Interleukin 3 (IL-3 or multi-CSF), a cytokine produced by activated T- lymphocytes, forms an important part of the host response to inflammatory stimuli. In addition to its physiological importance, the pharmacologic effects of IL-3 insure that it will play an important role in the treatment of hematologic, oncologic and infectious diseases in the near future. Despite intensive study, many questions remain about the role of IL-3 in physiologic and pathologic states. IL-3 acts by binding to high and possibly low affinity cell surface receptors. To understand its mechanism of action, the interaction between IL-3 and its cell surface receptor must be explored at a molecular level. Determining the precise site(s) of IL-3 binding to its cell surface receptor, it precise spatial relationships and how the IL-3 receptor interaction leads to signal transduction will be the prime focus of this proposal. Towards this end we will generate and test a series of interspecies chimera of primate and murine IL-3 map the site(s) of receptor-ligand binding. This approach to structure-function analysis has several theoretical and practical advantages. IL-3 and informative mutants will be tested in a number of biological and receptor binding assays to determine the functional domains of the molecule. In addition, the structures of proteins of moderate size (under Mr 30,000) can now be determined in solution. Our experience with comparable proteins indicates that IL-3 is an excellent candidate for structure determination by multidimensional NMR methods. A major collaboration has been arranged to perform this work. These studies will form the basis for manipulation of the IL-3 molecule to create reagents useful for further physiologic study. And, to better understand the site(s) of interaction with its ligand, we will clone human IL-3 receptor polypeptides and generate and analyze mutant forms of the receptor subunits. The molecular details of the IL-3-IL-3 receptor interaction will thus be determined. Based on a growing understanding of the structure-function relationships of the related cytokines, GM-CSF, an important corollary of our work will derive from understanding the relatedness and interaction of these two distinct, but functionally related hematopoietic growth factors, IL- 3 and GM-CSF. Once the critical interactive residues on the two growth factors and their receptors have been identified these regions will be compared within the context of their secondary and tertiary structures. Additional chimers and substitution mutants will be developed to test working hypotheses. In so doing we hope to better understand the principles of protein-protein interaction and the evolutionary relationships amongst similar but distinct proteins. Taken together, these studies will further our understanding of the cell biology and protein chemistry of hematopoiesis.
Showing the most recent 10 out of 11 publications
