Mutational Analysis of the Export Targeting Motif of Fibroblast Growth Factor 2, a Mediator of Tumor-Induced Angiogenesis
The majority of secretory proteins is exported from mammalian cells by the classical secretory pathway involving subcellular compartments such as the endoplasmic reticulum (ER) and the Golgi apparatus. However, basic fibroblast growth factor (FGF2), a potent mediator of tumor-induced angiogenesis, has been shown to be secreted by a non-classical pathway that does not depend on the functions of the ER and the Golgi apparatus. The molecular characterization of the FGF2 export mechanism is not only a fundamental problem in cell biology but also of great interest for biomedical research since it may pave the way for the development of a novel class of anti-angiogenic drugs. In this thesis, a robust model system designed to quantitatively assess FGF2 secretion under various experimental conditions was developed. A retroviral expression system was established in CHO cells that allows for a stable integration of reporter constructs whose expression can be induced by doxicycline. In order to monitor expression of FGF2 reporter molecules they were constructed as GFP fusion proteins. Based on this experimental system, secretion of FGF2-GFP can be quantified by flow cytometry, confocal microscopy and biochemical methods since exported FGF2-GFP binds to cell surface heparan sulfate proteoglycans and, therefore, is accessible by membrane-impermeable tools such as antibodies and biotinylation reagents. In the second part of this thesis, a systematic mutational analysis of the FGF2 open reading frame was conducted in order to identify cis elements that direct FGF2 to its export machinery. Initial experiments revealed the identification of FGF2 mutants that are defective in binding to heparan sulfate proteoglycans. Such mutants were neither detectable on the cell surface nor in the medium of cells suggesting that the interaction of FGF2 with heparan sulfate proteoglycans does not only play a role in FGF2 signaling but also in the overall process of FGF2 externalization from mammalian cells. A collection of more than a hundred FGF2 mutants and corresponding stable cell lines described in this thesis now provide a basis for future studies in order to conduct a detailed analysis of determinants required for FGF2 secretion.
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