Association of GAD-65, but not of GAD-67, with the Golgi complex of transfected Chinese hamster ovary cells mediated by the N-terminal region.

Solimena, Michele; Aggujaro, Diego; Muntzel, Christiana; Dirkx, Ronald; Butler, Margaret H.; Camilli, Pietro De; Hayday, Adrian

doi:10.1073/pnas.90.7.3073

Cited by 76 publications

(68 citation statements)

References 32 publications

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“…Surprisingly, a ,-galactosidase-GAD44 fusion protein exhibited activity equal to that of the fusion protein 0-20A (27), which includes almost the entire coding region of GAD (26). The leader peptide, which carries the signal mediating subcellular targeting of the enzyme (53), probably exerts regulatory functions. One of its primary roles could be related to the translation of GAD44 from ORF2.…”

Section: Discussionmentioning

confidence: 99%

Distinct protein forms are produced from alternatively spliced bicistronic glutamic acid decarboxylase mRNAs during development.

Szabó¹,

Katarova²,

Greenspan³

1994

Mol. Cell. Biol.

160

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It has been shown that the enzyme glutamic acid decarboxylase (GAD; EC 4.1.1.15), which catalyzes the conversion of L-glutamate to y-aminobutyric acid in the central nervous system of vertebrates, can be first detected in rodents at late embryonic stages. In contrast, we have found that the gene coding for the 67-kDa form of GAD is already transcriptionally active at embryonic day E10.5 in the mouse. In addition to the 3.5-kb adult-type mRNA, we have detected two 2-kb embryonic messages that contain alternatively spliced exons of 80 (1-80) and 86 (1-86) bp, respectively. The overlapping stop-start codon TGATG, found in the embryonic exons, converts the monocistronic adult-type transcript into a bicistronic one, coding for a 25-kDa leader peptide and a 44-kDa enzymatically active truncated GAD. A second stop codon at the 3' end of the 86-bp exon abolishes the expression of truncated GAD. The products of the two embryonic mRNAs were identified in a rabbit reticulocyte in vitro translation system, COS cells, and mouse embryos. The two GAD embryonic forms represent distinct functional domains and display characteristic developmental patterns, consistent with a possible role in the formation of the y-aminobutyric acid-ergic inhibitory synapses.

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Section: Discussionmentioning

confidence: 99%

Distinct protein forms are produced from alternatively spliced bicistronic glutamic acid decarboxylase mRNAs during development.

Szabó¹,

Katarova²,

Greenspan³

1994

Mol. Cell. Biol.

160

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“…Katarova, unpublished results). To reveal the spatial expression pattern of GAD65, we used GAD65-specific antibody N-65 (Solimena et al, 1993).…”

Section: Spatial Segregation Of Gad Forms During Lens Developmentmentioning

confidence: 99%

“…1096176, Roche Applied Sciences, Penzberg, Germany) in PBS. The sections were then incubated overnight at 4°C with rabbit anti-GAD serum #6799 (Katarova et al, 1990) at a dilution of 1:500 or rabbit anti-GAD65 serum N65 at 1:500 (a kind gift from Pietro de Camilli, Yale University; Solimena et al, 1993) in 1% bovine serum albumin (BSA, Sigma-Aldrich Co.) in PBT. Sections were washed 3 ϫ 10 min at room temperature with PBT, then incubated for 2 hr with a biotinylated anti-rabbit antibody (1: 500 in PBT, Vector Laboratories, Burlingame, CA), followed by 2 ϫ 10 min PBT, 1 ϫ 10 min PBS washes.…”

Section: Tissue Preparationmentioning

confidence: 99%

GAD isoforms exhibit distinct spatiotemporal expression patterns in the developing mouse lens: Correlation with Dlx2 and Dlx5

Kwakowsky

Schwirtlich

Zhang

et al. 2007

Developmental Dynamics

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Gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter of the adult nervous system and its biosynthetic enzyme glutamic acid decarboxylase (GAD) are abundantly expressed in the embryonic nervous system and are involved in the modulation of cell proliferation, migration, and differentiation. Here we describe for the first time the expression of GABA and embryonic and adult GAD isoforms in the developing mouse lens. We show that the GAD isoforms are sequentially induced with specific spatiotemporal profiles: GAD65 and embryonic GAD isoforms prevail in primary fibers, while GAD67 is the predominant GAD expressed in the postnatal secondary fibers. This pattern correlates well with the expression of Dlx2 and Dlx5, known as upstream regulators of GAD. GABA and GAD are most abundant at the tips of elongating fibers and are absent from organelle-free cells, suggesting their involvement is primarily in shaping of the cytoskeleton during fiber elongation stages. Developmental Dynamics 236: 3532-3544, 2007.

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“…(23) cell lines served as targets for the CTL assay. A line that expressed GAD65 (M12.C3.GAD) was produced by transfecting M12.C3 cells using electroporation (Bio-Rad, Hercules, CA) and the vector pRC/RSV (Invitrogen, Carlsbad, CA) containing the full-length rat GAD65 gene (obtained as a gift from M. Solimena, Yale University, New Haven, CT) (24). A line transfected with the pRC/RSV vector alone (M12.C3.RSV) was used as a control.…”

Section: Cd8mentioning

confidence: 99%

MHC Class I-Restricted Determinants on the Glutamic Acid Decarboxylase 65 Molecule Induce Spontaneous CTL Activity

Quinn

McInerney

Sercarz

2001

The Journal of Immunology

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CD4+ T cell responses to glutamic acid decarboxylase (GAD65) spontaneously arise in nonobese diabetic (NOD) mice before the onset of insulin-dependent diabetes mellitus (IDDM) and may be critical to the pathogenic process. However, since both CD4+ and CD8+ T cells are involved in autoimmune diabetes, we sought to determine whether GAD65-specific CD8+ T cells were also present in prediabetic NOD mice and contribute to IDDM. To refine the analysis, putative Kd-binding determinants that were proximal to previously described dominant Th determinants (206–220 and 524–543) were examined for their ability to elicit cytolytic activity in young NOD mice. Naive NOD spleen cells stimulated with GAD65 peptides 206–214 (p206) and 546–554 (p546) produced IFN-γ and showed Ag-specific CTL responses against targets pulsed with homologous peptide. Conversely, several GAD peptides distal to the Th determinants, and control Kd-binding peptides did not induce similar responses. Spontaneous CTL responses to p206 and p546 were mediated by CD8+ T cells that are capable of lysing GAD65-expressing target cells, and p546-specific T cells transferred insulitis to NOD.scid mice. Young NOD mice pretreated with p206 and p546 showed reduced CTL responses to homologous peptides and a delay in the onset of IDDM. Thus, MHC class I-restricted responses to GAD65 may provide an inflammatory focus for the generation of islet-specific pathogenesis and β cell destruction. This report reveals a potential therapeutic role for MHC class I-restricted peptides in treating autoimmune disease and revisits the notion that the CD4- and CD8-inducing determinants on some molecules may benefit from a proximal relationship.

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Association of GAD-65, but not of GAD-67, with the Golgi complex of transfected Chinese hamster ovary cells mediated by the N-terminal region.

Cited by 76 publications

References 32 publications

Distinct protein forms are produced from alternatively spliced bicistronic glutamic acid decarboxylase mRNAs during development.

Distinct protein forms are produced from alternatively spliced bicistronic glutamic acid decarboxylase mRNAs during development.

GAD isoforms exhibit distinct spatiotemporal expression patterns in the developing mouse lens: Correlation with Dlx2 and Dlx5

MHC Class I-Restricted Determinants on the Glutamic Acid Decarboxylase 65 Molecule Induce Spontaneous CTL Activity

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