Disease relevance of Gad2

• In this study we have investigated the targeting signal of the 65-kD isoform of glutamic acid decarboxylase (GAD65), a major autoantigen in two autoimmune diseases: Stiff-Man syndrome and insulin-dependent diabetes mellitus [1].
• GAD-65 is a dominant autoantigen in stiff-man syndrome and insulin-dependent diabetes mellitus [2].
• These findings suggest that increased GABA production in lateral nucleus of the hypothalamus induced by GAD65 gene transfer may reduce weight gain through reduced feeding [3].
• Defective herpes simplex virus (HSV) vectors containing glutamic acid decarboxylase (GAD) cDNAs, either GAD65 or GAD67, were used to examine GAD function and GABA synthesis in rat cortical astrocytes, CNS cells that do not endogenously synthesize GABA [4].
• In addition, hypoxia led to an increase in GAD65 and GAD67 protein levels and GAD enzymatic activity [5].

High impact information on Gad2

• These forms, GAD65 and GAD67, derive from two genes [6].
• Deletion of the first 23 amino acids at the NH2 terminus of the GAD65 30/45A mutant also does not affect the hydrophobicity and membrane anchoring of the GAD65 protein [7].
• The results suggest that amino acids 24-31 are required for hydrophobic modification and/or targeting of GAD65 to membrane compartments, whereas palmitoylation of Cys 30 and Cys 45 may rather serve to orient or fold the protein at synaptic vesicle membranes [7].
• Amino acid residues 24-31 but not palmitoylation of cysteines 30 and 45 are required for membrane anchoring of glutamic acid decarboxylase, GAD65 [7].
• Mutation of Cys 30 and Cys 45 to Ala results in a loss of palmitoylation but does not significantly alter membrane association of GAD65 in COS-7 cells [7].

Biological context of Gad2

• In situ hybridization of fluorescently labeled GAD probes to human chromosomes localizes the human GAD65 gene to chromosome 10p11.23 and the human GAD67 gene to chromosome 2q31 [8].
• Phosphorylation status does not affect membrane anchoring of GAD65, nor its Km or Vmax for glutamate [9].
• GAD65 mRNA upregulation was only observed in the BST and dentate gyrus [10].
• Moreover, 87 % of beta cells that have a high insulin secretory rate express GAD65 [11].
• The findings suggest that none of the single antibody specificities are as sensitive as islet cell antibodies, but that a combination of GAD65 antibodies and antibodies to 37,000/40,000 M(r) islet tryptic fragments has the potential to identify more than 90% of future cases of IDDM.(ABSTRACT TRUNCATED AT 250 WORDS)[12]

Anatomical context of Gad2

• The predominant form of GAD in pancreatic beta-cells, GAD65, is synthesized as a soluble hydrophilic molecule, which is modified to become firmly membrane anchored [13].
• However, c-fos induction was not affected in other forebrain GABAergic stress pathways, such as the lateral septum, medial preoptic area or dorsomedial hypothalamus. vSUB lesions increased GAD65 or GAD67 mRNA levels in several efferent targets, including anterior and posterior subnuclei of the bed nucleus of the stria terminalis and lateral septum [14].
• Lesions did not effect stress-induced increases in GAD65 expression in principal output nuclei of the amygdala [14].
• A signal located within amino acids 1-27 of GAD65 is required for its targeting to the Golgi complex region [1].
• In this study, the cloned genes for GAD-65 and GAD-67 were expressed separately in Chinese hamster ovary (CHO) cells and COS cells [2].

Associations of Gad2 with chemical compounds

• GAD65 and GAD67 are two isoforms of the enzyme glutamic acid decarboxylase which catalyze the production of GABA from glutamate, primarily in the brain [15].
• The NH2-terminal domain of GAD65 is the site of a two-step modification, the last of which results in a firm membrane anchoring that involves posttranslational hydroxylamine sensitive palmitoylation [13].
• GABA-C receptor subunits (rho1, rho2, rho3) and GABA synthesizing enzymes (GAD 65 and GAD 67) were shown to be expressed as assayed by RT-PCR, and GABA-C receptor stimulation by CACA obviously increased intracellular Ca2+ concentration in the anterior pituitary cells [16].
• Two isoforms of glutamic acid decarboxylase (GAD67 and GAD65) and their mRNAs were localized in the rat brain by immunohistochemistry and nonradioactive in situ hybridization methods with digoxigenin-labeled cRNA probes [17].
• CONCLUSION/INTERPRETATION: These results show that the most active beta cells, which secrete more insulin, also express GAD65 and that manipulating extracellular glucose may modify the expression of the enzyme and possibly the autoimmune attack in Type I diabetes [11].

Physical interactions of Gad2

• Here, we show that palmitoylated GAD65 colocalizes with the small GTP-binding protein Rab5a in Golgi membranes and in axons but not in dendrites [18].

Regulatory relationships of Gad2

• The results suggest that palmitoylation of GAD65 regulates the trafficking of the protein from Golgi membranes to an endosomal trafficking pathway in axons that is dependent on Rab5a and is required for the targeting of several synaptic vesicle proteins to presynaptic clusters [18].
• In summary: 1) IL-1 beta dramatically inhibits GAD-65 expression [19].
• Clones expressing GAD65 had higher enzymatic activity than those expressing GAD67 [20].

Other interactions of Gad2

• Palmitoylation controls trafficking of GAD65 from Golgi membranes to axon-specific endosomes and a Rab5a-dependent pathway to presynaptic clusters [18].
• In contrast, BDNF-up-regulated GABA release and GAD65/67 expression depended on MAPK [21].
• Expression of both GAD65 and GAD67 mRNA, measured by RNase protection assay, was also decreased by IL-1 beta and completely restored to baseline levels by NMMA [22].
• Neither the expression of preoptic GnRH mRNA nor the expression of GAD65 and of GnRH-R mRNA in both hypothalamic structures was affected by feed restriction [23].
• 3) IFN gamma at doses of 10, 100, 1000 U/ml had no effect on insulin secretion or on GAD-65 expression [19].

Analytical, diagnostic and therapeutic context of Gad2

• Here we show by immunogold electron microscopy that GAD65 is localized to the membrane of small vesicles which are identical in size to small synaptic-like microvesicles in pancreatic beta-cells [13].
• In this study we use site-directed mutagenesis to identify the first two cysteines in the NH2-terminal region, Cys 30 and Cys 45, as the sites of palmitoylation of the GAD65 molecule [7].
• Rat islets contain GAD65 and GAD67 autoantibody targets, but human islets express only GAD65, now confirmed by direct immunoprecipitation from radiolabeled rat and human islets [24].
• Quantitative immunoblotting showed that the decline in GAD activity was attributable to a 75-80% decrease in GAD67 levels, whereas the levels of GAD65 remained unchanged [25].
• Moreover, as detected by cDNA microarray analysis, PB treatment at low dose enhanced mRNA expression of glutamic acid decarboxylase (GAD65), an enzyme involved in the synthesis of gamma-aminobutyric acid (GABA), and suppressed MAP kinase p38 and other intracellular kinases gene expression [26].

References