Glycine transporter isoforms show differential subcellular localization in PC12 cells

Geerlings, Arjan; Núñez, Enrique; Rodenstein, Lara; López-Corcuera, Beatriz; Aragón, Carmen

doi:10.1046/j.1471-4159.2002.00930.x

Cited by 18 publications

(19 citation statements)

References 36 publications

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“…After truncation, intact GlyT2 could be replenished from a large intracellular pool of intact transporters, keeping the amount of surface GlyT2 more or less constant. This result is in good agreement with a previous report by Geerlings et al . (2002) who found only 5–10% of total GlyT2 N‐terminal immunoreactivity to be localized on the cell surface.…”

Section: Resultssupporting

confidence: 94%

Calpain‐mediated proteolytic cleavage of the neuronal glycine transporter, GlyT2

Baliova

Betz

Jursky

2003

Journal of Neurochemistry

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The glycine transporter 2 (GlyT2) belongs to the family of Na + /CL --dependent plasma membrane transporters and is localized on the presynaptic terminals of glycinergic neurons. GlyT2 differs from other family members by its extended N-terminal cytoplasmic region. We report that activation of a Ca 2+ -dependent protease, most likely calpain, in spinal cord synaptosomes or cultured spinal cord neurons, results in partial proteolysis of GlyT2. Regions sensitive to calpain cleavage in vivo are located in the N-terminal and, to a lesser extent, C-terminal regions of the transporter protein. Incubation of a GlyT2 N-terminal fusion protein with spinal cord extract in the presence of calcium followed by protein sequence analysis localized the major N-terminal cleavage site after methionine 156, with a second cleavage site being situated after glycine 164. Interestingly, the size of the N-terminally truncated GlyT2 protein (70 kDa) is similar to that of most other transporter family members, and truncated GlyT2 displayed full transport activity upon expression in HEK293 cells. Our data suggest that Ca 2+ -triggered proteolysis may contribute to the regulation of GlyT2 trafficking and/or function in the neuronal plasma membrane.

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Section: Resultssupporting

confidence: 94%

Calpain‐mediated proteolytic cleavage of the neuronal glycine transporter, GlyT2

Baliova

Betz

Jursky

2003

Journal of Neurochemistry

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“…As the antibody used in our study would recognize all currently known splice variants of NAT, the nuclear localization might be attributable to aberrant splice variant expression in phaeochromocytoma. Whereas this remains to be examined, differential subcellular localization has been demonstrated for isoforms of the glycine transporter in PC12 cells (Geerlings et al 2002) and the expression of c-terminal splice variants of human NAT has been shown to cause intracellular retention of the variant protein and loss of function (Bauman and Blakely 2002). The clinical significance of the differing patterns of NAT expression between normal tissue and phaeochromocytoma is unknown but may result in abnormal or non-functional expression of the transporter.…”

Section: Discussionmentioning

confidence: 99%

Expression of the noradrenaline transporter and phenylethanolamine N-methyltransferase in normal human adrenal gland and phaeochromocytoma

et al. 2005

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Expression of the noradrenaline transporter (NAT) was examined in normal human adrenal medulla and phaeochromocytoma by using immunohistochemistry and confocal microscopy. The enzymes tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) were used as catecholamine biosynthetic markers and chromogranin A (CGA) as a marker for secretory granules. Catecholamine content was measured by using high performance liquid chromatography (HPLC). In normal human adrenal medulla (n=5), all chromaffin cells demonstrated strong TH, PNMT and NAT immunoreactivity. NAT was co-localized with PNMT and was located within the cytoplasm with a punctate appearance. Human phaeochromocytomas demonstrated strong TH expression (n=20 samples tested) but variable NAT and PNMT expression (n=24). NAT immunoreactivity ranged from absent (n=3) to weak (n=10) and strong (n=11) and, in some cases, occupied an apparent nuclear location. Unlike the expression seen in normal human adrenal medullary tissue, NAT expression was not consistently co-localized with PNMT. PNMT also showed highly variable expression that was poorly correlated with tumour adrenaline content. Immunoreactivity for CGA was colocalized with NAT within the cytoplasm of normal human chromaffin cells (n=4). This co-localization was not consistent in phaeochromocytoma tumour cells (n=7). The altered pattern of expression for both NAT and PNMT in phaeochromocytoma indicates a significant disruption in the regulation and possibly in the function of these proteins in adrenal medullary tumours.

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“…The GFP-GlyT2 plasmid was constructed and characterized as described previously (30,31). The RFP and HA tags were fused in frame with GlyT2 at the N terminus, and they did not interfere either with the uptake capacity in [ 3 H]glycine uptake assays or its expression at the plasma membrane as assessed by biotinylation (see Fig.…”

Section: Methodsmentioning

confidence: 99%

Molecular Basis of the Dominant Negative Effect of a Glycine Transporter 2 Mutation Associated with Hyperekplexia

Arribas-González

Juan-Sanz

Aragón

et al. 2015

Journal of Biological Chemistry

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Background: Hyperekplexia is caused by defective glycinergic neurotransmission. Results: A dominant negative glycine transporter-2 mutant is trapped in a calnexin-bound state and retains wild type GlyT2 in the endoplasmic reticulum. Conclusion: Chemical chaperones rescue the folding defect of the mutant and overcome its dominant negative effect. Significance: This opens the way to revert the dominant negative effect exerted by the mutant associated with hyperekplexia in neurons.

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Glycine transporter isoforms show differential subcellular localization in PC12 cells

Cited by 18 publications

References 36 publications

Calpain‐mediated proteolytic cleavage of the neuronal glycine transporter, GlyT2

Calpain‐mediated proteolytic cleavage of the neuronal glycine transporter, GlyT2

Expression of the noradrenaline transporter and phenylethanolamine N-methyltransferase in normal human adrenal gland and phaeochromocytoma

Molecular Basis of the Dominant Negative Effect of a Glycine Transporter 2 Mutation Associated with Hyperekplexia

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