Investigations on the physiology and molecular biology of calcium and phosphoprotein-regulated signalling pathways have been initiated, with emphasis on the calmodulin (CaM)-dependent phosphatase, calcineurin (CN), and cyclic nucleotide phosphodiesterase (PDE). Immunocytochemical studies indicate that the calmodulin-dependent isoform of brain PDE is expressed predominantly in regional output neurons, consistent with a role in integration of synaptic input. Further, chemical destruction of climbing fiber afferents to cerebellar Purkinje cells causes loss of PDE immunoreactivity suggesting a """"""""transsynaptic"""""""" model for regulation of gene expression. Current studies on the cloning of this enzyme are designed to determine the control mechanism(s) for expression. Recent studies have shown that CN is the major cytosolic CaM-binding protein (BP) in lymphocytes and that it is differentially expressed in subpopulations of lymphoid cells. To examine its regulation in the immune system (and brain), cDNAs for the catalytic subunit of this phosphatase have been cloned using expression vector immunoscreening and a novel plaque hybridization method employing biotinylated restriction fragments. Tissue- specific mRNA species are now being characterized and in vitro mutagenesis will be carried out to examine important allosteric and catalytic domains on the enzyme, some of which are suggested by clear similarities to other recently cloned phosphatases. Ongoing studies with model phosphopeptides will be used to investigate immunologically the phosphorylation of receptors and proteins involved in control of intermediary metabolism and cytoskeletal organization.
