IGFBP-3 and IGFBP-5 associate with the cell binding domain (CBD) of fibronectin James Beattie * ,1 , Michaela Kreiner, Gordon J. Allan, David J. Flint, Diana Domingues, Christopher F. van der Walle Strathclyde Institute of Pharmacy and Biomedical Sciences (SIBPS), University of Strathclyde, 27 Taylor St., Glasgow G4 0NR, United Kingdom article info Article history: Received 17 February 2009 Available online 21 February 2009 Keywords: Fibronectin Insulin-like growth factor binding protein Surface plasmon resonance abstract We have used Surface Plasmon Resonance (SPR) – based biosensor technology to investigate the interac- tion of the six high affinity insulin-like growth factor binding proteins (IGFBP 1–6) with the cell binding domain (CBD) of fibronectin. Using a biotinylated derivative of the ninth and tenth TypeIII domains of FN ( 9–10 FNIII), we show that IGFBP-3 and -5 bind to FN-CBD. We show that this binding is inhibited by IGF-I and that, for IGFBP-5, binding occurs through the C-terminal heparin binding domain of the protein. Using site-directed mutagenesis of 9–10 FNIII, we show both the ‘‘synergy” and RGD sites within these FN domains are required for maximum binding of both IGFBPs. We discuss the possible biological conse- quences of our results. Ó 2009 Elsevier Inc. All rights reserved. Introduction IGFBP-5 is an important regulator of the activity of both insulin- like growth factors (IGF-I and IGF-II) [1]. The affinity of IGFBP-5 for both IGFs exceeds that of cell surface IGF-I and IGF-II receptors (IGF-IR and IGF-IIR) and as such most IGFs are sequestered in bind- ing complexes with IGFBP-5 and other high affinity IGFBPs [2]. This disparity in affinity of IGFBPs and IGFRs for IGFs requires the pro- teolysis of IGFBPs to release IGFs and allow binding to cell surface receptors [3]. IGFBP-5 and IGFBP-3 bind to other biomolecules such as components of the extracellular matrix – including hepa- rin, osteopontin, PAI-1 and vitronectin [4–7]. Previous yeast two- hybrid studies have suggested that the interaction between IGFBP-5 and FN is mediated via the C-terminal 10th–11th Type I domains of FN [8]. In this study we have used Surface Plasmon Resonance (SPR) biosensor technology to further characterise the interaction of IGFBP-5 and IGFBP-3 with FN. In contrast to previous studies, we demonstrate conclusively that both IGFBP-3 and -5 interact with the FN cell binding domain (CBD), comprising the 9th–10th FN Type III domains ( 9–10 FNIII), which harbour the integrin a5b1- binding synergy (PHSRN) and RGD sites, respectively [9]. Using C-terminal mutants we show that residues within the heparin binding domain of IGFBP-5 are important for binding, although there exist subtle differences in the residues which are required for interaction with 9–10 FNIII compared with those required for heparin binding. In contrast to previous studies we demonstrate that binding of IGFBP-3 and -5 to 9–10 FNIII is inhibited following co-incubation with IGF-I. RGD peptide had little effect on the bind- ing of either IGFBP-3 or -5 to 9–10 FNIII, although mutagenesis with- in the PHSRN or RGD motifs of 9–10 FNIII inhibited IGFBP binding. We discuss the possible biological consequences of our data in relation to regulation of IGF activity in the pericellular environment. Materials and methods Materials. Mouse (m) IGFBP-5 expression, mutagenesis and purification has been described previously [10,11]. mIGFBP-1, mIGFBP-2, mIGFBP-3, hIGFBP-4, and mIGFBP-6 were supplied by R&D Systems (Abingdon, UK) The 9–10 FNIII wild type cDNA cloned into pRSET was from Prof. H. Mardon, University of Oxford. Muta- tion of the 9–10 FNIII cDNA template as directed by the amino acid substitutions described in Table 1 was made following the Quick- change TM protocol (Stratagene, Amsterdam, Netherlands). The 9–10 FNIII construct used in this study was a stable mutant (substi- tuting Pro 1408 for Leu as described in [12]) extended at the C-termi- nus with a GGC tripeptide [13]. Using this 9–10 FNIII construct as a template, and showing only the sense strand, 9–10 FNIII-PHAAA was achieved using 5 0 -GAA GAT CGG GTG CCC CAC G CT GC G GC T TCC ATC ACC CTC ACC AAC C; 9–10 FNIII-KGD was achieved using 5 0 -GTG TAT GCT GTC ACT GGC AAA GGA GAC AGC CCC GCA AGC; and 9–10 FNIII-GG was achieved using 5 0 -CCC ATT GAT TGG CCA ACA ATC AAC AGG TGG C GT TTC TGA TGT TCC GAG GGA CC (muta- tions underlined). The 9–10 FNIII proteins were biotinylated via the sulfhydryl group of the C-terminal cysteine with PEO 2 -maleimide activated biotin (Pierce, UK) according to the manufacturer’s 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.02.088 * Corresponding author. E-mail address: J.Beattie@leeds.ac.uk (J. Beattie). 1 Present address: Dept. of Oral Biology, Leeds Dental Institute, Clarendon Way, Leeds LS2 9LU, United Kingdom. Biochemical and Biophysical Research Communications 381 (2009) 572–576 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc