Current understanding of the thrombospondin-1 interactome.

MATBIO-01037; No of Pages 9 Matrix Biology xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Matrix Biology journal homepage: www.elsevier.com/locate/matbio

Mini review

Current understanding of the thrombospondin-1 interactome Andrea Resovi a, Denise Pinessi a, Giovanna Chiorino b, Giulia Taraboletti a,⁎ a b

Tumor Angiogenesis Unit, Department of Oncology, IRCCS-Istituto di Ricerche Farmacologiche Mario Negri, 24126 Bergamo, Italy Fondo Edo ed Elvo Tempia Valenta, Laboratory of Cancer Genomics, 13900 Biella, Italy

a r t i c l e

i n f o

Available online xxxx Keywords: Thrombospondin-1 Protein–protein interaction Growth factors Angiogenesis Matricellular proteins Domains

a b s t r a c t The multifaceted action of thrombospondin-1 (TSP-1) depends on its ability to physically interact with different ligands, including structural components of the extracellular matrix, other matricellular proteins, cell receptors, growth factors, cytokines and proteases. Through this network, TSP-1 regulates the ligand activity, availability and structure, ultimately tuning the cell response to environmental stimuli in a context-dependent manner, contributing to physiological and pathological processes. Complete mapping of the TSP-1 interactome is needed to understand its diverse functions and to lay the basis for the rational design of TSP-1-based therapeutic approaches. So far, large-scale approaches to identify TSP-1 ligands have been rarely used, but many interactions have been identified in small-scale studies in defined biological systems. This review, based on information from protein interaction databases and the literature, illustrates current knowledge of the TSP-1 interactome map. © 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-SA license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

Contents 1. 2. 3.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Methodological approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Current knowledge of the TSP-1 interactome . . . . . . . . . . . . . . . . . . . . . . 3.1. Common recognition motifs. . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Complexity of the TSP-1 association system — the example of growth factor networks 3.3. Functional analysis and physiological relevance of the TSP-1 interaction network. . . 3.4. Extracellular or intracellular interactions . . . . . . . . . . . . . . . . . . . . . 4. Future prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction In his 1995 seminal paper, Paul Bornstein defined matricellular proteins as extracellular molecules which function by binding to multiple ligands (Bornstein, 1995). Although this definition has been updated and tuned in recent years (as discussed elsewhere in this issue), the establishment of multiple protein–ligand interactions is still considered a key trait of these molecules, explaining their typical range of functions. Thrombospondin-1 (TSP-1) is a prototype matricellular protein that can physically interact with a variety of ligands, including structural ⁎ Corresponding author at: Tumor Angiogenesis Unit, Department of Oncology, IRCCSIstituto di Ricerche Farmacologiche Mario Negri, Via Stezzano, 87 24126 Bergamo, Italy. Tel.: +39 035 421331. E-mail address: [email protected] (G. Taraboletti).

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components of the extracellular matrix, other matricellular proteins, cell receptors, growth factors, cytokines and proteases. These interactions involve distinct binding sites and can occur simultaneously, leading to a variety of scenarios, spanning from the activation of receptors and downstream signaling pathways, to the formation of multimolecular complexes, sequestration and inactivation of growth factors and enzymes, alterations of protein localization, proteolytic processing and internalization, and effects on the receptor/ligand equilibrium and downstream signaling. Comprehensive mapping of the TSP-1 interactome is needed to clarify its context-dependent functional role. Furthermore, the identification of new ligands and protein–protein interaction (PPI) networks might indicate new roles for TSP-1 in physiological and pathological processes, providing tools for designing TSP-1-based therapeutic agents to perturb or mimic these interactions (Zhang and Lawler, 2007; Taraboletti and Bonezzi, 2009; Belotti et al., 2011).

http://dx.doi.org/10.1016/j.matbio.2014.01.012 0945-053X/© 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-SA license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

Please cite this article as: Resovi, A., et al., Current understanding of the thrombospondin-1 interactome, Matrix Biol. (2014), http://dx.doi.org/ 10.1016/j.matbio.2014.01.012

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Name

Entry name UniProt Gene name

Methodology Binding site

Reference

Aggrecan Amyloid beta A4 protein Angiocidin (26S proteasome non-ATPase regulatory subunit 4) Apolipoprotein E receptor 2 (LDL receptor-related protein 8) ATF6α (cAMP-dependent transcription factor ATF-6α) Biglycan Calcium Calreticulin

PGCA A4 PSMD4 LRP8 ATF6A PGS1

ACAN APP PSMD4 47 nM LRP8 (APOER2) 32 nM ATF6 BGN

SP SPR SP, CoIP SP PD SPR

CALR

CALR

CoIP

Type III rep N-term (ELTGAARKGSGRRLVKGPD)

Kuznetsova et al., 2006 Faye et al., 2009 Zhou et al., 2004 Blake et al., 2008 Lynch et al., 2012 Faye et al., 2009 Kvansakul et al., 2004; Lawler et al., 1982 Goicoechea et al., 2000; Yan et al., 2010

CALU CATG PTPRJ CD36 CD47 CSPG CTR CO1A1 CO2A1 CO3A1 CO4A1 CO5A1 CO6A1 CO7A1 COBA1 COIA1 CFAH PGS2 ELNE MK ECM1 FBN2 FIBA FGF2 FINC LEG1 GPC

CALU CTSG PTPRJ CD36 CD47 CSPG CTR COL1A1 COL2A1 COL3A1 COL4A1 COL5A1 COL6A1 COL7A1 COL11A1 COL18A1 CFH DCN ELANE MAPK/ERK ECM1 FBN2 FGA FGF2 FN1 LGALS1 GPC

HGF HRG IBP5

HGF HRG IGFBP5

AC, SPR SP CoIP SP CoIP, CBA ACE SP SPR SP SP SP SP SPR Y2H SPR SPR SPR SP SP CoIP Y2H Y2H SP SP, SPR, NMR SP SP ACE ACE ACE SP SP CoIP

Hansen et al., 2009 Hogg et al., 1993a Takahashi et al., 2012 Type I rep Asch et al., 1992; Dawson et al., 1997; Klenotic et al., 2013 C-term G domain (RFYVVMWK) Gao et al., 1996; Isenberg et al., 2009 Herndon et al., 1999 Lawler et al., 1986 Faye et al., 2009; Galvin et al., 1987 Galvin et al., 1987 Galvin et al., 1987 Galvin et al., 1987 Type I rep Galvin et al., 1987; Mumby et al., 1984 Faye et al., 2009 Type I rep Aho and Uitto., 1998 Faye et al., 2009 Faye et al., 2009 Vaziri-Sani et al., 2005 Main site: N-term (KKTR) Merle et al., 1997 Type III rep (NCQYVYNV) Hogg et al., 1993b Baek et al., 2013 Sercu et al., 2009 Aho and Uitto., 1998 Type I rep and N-term Lahav et al., 1984; Leung and Nachman, 1982; Bonnefoy et al., 2001 Type III rep (DDDDDNDKIPDDRDN) Margosio et al., 2003, 2008; Taraboletti et al., 1997 70 kDa stalk region Dardik and Lahav, 1989 Moiseeva et al., 2000 Herndon et al., 1999 N-term (R42, R76 and R77) Feitsma et al., 2000; Herndon et al.1999; Sun et al.1989 N-term Dixit et al., 1984; Herndon et al., 1999 Lamszus et al., 1996 Type I rep (CSVTCG) Silverstein et al., 1985; Simantov et al., 2001 Nam et al., 2000

Calumenin Cathepsin G CD148 (R-PTP-eta) CD36 (Platelet glycoprotein 4) CD47 (Integrin associated protein, IAP) Chondroitin sulfate PG Chymotrypsin Collagen I Collagen II Collagen III Collagen IV Collagen V Collagen VI Collagen VII Collagen XI Collagen XVIII (endostatin) Complement factor H Decorin Elastase, neutrophil ERK (Extracellular signal-regulated kinase) Extracellular matrix protein 1 Fibrillin-2 Fibrinogen/fibrin Fibroblast growth factor 2 Fibronectin Galectin-1, Gal-1 Glypican (cerebroglycan) Heparan sulfate proteoglycans Heparin Hepatocyte growth factor Histidine-rich glycoprotein Insulin-like growth factor binding protein 5

Affinity (Kd)

0.4 μM 2 nM 13 nM nM range pM range 235–648nM

49 nM 17 nM

3–127 nM 112 nM 33 nM 180–260 nM 41 nM

7 nM

N-term Type I rep (CSVTCG) Type III rep

Type III rep (NCPFHYNP)

A. Resovi et al. / Matrix Biology xxx (2014) xxx–xxx


Table 1 TSP-1 ligands.

Name Gene name

Affinity (Kd)

Integrin α2ß1 Integrin α3ß1 Integrin α4ß1 Integrin α5ß1 Integrin α6ß1 Integrin α9ß1 Integrin αIIb/beta-3 Integrin αv/beta-3 Integrin ß1 Jagged1 Kininogen 1 Laminin, ß3 chain Low-density lipoprotein (LDL) receptor-related protein Lysosomal membrane protein II (LIMPII) Matrix metalloproteinase-2 (72 kDa type IV collagenase) Matrix metalloproteinase-9 Neuroligin-1 Notch 3 (Neurogenic locus notch homolog protein 3) Osteoprotegerin (TNF receptor superfamily member 11B, OPG) Phospholipid scramblase Plasminogen Platelet-derived growth factor Pleckstrin Procollagen C-proteinase enhancer 1 (PCPE1)

ITA2/ITB1 ITA3/ITB1 ITA4/ITB1 ITA5/ITB1 ITA6/ITB1 ITA9/ITB1 ITA2B/ITB3 ITAV/ITB3 ITB1 JAG1 KNG1 LAMB3 LRP1 SCRB2 MMP2 MMP9 NLGN1 NOTC3 TR11B PLS PLMN PDGF PLEK PCOC1

ITGA2/ITGB1 ITGA3/ITGB1 ITGA4/ITGB1 ITGA5/ITGB1 ITGA6/ITGB1 ITGA9/ITGB1 ITGA2B/ITGB3 ITGAV/ITGB3 ITGB1 JAG1 KNG1 LAMB3 LRP1 SCARB2 MMP2 MMP9 NL1 NOTCH3 TNFRSF11B PLSCR PLG PDGF PLEK PCOLCE

Sclerostin Secreted frizzled-related protein (sFRP)-1 SPARC (secreted protein, acidic, cysteine-rich, osteonectin) Stromal interaction molecule 1 Sulfated glycolipids Syndecan-3 Thrombin (Factor II) Thrombospondin-1 Tissue factor pathway inhibitor Transforming growth factor, beta (TGFß) Transglutaminase-2 Tubulin Tumor necrosis factor-stimulated gene-6 (TSG-6) Urokinase-type plasminogen activator Vascular endothelial growth factor A Versican Very low-density lipoprotein receptor Voltage-dependent calcium channel subunit α2δ-1 Von Willebrand Factor

SOST SFRP SPRC STIM1

SOST SFRP SPARC STIM1

SDC3 THRB TSP1 TFPI TGFB TGM2 TBB TSG6 UROK VEGFA CSPG VLDLR CA2D1 VWF

SDC3 F2 THBS1 TFPI TGFB TGM2 TUBB TNFAIP6 PLAU VEGFA VCAN VLDLR CACNA2D1 VWF

Methodology Binding site 72 nM

23 nM

17–52 nM 3–20 nM 70–100 nM

35 nM 11–32 nM 20 pM, 14 nM 48 nM 1 nM

48 nM 480 nM 7 nM

310 nM 40–50 nM

14 nM

CBA CoIP, CBA AC, CBA AC CBA SP PD CBA CBA Y2H, CoIP, SP SP Y2H, PD SP SP Y2H, CoIP Y2H, PD CoIP, SP SP SP Y2H RI, SP SP PD SPR AC SP SP CoIP SP ACE SP SP SP SP SPR Y2H PD SP ACE, CoIP SP SP CoIP SP

Reference N-term (FQGVLQNVRFVF) N-term (AELDVP) N-term (LALERKDHSG) N-term Type III rep Type III rep (RGD) Type I and type II rep Type I rep Type I rep N-term Type I rep (CSVTCG) Type I rep Type I rep

N-term

N-term

Type I rep (KRFK and WxxW)

N-term Type I rep? N-term Type II rep Type I rep (CSVTCG, CSTSCG)

Tuszynski and Kowalska, 1991 Chandrasekaran et al., 2000, 1999; DeFreitas et al., 1995 Calzada et al., 2004b; Yabkowitz et al., 1993 Yabkowitz et al., 1993 Calzada et al., 2003 Staniszewska et al., 2007 Lawler and Hynes, 1989 Lawler and Hynes, 1989 Calzada et al., 2004a Aho and Uitto., 1998; Meng et al., 2009 DeLa Cadena et al., 1994 Aho and Uitto., 1998 Godyna et al., 1995; Mikhailenko et al., 1995; Wang et al., 2004 Crombie and Silverstein, 1998 Bein and Simons, 2000; Yang et al., 2001 Bein and Simons, 2000 Xu et al., 2010 Meng et al., 2009 Zannettino et al., 2005 Aho and Uitto, 1998 Silverstein et al., 1984 Hogg et al., 1997 Baig et al., 2009 Salza et al., 2014 Devarajan-Ketha et al., 2012 Martin-Manso et al., 2011 Clezardin et al., 1988; Sage et al., 1989 Ambily et al., 2013 Roberts et al., 1985 Herndon et al., 1999 Lawler et al., 1986; Milev and Essex, 1999 Lahav et al., 1984 Mast et al., 2000 Murphy-Ullrich et al., 1992; Schultz-Cherry et al., 1995 Faye et al., 2009 Mitocheck Database http://www.mitocheck.org Kuznetsova et al., 2005 Silverstein et al., 1990 Greenaway et al., 2007; Gupta et al., 1999; Laklai et al., 2009 Kuznetsova et al., 2006 Blake et al., 2008 Eroglu et al., 2009 Xie et al., 2001



Entry name UniProt

For each TSP-1 ligand the name, entry name (according to UniProt), gene name, affinity (as Kd), binding site identified on the TSP-1 molecule (rep: repeats) and reference are given. Methodology: SP, solid phase binding assay; SPR, surface plasmon resonance; CoIP, co-immunoprecipitation; PD, pull down assay; CBA, cell binding assay; ACE, affinity coelectrophoresis; Y2H, yeast two-hybrid; NMR, nuclear magnetic resonance; AC, affinity chromatography; RI, rocket immunoelectrophoresis.

3

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interactions, and added new interactions published in the literature, as of December 2013.

2. Methodological approaches The TSP-1 interaction network is still not fully delineated. Most information comes from small-scale experimental studies on single binary interactions in specific biological systems, based on approaches such as solid-phase binding experiments, surface plasmon resonance (SPR), and nuclear magnetic resonance (NMR). Pull-down or coimmunoprecitation assays have been used to identify molecules forming complexes with TSP-1, though in this case, other assays are usually required to verify whether the identified interactions are direct or indirect, mediated by cross-bridging ligands. Large-scale approaches to identify novel TSP-1 ligands have been used less. The classic yeast two-hybrid screen (Y2H), optimized to detect protein interactions in the reducing intracellular environment, is not the appropriate setting for extracellular proteins such as TSP-1. In addition, it is not optimal with such a large molecule, with multiple binding sites — sometimes cryptic and profoundly affected by the environmental conditions. However, the use of single TSP-1 domains has overcome these limits and allowed the identification of several ligands by Y2H screening (Aho and Uitto, 1998; Bein and Simons, 2000; Aho, 2004). A number of public, manually curated repositories are available that collect PPI information from large- and small-scale experimental studies, including the Human Protein Reference Database (HPRD, http:// www.hprd.org), the Biological General Repository for Interaction Datasets (BioGRID, http://thebiogrid.org), MatrixDB (http://matrixdb. ibcp.fr), the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING, http://string-db.org), the International Molecular Exchange Consortium (IMEx, http://www.imexconsortium.org) and IntAct (http://www.ebi.ac.uk/intact/). In this review, we have collected information from different PPI databases, verified the evidence of the

3. Current knowledge of the TSP-1 interactome Table 1 lists 83 TSP-1 ligands. Some of these interactions have been well characterized in terms of binding parameters, involved domain and physiological relevance. In other cases the occurrence and relevance of the interaction in physiological conditions still need to be confirmed. The largest classes are extracellular matrix molecules, cell receptors, growth regulatory factors and proteases (Fig. 1). TSP-1 interacts with proteins and non-protein ligands. Among the latter, TSP-1 characteristically binds calcium and glycosaminoglycans, including heparin, heparan sulfate, dermatan sulfate, and chondroitin sulfate. Calcium and heparin/ heparan-sulfate are regulators of TSP-1 conformation, exposure of cryptic binding sites, and location (Dardik and Lahav, 1999; Kvansakul et al., 2004; Tan et al., 2008). Many interactions are inhibited by heparin and influenced by calcium concentration (Fig. 2), indicating that these interactions will not only be dictated by TSP-1 and ligand availability, but will be restricted to defined biological contexts with optimal calcium concentration and appropriate involvement or non-involvement of heparin/heparan sulfate. The exact binding domain and sequence on the TSP-1 molecule have been identified for many ligands (Table 1 and Fig. 3). The active sequence can be exposed – and both the whole molecule and the isolated fragments/peptides maintain the binding ability – or can be cryptic, becoming exposed following structural alteration of the protein caused by context-dependent factors, such as changes in calcium concentration, agents causing reduction or disulfide interchange events, interaction with

Fig. 1. Schematic representation of the main classes of TSP-1 interacting molecules.



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• polyhistidine stretches, recognized by the heparin-binding domain of TSP-1 (Vanguri et al., 2000). TSP-1 also recognizes the polyhistidine tag in recombinant protein constructs. This has led to the incorrect inclusion of dihydrofolate reductase (DHFR) and coronin as TSP-1 binding proteins in some PPI databases, although they were only used as prototype recombinant proteins interacting with TSP-1 through their his-tag (Vanguri et al., 2000); • the netrin domain, recognized by TSP-1 on sFRP-1 (Martin-Manso et al., 2011). There is also a netrin domain in another recently identified TSP-1 ligand, PCPE-1 (Salza et al., 2014), though its role in PCPE-1/ TSP-1 recognition is still not known. Fig. 2. TSP-1 interactions inhibited by heparin, modulated by calcium concentrations, or both.

other ligands or proteolytic processing (Hotchkiss et al., 1998; Taraboletti et al., 2000; Kvansakul et al., 2004; Lee et al., 2006; Margosio et al., 2008). In some cases, peptides and recombinant fragments reportedly behave in opposite ways. For example, interaction with heparin was observed with peptides based on sequences from the type I repeat (Guo et al., 1992), but not with a recombinant fragment containing the entire type I repeat (Panetti et al., 1999). This, as well as some discrepancies in binding results, might be explained by differences in the experimental and biological settings, once again underscoring the importance of the biological context in determining the pattern of TSP-1 associations. The local composition of the TSP-1 association network is also regulated by co-expression/co-secretion by the same cells, organs and tissues. For example, many TSP-1 binding molecules, such as TGFß, TFPI, calumenin, and PDGF, are secreted by activated platelets together with TSP-1, favoring focal aggregation of selected ligands in an environmentspecific functional complex. 3.1. Common recognition motifs TSP-1 binds to proteins belonging to the same family, or sharing common interacting domains, including: • CLESH-1 homology motifs, common to CD36, LIMP-II and HRGP (Crombie and Silverstein, 1998; Simantov et al., 2001), interacting with the type I repeat; • the LINK module, present in TSG-6 (Kuznetsova et al., 2005), versican and aggrecan (Kuznetsova et al., 2006), which binds the N-terminal domain of TSP in a heparin-dependent manner; • EGF-like repeats, representing the interacting domains of laminin, jagged-1 and fibrillin-2, considered a common target for the type I repeats of TSP-1 (Aho and Uitto, 1998);

3.2. Complexity of the TSP-1 association system — the example of growth factor networks Only binary interactions are reported in Table 1. However, an essential characteristic of TSP-1 is its ability to aggregate multi-molecular complexes, such as the well-characterized complexes composed of TSP-1/calreticulin/LPR1 (Orr et al., 2003) and TSP-1/HRG/plasminogen (Silverstein et al., 1985), expanding the range of complexity of the TSP-1 interaction network. This is further increased by the ability of many TSP-1 ligands to interact – directly or indirectly – with each other. Even further, the interaction of TSP-1 with some of its ligands reciprocally changes the binding ability for other molecules, due to active site occupancy and spatial hindrance or changes in molecular conformation, as in the case of the TSP-1 interaction with heparin, calcium, fibronectin and calreticulin (Dardik and Lahav, 1999; Kvansakul et al., 2004; Tan et al., 2008; Yan et al., 2010). The interaction of TSP-1 with factors and receptors involved in the regulation of cell proliferation is an example of how these complex interactions generate a variety of functional effects, with opposite outcomes on proliferation. TSP-1 directly binds growth factors, receptors and coreceptors. Following the interaction with TSP-1, these ligands can undergo different fates. TSP-1 binding to the latent TGF-β complex induces activation by stimulating a conformation change (Schultz-Cherry et al., 1995; Sweetwyne and Murphy-Ullrich, 2012). PDGF is protected from proteolytic degradation by TSP-1 binding (Krishnaswami et al., 2002) and endogenous TSP-1 promotes the growth factor's ability to interact with PDGFR (Hogg et al., 1997; Isenberg et al., 2005). By binding to the FGF-2 heparin-binding site, TSP-1 prevents FGF-2 interaction with HSPG in the extracellular matrix and on the cell surface (Taraboletti et al., 1997; Margosio et al., 2003; Colombo et al., 2010). Binding of a TSP-1-mimetic small molecule to FGF-2 not only prevented FGF-2 binding to HSPG by direct competition, but also affected FGF-2 internal dynamics, causing allosteric inhibition of FGF-2 binding to the highaffinity receptor FGFR1 (Pagano et al., 2012). TSP-1 binds VEGF (Gupta et al., 1999), inhibiting its activity in different ways: preventing its binding to cell receptors (Rodriguez-Manzaneque et al., 2001; Laklai et al., 2009), causing internalization (Greenaway et al., 2007) and by an indirect mechanism, through the interaction of the TSP-1 receptors CD36, CD47 and ß1 integrins with VEGFR2 (Zhang et al., 2009; Kaur et al., 2010; Kazerounian et al., 2011; Chu et al., 2013). Finally, although TSP-1 does not apparently bind to EGFR, the EGF-like type II repeats activate EGFR tyrosine phosphorylation and downstream signaling, by an indirect mechanism involving MMP-9-mediated release of EGFR ligands (Liu et al., 2009). 3.3. Functional analysis and physiological relevance of the TSP-1 interaction network

Fig. 3. Interaction of each domain of TSP-1 (monomer) with receptors and ligands. For many ligands, the binding site on TSP-1 has not yet been identified. *In parenthesis: number of bound calciums.

Bioinformatics analysis, performed with DAVID Bioinformatics Resources 6.7 (http://david.abcc.ncifcrf.gov/) identified GO terms that are significantly over-represented in TSP-1 ligands. The functional enrichment analysis based on GO annotation (biological processes level 5) indicated that TSP-1 ligands were involved in processes related to the regulation of matrix composition and remodeling, cell/matrix


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interaction, cell adhesion and motility, cell proliferation, and complex biological processes including angiogenesis, coagulation, tissue development in several systems (bone, skin, cartilage, nervous system), proteolysis and ion homeostasis (Figs. 4 and 5), in agreement with the known involvement of TSP-1 in these processes. A comprehensive analysis of the physiological relevance of the interactions is beyond the scope of the review. Excellent recent reviews discuss the involvement of TSP-1 interaction networks in specific physiological processes, including the regulation of tissue repair and fibrosis through the interaction of TSP-1 with TGFß, EGFR and the CRT–LRP1 co-complex (Sweetwyne and Murphy-Ullrich, 2012), the role of TSP-1 and a number of binding partners in synapse formation and CNS functions (Risher and Eroglu, 2012), and the complex regulation of angiogenesis through the interaction of TSP-1 with cell receptors, soluble factors and proteases (Lawler and Lawler, 2012).

3.4. Extracellular or intracellular interactions Not surprisingly, most TSP-1 interactions occur in the extracellular space, within the extracellular matrix or in proximity of the plasma membrane. Recently however, intracellular interactions have also been described, opening up a completely new picture of TSP activity. TSP-1 forms complexes with ATF6α (Lynch et al., 2012) and calumenin (Hansen et al., 2009) in the ER, where TSP-1 resides before being secreted and, depending on the cell type or calcium levels, can be retained for variable periods (Kuznetsov et al., 1997; Veliceasa et al., 2007). These studies provocatively propose a role for TSP-1 in the ER stress response and in disease, by assisting protein processing and secretion and contributing to reconstruction of the extracellular matrix after injury (Lynch et al., 2012). Notably, TSP-1 has been reported to bind – on the plasma membrane – to proteins typically associated with intracellular vesicular compartments, such as calreticulin (Goicoechea et al., 2000), LIMPII (Crombie and Silverstein, 1998), and STIM1 (Ambily et al., 2013). Although no intracellular interactions between TSP-1 and these proteins have been reported so far, it would be interesting to investigate the formation and role of such complexes in intracellular compartments. Even more striking is the recent report of an interaction of TSP-1 with ERK, in the cytoplasm (Baek et al., 2013). TSP-1 complexes with pERK, though not with inactive ERK, and tethers it to the cytosol, preventing its translocation to the nucleus and activity on nuclear

Fig. 5. Involvement of each TSP-1 ligand in GO biological processes (blue rectangle). Functional annotations based on DAVID Bioinformatics Resources were subjectively grouped in related classes, and manually integrated with literature data.

Fig. 4. Significantly enriched GO biological processes (level 5) in the list of TSP-1 ligands. Data, from the Functional Annotation Chart (DAVID Bioinformatics Resources) after removal of redundant terms, are expressed as −log (enrichment p-value).

substrates, ultimately mediating oncogenic Ras-induced senescence and functioning as a tumor suppressor (Baek et al., 2013). Both these intracellular activities are due to endogenous TSP-1, and are not reproduced by exogenous, extracellular TSP-1 (Lynch et al., 2012; Baek et al., 2013). These findings provide a new key to the interpretation of past reports of different activities for endogenous and exogenous TSP-1, and might drastically change our view of its role in



physiological and pathological conditions, particularly in the regulation of tumor progression, angiogenesis, and metastasis. 4. Future prospects The TSP-1 interactome is still not completely known. Large-scale approaches, including new-generation Y2H, array-based SPR and computational methods might well lead to the identification of new TSP-1 ligands. Specific approaches, such as tandem-affinity purification coupled to mass spectrometry, might expand the analysis from single interactions to entire pathways, networks and TSP-1-based multimolecular complexes. The correct identification of bona fide TSP-1 binding partners can unveil new functional properties of TSP-1. A fuller definition of the TSP-1 interaction networks will give us a better understanding of its role and mechanisms of action, and of how it acts in concert with interacting molecules to affect biological and pathological processes in a tissue-specific manner. This knowledge will lay the ground for the development of agents designed to perturb these interactions, to be used as investigational tools as well as for therapeutic purposes. It is essential to identify the relevant hubs and nodes in the TSP-1 association network as well as to obtain a detailed molecular characterization of the TSP-1– ligand interface in order to develop agents to perturb (or mimic) these interactions (Zhang and Lawler, 2007; Rusnati et al., 2010; Taraboletti et al., 2010). As an example of the potential of this approach, the identification and molecular/functional characterization of the interactive network of TSP-1 with FGF-2 and its cell receptors (HSPG and FGFR1) enabled us to make a pharmacophore-based search for small molecules which retain the structural and functional activity of the FGF-binding sequence of TSP-1, resulting in the identification of FGF2-binding, antiangiogenic leads (Colombo et al., 2010; Pagano et al., 2012). Acknowledgments We thank G. Bazzoni (IRCCS-Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy) for constructive discussion and suggestions and J. Baggott for editing the manuscript. Cited work from our laboratory was supported by Associazione Italiana per la Ricerca sul Cancro (AIRC, grant IG-13272). References Aho, S., 2004. Soluble form of Jagged1: unique product of epithelial keratinocytes and a regulator of keratinocyte differentiation. J. Cell. Biochem. 92, 1271–1281. Aho, S., Uitto, J., 1998. Two-hybrid analysis reveals multiple direct interactions for thrombospondin 1. Matrix Biol. 17, 401–412. Ambily, A., Kaiser, W.J., Pierro, C., Chamberlain, E.V., Li, Z., Jones, C.I., Kassouf, N., Gibbins, J.M., Authi, K.S., 2013. The role of plasma membrane STIM1 and Ca2+ entry in platelet aggregation. STIM1 binds to novel proteins in human platelets. Cell. Signal. 26, 502–511. Asch, A.S., Silbiger, S., Heimer, E., Nachman, R.L., 1992. Thrombospondin sequence motif (CSVTCG) is responsible for CD36 binding. Biochem. Biophys. Res. Commun. 182, 1208–1217. Baek, K.H., Bhang, D., Zaslavsky, A., Wang, L.C., Vachani, A., Kim, C.F., Albelda, S.M., Evan, G.I., Ryeom, S., 2013. Thrombospondin-1 mediates oncogenic Ras-induced senescence in premalignant lung tumors. J. Clin. Invest. 123, 4375–4389. Baig, A., Bao, X., Haslam, R.J., 2009. Proteomic identification of pleckstrin-associated proteins in platelets: possible interactions with actin. Proteomics 9, 4254–4258. Bein, K., Simons, M., 2000. Thrombospondin type 1 repeats interact with matrix metalloproteinase 2. Regulation of metalloproteinase activity. J. Biol. Chem. 275, 32167–32173. Belotti, D., Foglieni, C., Resovi, A., Giavazzi, R., Taraboletti, G., 2011. Targeting angiogenesis with compounds from the extracellular matrix. Int. J. Biochem. Cell Biol. 43, 1674–1685. Blake, S.M., Strasser, V., Andrade, N., Duit, S., Hofbauer, R., Schneider, W.J., Nimpf, J., 2008. Thrombospondin-1 binds to ApoER2 and VLDL receptor and functions in postnatal neuronal migration. EMBO J. 27, 3069–3080. Bonnefoy, A., Hantgan, R., Legrand, C., Frojmovic, M.M., 2001. A model of platelet aggregation involving multiple interactions of thrombospondin-1, fibrinogen, and GPIIbIIIa receptor. J. Biol. Chem. 276, 5605–5612. Bornstein, P., 1995. Diversity of function is inherent in matricellular proteins: an appraisal of thrombospondin 1. J. Cell Biol. 130, 503–506. Calzada, M.J., Sipes, J.M., Krutzsch, H.C., Yurchenco, P.D., Annis, D.S., Mosher, D.F., Roberts, D.D., 2003. Recognition of the N-terminal modules of thrombospondin-1 and thrombospondin-2 by alpha6beta1 integrin. J. Biol. Chem. 278, 40679–40687.

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