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UBC Theses and Dissertations
Studies into the mechanism of action of interleukin 3 : purification of interleukin 3 and characterization of its cell surface receptor Murthy, Sudish C.
Hemopoiesis is regulated, in part, by a group of potent, soluble hemopoietic growth factors. One of these growth factors, interleukin 3, stimulates the proliferation and differentiation of both multi-potential hemopoietic stem cells and maturing committed progenitors, and may thus play a central role in regulating hemopoiesis in vivo. Studies of the mechanism of action of this hemopoietic growth factor may therefore yield valuable insights into normal hemopoietic differentiation and provide an understanding of the pathogenesis of hemopoietic malignancies. In order to begin these studies, it was first necessary to devise a simple high yield purification procedure for murine interleukin 3, since published purification protocols were lengthy and resulted in unacceptably low yields. A simple and rapid 3-step procedure was therefore devised which greatly facilitated our studies. The novel aspect of this procedure involved the treatment of B6SUtA₁ cells with 0.01% glutaraldehyde which transformed them into mechanically resistant spheres. This made it possible to use these high interleukin 3 receptor-expressing cells as a solid phase reagent suitable for the large scale purification of interleukin 3. Purification, using these fixed cells and two standard purification steps, resulted in a 16,000-fold enrichment and purification to apparent homogeneity of interleukin 3 from serum-free pokeweed mitogen stimulated spleen cell conditioned medium. The effects of pure interleukin 3 on interleukin 3 receptor internalization and re-expression and the relationship between interleukin 3 receptor surface density and growth factor sensitivity were then examined. From these studies, it was demonstrated that surface bound interleukin 3 is rapidly internalized and cleaved at two specific sites, before being completely digested within lysosomes. However, unlike its ligand, the interleukin 3 receptor appears to be recycled to the cell surface without proteolytic cleavage. Furthermore, receptor re-expression is critically dependent upon the extracellular concentration of interleukin 3. More importantly, in the absence of interleukin 3, target cells express extremely high levels of surface receptor and become exquisitely sensitive to interleukin 3. iii Finally, the structure of the interleukin 3 receptor was examined. Previous the precise nature of the interleukin 3 receptor have proven particularly molecular weight estimates for the receptor have been forwarded. Through crosslinking agents, this controversy appears to have been resolved. A model generated suggests that the interleukin 3 receptor is a 140 kd membrane 3 binding and chemical crosslinking, becomes proteolytically cleaved to a 70kd surface protein.
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