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      Cyr61/CCN1 Displays High-Affinity Binding to the Somatomedin B 1–44 Domain of Vitronectin

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          Abstract

          Background

          Cyr61 is a member of the CCN ( Cyr61, connective tissue growth, NOV) family of extracellular-associated (matricellular) proteins that present four distinct functional modules, namely insulin-like growth factor binding protein (IGFBP), von Willebrand factor type C (vWF), thrombospondin type 1 (TSP), and C-terminal growth factor cysteine knot (CT) domain. While heparin sulphate proteoglycans reportedly mediate the interaction of Cyr61 with the matrix and cell surface, the role of other extracellular associated proteins has not been revealed.

          Methods and Findings

          In this report, surface plasmon resonance (SPR) experiments and solid-phase binding assays demonstrate that recombinant Cyr61 interacts with immobilized monomeric or multimeric vitronectin (VTNC) with K D in the nanomolar range. Notably, the binding site for Cyr61 was identified as the somatomedin B domain (SMTB  1–44) of VTNC, which mediates its interaction with PAI-1, uPAR, and integrin αvβ3. Accordingly, PAI-1 outcompetes Cyr61 for binding to immobilized SMTB  1–44, and Cyr61 attenuates uPAR-mediated U937 adhesion to VTNC. In contrast, isothermal titration calorimetry shows that Cyr61 does not display high-affinity binding for SMTB  1-44 in solution. Nevertheless, competitive ELISA revealed that multimeric VTNC, heat-modified monomeric VTNC, or SMTB  1–44 at high concentrations attenuate Cyr61 binding to immobilized VTNC, while monomeric VTNC was ineffective. Therefore, immobilization of VTNC exposes cryptic epitopes that recognize Cyr61 with high affinity, as reported for a number of antibodies, β-endorphin, and other molecules.

          Conclusions

          The finding that Cyr61 interacts with the SMTB 1–44 domain suggests that VTNC represent a point of anchorage for CCN family members to the matrix. Results are discussed in the context of the role of CCN and VTNC in matrix biology and angiogenesis.

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          Most cited references51

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          Crystal structure of the extracellular segment of integrin alpha Vbeta3 in complex with an Arg-Gly-Asp ligand.

          The structural basis for the divalent cation-dependent binding of heterodimeric alphabeta integrins to their ligands, which contain the prototypical Arg-Gly-Asp sequence, is unknown. Interaction with ligands triggers tertiary and quaternary structural rearrangements in integrins that are needed for cell signaling. Here we report the crystal structure of the extracellular segment of integrin alphaVbeta3 in complex with a cyclic peptide presenting the Arg-Gly-Asp sequence. The ligand binds at the major interface between the alphaV and beta3 subunits and makes extensive contacts with both. Both tertiary and quaternary changes are observed in the presence of ligand. The tertiary rearrangements take place in betaA, the ligand-binding domain of beta3; in the complex, betaA acquires two cations, one of which contacts the ligand Asp directly and the other stabilizes the ligand-binding surface. Ligand binding induces small changes in the orientation of alphaV relative to beta3.
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            Requirement of vascular integrin alpha v beta 3 for angiogenesis.

            Angiogenesis depends on the adhesive interactions of vascular cells. The adhesion receptor integrin alpha v beta 3 was identified as a marker of angiogenic vascular tissue. Integrin alpha v beta 3 was expressed on blood vessels in human wound granulation tissue but not in normal skin, and it showed a fourfold increase in expression during angiogenesis on the chick chorioallantoic membrane. In the latter assay, a monoclonal antibody to alpha v beta 3 blocked angiogenesis induced by basic fibroblast growth factor, tumor necrosis factor-alpha, and human melanoma fragments but had no effect on preexisting vessels. These findings suggest that alpha v beta 3 may be a useful therapeutic target for diseases characterized by neovascularization.
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              All in the CCN family: essential matricellular signaling modulators emerge from the bunker.

              The CCN family is a group of six secreted proteins that specifically associate with the extracellular matrix. Structurally, CCN proteins are modular, containing up to four distinct functional domains. CCN family members are induced by growth factors and cytokines such as TGFbeta and endothelin 1 and cellular stress such as hypoxia, and are overexpressed in pathological conditions that affect connective tissues, including scarring, fibrosis and cancer. Although CCN family members were discovered over a decade ago, the precise biological role, mechanism of action and physiological function of these proteins has remained elusive until recently, when several key mechanistic insights into the CCN family emerged. The CCNs have been shown to have key roles as matricellular proteins, serving as adaptor molecules connecting the cell surface and extracellular matrix (ECM). Although they appear not to have specific high-affinity receptors, they signal through integrins and proteoglycans. Furthermore, in addition to having inherent adhesive abilities that modulate focal adhesions and control cell attachment and migration, they execute their functions by modulating the activity of a variety of different growth factors, such as TGFbeta. CCN proteins not only regulate crucial biological processes including cell differentiation, proliferation, adhesion, migration, apoptosis, ECM production, chondrogenesis and angiogenesis, but also have more sinister roles promoting conditions such as fibrogenesis.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2010
                26 February 2010
                : 5
                : 2
                : e9356
                Affiliations
                [1 ]Section of Vector Biology, Laboratory of Malaria and Vector Research, Research Technologies Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America
                [2 ]Peptide Synthesis and Analysis Laboratory, Research Technologies Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America
                [3 ]Institute of Parasitology, Biology Centre, National Academy of Sciences of Czech Republic, Ceske Budejovice, Czech Republic
                University of Nebraska Medical Center, United States of America
                Author notes

                Conceived and designed the experiments: IF. Performed the experiments: IF MK JFA JL. Analyzed the data: IF MK JFA JL. Contributed reagents/materials/analysis tools: IF. Wrote the paper: IF MK JFA JL.

                Article
                09-PONE-RA-13848R1
                10.1371/journal.pone.0009356
                2829074
                20195466
                34f78016-0c31-446a-b288-2b9a3747c10d
                This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.
                History
                : 27 October 2009
                : 22 January 2010
                Page count
                Pages: 12
                Categories
                Research Article
                Biochemistry
                Molecular Biology
                Cell Biology/Cell Adhesion

                Uncategorized
                Uncategorized

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