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Journal of Cell Communication and Signaling

, Volume 5, Issue 4, pp 277–289

First Online: 23 March 2011Received: 03 February 2011Accepted: 28 February 2011DOI: 10.1007-s12079-011-0126-2

Cite this article as: Taferner, A., Micutkova, L., Hermann, M. et al. J. Cell Commun. Signal. 2011 5: 277. doi:10.1007-s12079-011-0126-2

Abstract

Insulin-like growth factor binding proteins IGFBPs are key regulators of insulin-like growth factor IGF mediated signal transduction and thereby can profoundly influence cellular phenotypes and cell fate. Whereas IGFBPs are extracellular proteins, intracellular activities were described for several IGFBP family members, such as IGFBP-3, which can be reinternalized by endocytosis and reaches the nucleus through routes that remain to be fully established. Within the family of IGFBPs, IGFBP-6 is unique for its specific binding to IGF-II. IGFBP-6 was described to possess additional IGF-independent activities, which have in part been attributed to its translocation to the nucleus; however, cellular uptake of IGFBP-6 was not described. To further explore IGFBP-6 functions, we developed a new method for the purification of native human IGFBP-6 from cell culture supernatants, involving a four-step affinity purification procedure, which yields highly enriched IGFBP-6. Whereas protein purified in this way retained the capacity to interact with IGF-II and modulate IGF-dependent signal transduction, our data suggest that, unlike IGFBP-3, human IGFBP-6 is not readily internalized by human tumor cells. To summarize, this work describes a novel and efficient method for the purification of native human insulin-like growth factor binding protein 6 IGFBP-6 from human cell culture supernatants, applying a four-step chromatography procedure. Intactness of purified IGFBP-6 was confirmed by IGF ligand Western blot and ability to modulate IGF-dependent signal transduction. Cellular uptake studies were performed to further characterize the purified protein, showing no short-term uptake of IGFBP-6, in contrast to IGFBP-3.

KeywordsIGF-binding protein Purification Chromatography Cellular uptake AbbreviationsHDFHuman diploid fibroblast

HICHydrophobic interaction chromatography

IGFInsulin-like growth factor

IGFBPInsulin-like growth factor binding protein

MWMolecular weight

Electronic supplementary materialThe online version of this article doi:10.1007-s12079-011-0126-2 contains supplementary material, which is available to authorized users.

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Autor: Andrea Taferner - Lucia Micutkova - Martin Hermann - Pidder Jansen-Dürr - Haymo Pircher

Fuente: https://link.springer.com/







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