Cyr61/CCN1 displays high-affinity binding to the somatomedin B(1-44) domain of vitronectin.
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.


RESULTS
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 alphav beta3. 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, beta-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.
Francischetti
Ivo M B
IM
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. ifrancischetti@niaid.nih.gov
Kotsyfakis
Michalis
M
Andersen
John F
JF
Lukszo
Jan
J
D000595:Amino Acid Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D055515:Cysteine-Rich Protein 61 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D039302:Integrin alphaVbeta3 / Q000378:metabolism; D008969:Molecular Sequence Data; D017395:Plasminogen Activator Inhibitor 1 / Q000378:metabolism; D011485:Protein Binding; D055503:Protein Multimerization; D017386:Sequence Homology, Amino Acid; D013002:Somatomedins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020349:Surface Plasmon Resonance; D020298:U937 Cells; D019096:Vitronectin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D055515:Cysteine-Rich Protein 61; D039302:Integrin alphaVbeta3; D017395:Plasminogen Activator Inhibitor 1; D013002:Somatomedins; D019096:Vitronectin; C016756:somatomedin B