A central goal of this research program is to determine how eukaryotic cells control the localization and expression of cAMP-dependent protein kinases (cA-PKs). The predominant cA-PK in the mammalian central nervous system (CNS) contains an R subunit isoform (RIIBeta) that appears to adapt the holoenzyme for specialized functions in brain. A striking characteristic of RIIBeta is its ability to bind tightly to intracellular structures in neurons. Thus, signals carried by cAMP in the CNS might be targeted to specialized intraneuronal compartments by high-affinity RIIBeta binding proteins. We have discovered two novel particulate proteins, bovine brain P75 and rat brain P150, that bind RIIBeta with high-affinity. The putative P75 and P150 anchor proteins are expressed almost exclusively in brain. We will now clone and characterize a full-length cDNA for P75. The RIIBeta binding domain of P75 will be characterized by in vitro mutagenesis, expression and functional analysis. The regulation of the expression of RIIBeta and P150 by cAMP and steroid hormones will be examined in the neuronal cell line E5. Regulatory elements in the P75 and RIIBeta genes that govern cell-specific expression will be determined. The regional, cellular and intraneuronal localization of P150 and RIIBeta in brain will be determined by immunocytochemistry in combination with light and electron microscopy. The RIIBeta binding domain of P75 will be used to explore the intracellular localization of RII subunits in nonneuronal cells. We recently identified Caenorhabditis elegans as an excellent system for studying (a) the regulation of R and C (catalytic subunit) expression and (b) the functions of cA-PKs in development and differentiation. We have cloned and characterized the C. elegans Rce, Cce and metallothionein (MT) genes and flanking regions. We will now investigate temporal, cell-specific and developmentally-controlled aspects of Rce and Cce promoter activation (gene expression) by using their promoter/enhancer regions to drive expression of the Beta-galactosidase reporter gene in developing, transgenic C. elegans. Finally, we will target the overexpression of the Cce subunit of cA-PK to intestinal cells via the gut-specific MT promoter/enhancer. High level gene expression will be obtained by adding cadmium to the medium. cDNAs corresponding to cell-specific genes regulated by cA-PK will be cloned and characterized.
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