L-Glutamate decarboxylase (GAD) is the enzyme involved in the synthesis of GABA, a major inhibitory neurotransmitter. There are two well characterized GAD isoforms in the brain, namely, GAD65 and GAD67 [(Erlander and Tobin, 1991)]. Recently, we have shown that soluble GAD (SGAD) is activated by dephosphorylation, mediated by calcineurin, and is inhibited by phosphorylation, mediated by PKA (Bao et al., 1994, 1995), whereas the membrane associated GAD (MGAD) is activated by protein phosphorylation which depends on the integrity of synaptic vesicles (Hsu et al., 1999). In addition, we have identified MGAD from synaptosomes as GAD65 (Hsu et al., 1999). Thus, GAD activity appears to be regulated also depending on the partition of GAD in soluble and membrane associated form. In addition, a detailed crystal structure of GAD is essential for understanding the regulation of GAD activity. Hence, we propose to perform the following studies: (1) To determine the three dimensional structure of GAD. This aspect of studies will be carried out in collaboration with Dr. F. Takusagawa (letter of collaboration is attached) who has recently determined the crystal structure of S- adenosylhomocysteine hydrolase (Hu et al., 1999). (2) To elucidate the mechanisms involved in anchoring GAD to synaptic vesicles with special focus on the role of heat shock cognate protein (HSC70) since our results indicate that GAD65, but not GAD67 forms complex with HSC70 (see Preliminary Results). (3) Characterization of GAD65 and GAD67 phosphorylation sites in terms of the identity of phosphoamino acids, the location (sequence), and the number of phosphorylation sites. In addition, site-directed mutagenesis will be used to determine which amino acid residues are important for phosphorylation and regulation of GAD activity. (4) To elucidate the physiological significance of GAD65 and GAD67 phosphorylation. (5) To address whether GAD65 and GAD67 activities are sensitive to intracellular GABA concentrations. The success of a large scale purification of recombinant human brain GAD65 and GAD67 and the availability of subtype specific antibodies for GAD65 and GAD67, ensure the high feasibility of this proposal.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
1R01NS037851-01A2
Application #
6199733
Study Section
Special Emphasis Panel (ZRG1-MDCN-5 (01))
Program Officer
Jacobs, Tom P
Project Start
2000-08-04
Project End
2004-07-31
Budget Start
2000-08-04
Budget End
2001-07-31
Support Year
1
Fiscal Year
2000
Total Cost
$254,625
Indirect Cost
Name
University of Kansas Lawrence
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
072933393
City
Lawrence
State
KS
Country
United States
Zip Code
66045
Leon, Rebecca; Wu, Heng; Jin, Ying et al. (2009) Protective function of taurine in glutamate-induced apoptosis in cultured neurons. J Neurosci Res 87:1185-94
Buddhala, Chandana; Hsu, Che-Chang; Wu, Jang-Yen (2009) A novel mechanism for GABA synthesis and packaging into synaptic vesicles. Neurochem Int 55:9-12
Wei, Jianning; Wu, Jang-Yen (2008) Post-translational regulation of L-glutamic acid decarboxylase in the brain. Neurochem Res 33:1459-65
Sha, Di; Jin, Ying; Wu, Heng et al. (2008) Role of mu-calpain in proteolytic cleavage of brain L-glutamic acid decarboxylase. Brain Res 1207:9-18
Jin, Ying; Wu, Heng; Cohen, Eric M et al. (2007) Genistein and daidzein induce neurotoxicity at high concentrations in primary rat neuronal cultures. J Biomed Sci 14:275-84
Wu, Heng; Jin, Ying; Buddhala, Chandana et al. (2007) Role of glutamate decarboxylase (GAD) isoform, GAD65, in GABA synthesis and transport into synaptic vesicles-Evidence from GAD65-knockout mice studies. Brain Res 1154:80-3
Wei, Jianning; Lin, Chun-Hua; Wu, Heng et al. (2006) Activity-dependent cleavage of brain glutamic acid decarboxylase 65 by calpain. J Neurochem 98:1688-95
Jin, Hong; Sha, Di; Wei, Jianning et al. (2005) Effect of apocalmodulin on recombinant human brain glutamic acid decarboxylase. J Neurochem 92:739-48
Wu, Heng; Jin, Ying; Wei, Jianning et al. (2005) Mode of action of taurine as a neuroprotector. Brain Res 1038:123-31
Sha, Di; Jin, Hong; Kopke, Richard D et al. (2004) Choline acetyltransferase: regulation and coupling with protein kinase and vesicular acetylcholine transporter on synaptic vesicles. Neurochem Res 29:199-207

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