CELLULAR REGULATION OF TYROSINE HYDROXYLASE

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DESCRIPTION: (from applicant's abstract): The broad goals of this project are to understand the molecular mechanisms whereby catecholaminergic (CA)cells replenish those stores of CA lost via secretion and, ultimately, to discover the governances thereof by which homeostasis or plasticity ensues. These CA stores can be rapidly repleted by an acceleration of de novo CA biosynthesis resulting from an increase in the activity of tyrosine hydroxylase (TH) in association with its phosphorylation at three different sites (Ser19, Ser3l, Ser4O). Based upon the increases in TH activity produced in vitro by selective phosphorylation of Ser3l or Ser4O, but not of Serl 9, an analogous relative participation of sites in situ has been inferred. This inference, however, makes the as yet untested assumption that interactions among the multiple sites do not occur when they are phosphorylated in concert. The potential presence, nature, and influence of such interactions forms a central hypothesis to be examined and tested in the project's specific aims: (1) Biochemical studies of multiple, site-specific TH phosphorylations using purified protein kinases (PI(s) and TH (native and recombinant isoforms). The kinetics of phosphorylation will be determined in TH isoforms and Ser mutants (plus or minus P04, plus or minus catecholate/iron) and dissociation of ternary catecholate complexes by phosphorylation will be determined for each site, separately and in combination. TH activity will be measured under ranges of conditions selected for the examination of site-specific modulation of TH activity and for evaluation of ternary complex dissociation as the underlying mechanism. (2) Cellular and molecular studies to identify PKs which mediate Serl 9 and Ser4O phosphorylation in situ and to distinguish the contributions of ERK1 vs ERK2 to Ser3l phosphorylation. Experiments will employ cell-permeable, peptide-based and non-peptide PK inhibitors (PDO98059 and SB203580) in isolated CA cells and antisense oligonucleotides in intact rat brain. PC1 2 cells expressing specific PKs or dominant-negative signaling macromolecules and PKA-deficient PC 12 cells will also be used. (3) Neurochemical studies of the involvement of site-specific TH phosphorylation in situ in CA biosynthesis rates. Striatal slices, CA cells expressing recombinant dopamine (DA) receptors, and AtT-20 cells expressing wild type TH and Ser mutants will be used to study the effects of depolarizing and/or autoreceptor agents concurrently upon CA biosynthesis and the phosphorylation states of Serl 9, Ser3l, and Ser4O, as determined immunochemically using siteand phospho-specific antibodies. (4) Combined neuroanatomical/chemical studies of multiple- site TH phosphorylation in vivo in regions of rat brain. The effects of haloperidol on Ser19, Ser3l, and Ser4O phosphorylation will be studied by immunohistochemical and blot immunolabeling procedures using site- and phosphorylation state-specific antibodies.