Molecular basis of the interaction between IGFBP-3 and retinoid X receptor: Role in modulation of RAR-signaling Lynette J. Schedlich a, * , Lloyd D. Graham b , Michelle K. O’Han a , Anita Muthukaruppan a,1 , Xiaolang Yan a , Sue M. Firth a , Robert C. Baxter a a Kolling Institute of Medical Research, University of Sydney, Royal North Shore Hospital, St. Leonards, NSW 2065, Australia b CSIRO Molecular & Health Technologies, Sydney Laboratory, P.O. Box 184, North Ryde, NSW 1670, Australia Received 16 March 2007, and in revised form 18 June 2007 Available online 26 June 2007 Abstract IGFBP-3 interacts with the retinoid X receptor-a (RXRa) and retinoic acid receptor-a (RARa) and thereby interferes with the for- mation of RXR:RAR heterodimers. Here we identify the domains in RXRa and IGFBP-3 that participate in this interaction. When different regions of RXRa were expressed independently, we found that only the DNA-binding domain (C-domain) bound IGFBP-3. Residues in the second Zn-finger loop (Gln49, Arg52), which contribute to C-domain dimerization on DR1 response elements, proved essential to IGFBP-3 binding. In complementary studies, we found that residues within the N-terminal domain of IGFBP-3 (Thr58, Arg60) and motifs in its C-terminal domain ( 220 LysLysLys, 228 LysGlyArgLysArg) were required for interaction with RXRa and RARa. Unlike wild-type IGFBP-3, the non-retinoid receptor-binding mutants of IGFBP-3 were unable to attenuate all-trans-retinoic acid- induced transactivation of the RAR response element by RXR:RAR heterodimers. We conclude that residues in both the N- and C-ter- minal domains of IGFBP-3 are involved in binding the retinoid receptors, and that this interaction is essential to the modulation of RAR-signaling by IGFBP-3. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Insulin-like growth factor binding protein-3; Retinoid receptors; Protein:protein interaction; Mutagenesis; Nuclear receptor signaling Insulin-like growth factor binding protein (IGFBP)-3 belongs to a family of IGFBPs which have a well character- ized role in regulating the bioavailability of insulin-like growth factors (IGFs) in the extracellular environment [1]. However, there is increasing evidence that several of these IGFBPs, including IGFBP-3, have IGF-independent effects on cell growth, differentiation and apoptosis [2,3]. The mechanisms for these effects are not fully understood, but may involve an interaction between the IGFBPs and different cell signaling pathways both at the cell surface and within the cell. For example, we have shown that IGFBP-3 can potentiate the mitogenic effects of epidermal growth factor, a ligand that activates a transmembrane receptor [4]. In this context IGFBP-3 is acting as an inde- pendent growth stimulator. We and others have also shown that IGFBP-3 functions at an intracellular level to modu- late signaling mediated by nuclear retinoid X receptor (RXR), retinoic acid receptor (RAR) [5,6] and vitamin D receptor (VDR) (our unpublished results). Retinoic acid is a non-steroid hormone essential for the maintenance of normal growth and differentiation of epi- thelial cells [7]. In addition, the retinoids are potent growth-inhibitory and pro-apoptotic factors for many nor- mal and malignant cell types. The actions of retinoic acid are mediated by a family of nuclear proteins, RAR-a,-b and -c, which heterodimerize with one of the three isoforms of RXR, RXR-a,-b or -c, to form ligand-activated tran- scription factor complexes [8]. Ligand activation of the het- erodimer initiates gene transcription when it is bound to 0003-9861/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2007.06.013 * Corresponding author. Fax: +61 2 9926 8484. E-mail address: lyns@med.usyd.edu.au (L.J. Schedlich). 1 Present address: Faculty of Medical and Health Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand. www.elsevier.com/locate/yabbi ABB Archives of Biochemistry and Biophysics 465 (2007) 359–369