Abstract
Insulin-like growth factor-binding proteins (IGFBPs) play an integral role in modifying insulin-like growth factor actions in a wide variety of cell types. Recent evidence suggests that IGFBP-3 and IGFBP-5 also have effects on cell growth that are insulin-like growth factor-independent. In investigating possible mechanisms for this effect, the intracellular trafficking of IGFBP-3 and IGFBP-5, both of which contain sequences with the potential for nuclear localization, was studied in T47D cells. Nuclear uptake of fluorescently labeled IGFBP-3 and IGFBP-5 was observed in a proportion of T47D cells that appeared to be rapidly dividing. IGFBP-1 and IGFBP-2, which do not possess the putative domain for nuclear translocation, were not transported to the nuclei of T47D cells. When T47D cells were preincubated with excess unlabeled IGFBP-3, nuclear localization of labeled IGFBP-3 or IGFBP-5 was not detected, indicating that their nuclear translocation involves a common pathway. Inhibition of receptor-mediated endocytosis did not affect nuclear uptake of IGFBP-3, suggesting that it uses an alternative non-classical import pathway for transport across the plasma membrane. In addition, a variant form of IGFBP-3 with a mutation in the putative nuclear localization sequence was unable to translocate to the nuclei of T47D cells, suggesting that nuclear translocation of IGFBP-3 was dependent on these carboxyl-terminal basic residues.
Highlights
The insulin-like growth factors (IGF-I and IGF-II)1 are potent mitogens, which stimulate proliferation in many normal and malignant cell types [1]
As part of our investigations into the mechanisms that regulate breast cancer cell growth, we have studied the nuclear localization of Insulin-like growth factor-binding proteins (IGFBPs)-3 and IGFBP-5 in the T47D cell line, an estrogen receptor-positive human breast cancer cell line that is reported to express IGFBP-2, -4, and -5 [19]
Based on a common putative nuclear localization signal in the carboxyl-terminal domains of IGFBP-3 and IGFBP-5, we hypothesized that IGFBP-5 would translocate to cell nuclei
Summary
Materials—Natural, glycosylated IGFBP-3 was isolated from Cohn fraction IV of human plasma [20], IGFBP-1 was purified from human amniotic fluid [21], and recombinant human IGFBP-2 was provided by Sandoz, Basel, Switzerland. Labeled binding proteins were separated from free Cy3 dye by size exclusion chromatography on a 1 ϫ 50-cm column of Sephadex G-100 (Amersham Pharmacia Biotech) in 50 mM sodium phosphate buffer, pH 6.8, 0.5 M NaCl, and 1 g/liter bovine serum albumin. Transferrin (Sigma) was labeled without the addition of bovine serum albumin, and the protein concentration was determined using the Bradford assay. Labeled binding proteins (0.5 g/ml) were added and the cells incubated for 60 min at 22 °C. 5 M calcein AM (Molecular Probes, Eugene, OR) was added [25] and after another 30 min of incubation the cells were washed and fixed with Histochoice (Amresco, Solon, OH), mounted in an antifade medium, and examined using fluorescent microscopy. The residues in IGFBP-3 and IGFBP-5 that conform to this consensus pattern are indicated in bold type
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