The long term goal of our research is to determine what cellular processes are regulated by the neuronal calcium-binding protein S100alpha and how S100alpha influences the expression of the neuronal cell phenotype. Studies in our laboratory have directly demonstrated that decreased S100alpha expression blocks NGF-induced neurite extension in PC12 cells. In order to determine how changes in S100alpha expression result in altered phenotypes, one must first determine how S100alpha expression is regulated and what cellular process/target proteins are controlled by S100alpha. The studies proposed in this application utilize a molecular approach directly address both of these questions. The first specific aim is to utilize radioimmunoassays, indirect immunofluorescence microscopy, and dot blot hybridization to quantitate the effects of differentiating agents (e.g. NGF, cAMP, and dexamethasone) on S100alpha protein levels, subcellular distribution, binding protein profiles, and steady state mRNA levels. Nuclear runoff transcription, mRNA stability, and pulse-chase assays will be used to determine if transcriptional and/or post-transcriptional mechanisms regulate S100alpha expression. The second specific aim is to complete the isolation and characterization of the rat S100alpha gene locus(s) and to utilize CAT constructs in transient transfection experiments to determine which S100alpha promoter sequences are involved in transcriptional regulation of S100alpha expression in PC12 cells. In the third specific aim, the level of S100alpha protein will be altered by transfection with S100alpha sense and anti-sense expression vectors to determine the step in the NGF differentiation pathway which is controlled by S100alpha. These same clones will be used in specific aim four to determine if tubulin and/or the microtubule associated gamma protein are regulated by S100alpha in living PC12 cells. These studies will provide the first direct information regarding S100alpha expression and function in neuronal cells and represent a major step in ascertaining the role of S100alpha in neurological disease and its therapeutic potential.
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