dispensable for ECM retention, but ␤1 integrins enhance activity. These novel data advance concepts of the molecular processes that lead to ECM retention of TSP1. Journal of Cell Science Matrix retention of thrombospondinsTSP2 is present in cartilage and various connective tissues (Bornstein et al., 2004). TSP4 is present in the ECM of the nervous system, at neuromuscular junctions, in the developing retina, adult eye and tendon (Arber and Caroni, 1995; Hauser et al., 1995;Stenina et al., 2003; Dunkle et al., 2007). TSP5 or COMP (cartilage oligomeric matrix protein) is a component of ECM in cartilage, synovium and tendon, which associates with collagen fibrils and binds collagens II and IX, matrilin-3 and fibronectin in vitro (Hedbom et al., 1992; DiCesare et al., 1995; DiCesare et al., 1997; DiCesare et al., 2002;Sodersten et al., 2005). In vivo models and human diseases provide evidence that TSPs contribute functionally to ECM organisation and can modulate the adhesive or structural properties of ECM. For example, TSP2-null mice have aberrant assembly of collagen fibrils because of reduced cell surface tissue transglutaminase, resulting in lax tendons and decreased tensile strength of the skin (Kyriakides et al., 1998; Agah et al., 2005). Interaction of Drosophila TSP (D-TSP) with ␣PS2 integrin on muscle cells is needed for stable tendon and muscle attachment during development (Chanana et al., 2007;Subramanian et al., 2007). Polymorphisms in the coding sequences of human THBS1 and THBS4, which predispose to cardiovascular disease, enhance platelet aggregation in the case of TSP1 and affect endothelial cell adhesion in the case of TSP4 (Stenina et al., 2007).In vitro binding assays with purified proteins have demonstrated that TSPs can bind to multiple structural ECM components and to various growth factors that may themselves bind ECM (reviewed by Adams, 2001). These findings indicate targets for binding by extracellular TSPs, but do not address the molecular processes by which TSPs come to be retained within ECM. Knowledge of these processes is important with regard to the clear biological significance of TSPs in connective ECM, tumour biology, immune response, synaptogenesis and vascular function. Prospective therapeutic modulations or construction of synthetic ECM would also benefit from this knowledge. Until recently, the ability to analyse these mechanisms was hampered by a lack of understanding of the complex structure of TSP polypeptides, particularly the large and highly conserved C-terminal region. In recent years, structures for all the major domains of TSP1 and TSP2 have been solved at the atomic level (Tan et al., 2002;Tan et al., 2006;Kvansakul et al., 2004; Carlson et al., 2005). We demonstrate here that ECM retention...

Heparan Sulfate Modification of the Transmembrane Receptor CD47 Is Necessary for Inhibition of T Cell Receptor Signaling by Thrombospondin-1

“…Further studies have confirmed this binding and binding of recombinant C-terminal regions of TSP1 to cell surface CD47 (70). However, a recombinant extracellular domain of CD47 expressed in bacteria failed to bind to TSP1 (71). This may be explained by incorrect folding of the recombinant CD47 domain or involvement of a long range disulfide bond not present in that construct (72,73), but our results suggest that post-translational modification with heparan sulfate may also be required for high affinity binding of TSP1 to CD47.…”

Signal regulatory protein alpha (SIRPα)/CD47 interaction and function

“…One puzzling aspect concerning CD47 is that much of the data on thrombospondin binding is from peptide binding analysis but the structure of the relevant thrombospondin domain shows that the peptides are in the core of the domain and not readily accessible [11]. This domain shows no affinity for the extracellular IgSF domain of CD47 [12] but direct binding of comparable proteins to CD47 at the cell surface has recently been shown [13] which might indicate that other parts of CD47 are involved. CD47 interactions and signalling are complex and beyond detailed analysis in this review.…”

Differential Interactions of Thrombospondin-1, -2, and -4 with CD47 and Effects on cGMP Signaling and Ischemic Injury Responses

“…Of the two CD47 binding VVM motifs identified in this domain of TSP1, the first is conserved among all five TSPs, suggesting that CD47 binding could be a universal attribute of this family (21). Based on structural evidence that the VVM motifs may not be accessible (22,23), however, conservation of VVM motifs may not be sufficient to predict CD47 binding. Uncertainty regarding the location of the CD47 binding site in the G domain of TSP1 therefore limits interpretation of the known sequence homology to predict CD47 binding to other TSP family members.…”