The proposed research will examine various aspects of the posttranscriptional regulation by Ca2+ or PRL gene expression in the GH3 cell. The basic experimental paradigm for the proposed research is the culture of GH3 cells in a chemically-defined, low Ca2+, serum-free medium (SFM) with or without 0.5 mM CaCl2, which results in low and high levels, respectively, of cytoplasmic PRL mRNA. The effects of Ca2+ on the levels of spliced and unspliced PRL mRNA, and on the rats of transcription from the endogenous PRL gene, and from several PRL gene constructs in transient transfection assays. These experiments will determine whether the posttranscriptional effects of Ca2+ on PRL mRNA occur in the nucleus, and whether Ca2+ influences the rate of splicing of the PRL precursor mRNA. The effects of Ca2+ on cytoplasmic PRL mRNA degradation will be assessed by 3H-uridine pulse-chase experiments. The RNA sequence which confers the highly-specific effect of Ca2+ on PRL mRNA, referred to as the Ca2+-dependent RNA regulatory element (CaRRE), will be searched for by transient transfection assays of PRL-globin fusion gene constructs. Preliminary studies indicate that Ca2+ induces constructs containing the 5' end of the PRL gene, and thus, the proposed studies will concentrate on searching for the CaRRe within this region of the corresponding mRNA. The RNA gel shift and UV crosslinking approaches will be used to search for either nuclear or cytosolic factors which bind specifically to the CaRRE. The effects of Ca2+ on possible modifications of this factor will then be examined. Finally, the role of the ubiquitous Ca2+- binding protein, calmodulin, in the Ca2+ regulation of the PRL gene expression will be examined by establishing permanent transfected cell lines which harbor an inducible calmodulin gene in the """"""""sense"""""""" and """"""""antisense"""""""" orientations. These experiments will allow us to examine the effects, if any, of the overproduction and underproduction of calmodulin on the ability of Ca2+ to stimulate PRL gene expression.
