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Interleukin-3 signal transduction : purification and characterization of the murine interleukin-3 receptor

University of British Columbia

Murine interleukin-3 (mIL-3) is a potent hemopoietic growth factor that is produced primarily by activated T lymphocytes and stimulates the proliferation and differentiation of pluripotent stem cells and committed myeloid and early lymphoid progenitors. In order to gain some insight into its mechanism of action, we set out to purify and characterize its cell surface receptor. To monitor this purification, an assay capable of detecting detergent solubilized mIL-3R's was devised. With this assay, a simple two step purification protocol, involving anti-phosphotyrosine Sepharose and biotinylated mIL-3/streptavidin agarose, was developed to purify the mIL-3R This protocol was based, in part, on a previous observation in our laboratory that the mIL-3R becomes tyrosine phosphorylated upon ligand binding. Two formsof the receptor were obtained using this procedure; a tyrosine/serine phosphorylated 140 kD form that was greater that 98% pure, and a less pure, serine phosphorylated 120 kD form. Alkaline phosphatase treatment, chymotrypsin digestion and Western analysis using antibodies to the N-terminus of the 120 kD form established that, apart from phosphorylation differences, these two proteins were identical. Intriguingly, the 140 kD tyrosine/serine phosphorylated mIL-3R was exquisitely sensitive to proteolysis whereas the 120 kD receptor was not. This proteolysis was also shown to occur in intact cells in response to mIL-3 and took place rapidly at 37°C in the presence of lysosomal inhibitors. These observations suggest that this mIL-3 stimulated proteolysis occurs at the cell surface and could play an important role in the mechanism of action of mIL-3. N-terminal amino acid sequencing and amino acid composition analysis of the purifiedmIL-3R showed it to be identical to an mIL-3 binding protein subsequently cloned by expression in COS cells. Examination of this sequence revealed no consensus kinase domains, indicating the tyrosine phosphorylation of the mIL-3R must be mediated by an associated kinase. Studies directed towards the identification and purification of this mIL-3R associated tyrosine kinase, as well as the characterization of other mIL-3R associated proteins that we have identified, are presented. Our results to date have led us to propose a model of mIL-3Rinduced signal transduction that may also apply, to some extent, to other members of the hemopoietic receptor super family.

Thesis/Dissertation

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2009-01-06

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http://hdl.handle.net/2429/3385

Pathology

University of British Columbia

UBCV

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