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Identification of a molecular target of psychosine and its role in globoid cell formation.

Im DS, Heise CE, Nguyen T, O'Dowd BF, Lynch KR - J. Cell Biol. (2001)

Bottom Line: The glycosphingolipid, psychosine (d-galactosyl-beta-1,1' sphingosine), accumulates to micromolar levels in GLD patients who lack the degradative enzyme galactosyl ceramidase.Here we document that an orphan G protein-coupled receptor, T cell death-associated gene 8, is a specific psychosine receptor.Treatment of cultured cells expressing this receptor with psychosine or structurally related glycosphingolipids results in the formation of globoid, multinuclear cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA.

ABSTRACT
Globoid cell leukodystrophy (GLD) is characterized histopathologically by apoptosis of oligodendrocytes, progressive demyelination, and the existence of large, multinuclear (globoid) cells derived from perivascular microglia. The glycosphingolipid, psychosine (d-galactosyl-beta-1,1' sphingosine), accumulates to micromolar levels in GLD patients who lack the degradative enzyme galactosyl ceramidase. Here we document that an orphan G protein-coupled receptor, T cell death-associated gene 8, is a specific psychosine receptor. Treatment of cultured cells expressing this receptor with psychosine or structurally related glycosphingolipids results in the formation of globoid, multinuclear cells. Our discovery of a molecular target for psychosine suggests a mechanism for the globoid cell histology characteristic of GLD, provides a tool with which to explore the disjunction of mitosis and cytokinesis in cell cultures, and provides a platform for developing a medicinal chemistry for psychosine.

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Inhibition of forskolin-evoked cAMP accumulation in TDAG8 DNA-transfected RH7777 cells. Clonal populations of RH7777 cells transfected with the TDAG8 receptor DNA (B and C) or plasmid vector (A) were treated with forskolin and isobutylmethylxanthine and challenged with varying concentrations of PSY. After 15 min, cells were solubilized and the cAMP content was determined. The cAMP content per well in the absence of PSY was 115.3 ± 15.8 pmol. (A) Lack of response in mock-transfected RH7777 cells treated with 10 μM PSY. (B) Dose–response curve of PSY for inhibition of cAMP accumulation (open circles, PTX [100 ng/ml for 24 h] treated; filled circles, PTX untreated). (C) Activity of various sphingoid-based lipids (10 μM) in this assay.
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Figure 1: Inhibition of forskolin-evoked cAMP accumulation in TDAG8 DNA-transfected RH7777 cells. Clonal populations of RH7777 cells transfected with the TDAG8 receptor DNA (B and C) or plasmid vector (A) were treated with forskolin and isobutylmethylxanthine and challenged with varying concentrations of PSY. After 15 min, cells were solubilized and the cAMP content was determined. The cAMP content per well in the absence of PSY was 115.3 ± 15.8 pmol. (A) Lack of response in mock-transfected RH7777 cells treated with 10 μM PSY. (B) Dose–response curve of PSY for inhibition of cAMP accumulation (open circles, PTX [100 ng/ml for 24 h] treated; filled circles, PTX untreated). (C) Activity of various sphingoid-based lipids (10 μM) in this assay.

Mentions: When expressed in RH7777 hepatoma cells, human TDAG8 mediated PSY-induced inhibition of forskolin-driven cAMP rise in a concentration-dependent manner (EC50 = 3.4 μM) (Fig. 1A and Fig. B). This response was evoked also by structurally related lysolipids, e.g., d-glucosyl-β-1,1′ sphingosine (GlcPSY), LacPSY, and lysosulfatide, but not by N-acetyl PSY, sphingosine 1-phosphate, lysophosphatidic acid, ceramide 1-phosphate, or lysophosphatidylcholine (Fig. 1 C). Similar results were found using the orthologous mouse TDAG8 DNA (data not shown). The PSY response was not blocked by pretreatment of RH7777 cultures with pertussis toxin (PTX; 100 ng/ml for 24 h), suggesting the involvement of PTX-insensitive G proteins, perhaps Gαz (Kozasa and Gilman 1995). SPC also was active in this assay, but this response, which was PTX sensitive (not shown), probably proceeds through an endogenous receptor in RH7777 cells (Im et al. 2000a). This suspicion was confirmed when SPC failed in further assays (see below). The ability of PSY to activate TDAG8 was confirmed by this lipid evoking Ca2+ transients in TDAG8/HEK293 cells (Fig. 2) and the PSY-driven internalization of a TDAG8/GFP fusion protein in HEK293T cells (Fig. 3). Both actions were mimicked by related lysoglycolipids (e.g., GlcPSY and lysosulfatide), but not by SPC or N-acetyl PSY at concentrations up to 10 μM (data not shown).


Identification of a molecular target of psychosine and its role in globoid cell formation.

Im DS, Heise CE, Nguyen T, O'Dowd BF, Lynch KR - J. Cell Biol. (2001)

Inhibition of forskolin-evoked cAMP accumulation in TDAG8 DNA-transfected RH7777 cells. Clonal populations of RH7777 cells transfected with the TDAG8 receptor DNA (B and C) or plasmid vector (A) were treated with forskolin and isobutylmethylxanthine and challenged with varying concentrations of PSY. After 15 min, cells were solubilized and the cAMP content was determined. The cAMP content per well in the absence of PSY was 115.3 ± 15.8 pmol. (A) Lack of response in mock-transfected RH7777 cells treated with 10 μM PSY. (B) Dose–response curve of PSY for inhibition of cAMP accumulation (open circles, PTX [100 ng/ml for 24 h] treated; filled circles, PTX untreated). (C) Activity of various sphingoid-based lipids (10 μM) in this assay.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169470&req=5

Figure 1: Inhibition of forskolin-evoked cAMP accumulation in TDAG8 DNA-transfected RH7777 cells. Clonal populations of RH7777 cells transfected with the TDAG8 receptor DNA (B and C) or plasmid vector (A) were treated with forskolin and isobutylmethylxanthine and challenged with varying concentrations of PSY. After 15 min, cells were solubilized and the cAMP content was determined. The cAMP content per well in the absence of PSY was 115.3 ± 15.8 pmol. (A) Lack of response in mock-transfected RH7777 cells treated with 10 μM PSY. (B) Dose–response curve of PSY for inhibition of cAMP accumulation (open circles, PTX [100 ng/ml for 24 h] treated; filled circles, PTX untreated). (C) Activity of various sphingoid-based lipids (10 μM) in this assay.
Mentions: When expressed in RH7777 hepatoma cells, human TDAG8 mediated PSY-induced inhibition of forskolin-driven cAMP rise in a concentration-dependent manner (EC50 = 3.4 μM) (Fig. 1A and Fig. B). This response was evoked also by structurally related lysolipids, e.g., d-glucosyl-β-1,1′ sphingosine (GlcPSY), LacPSY, and lysosulfatide, but not by N-acetyl PSY, sphingosine 1-phosphate, lysophosphatidic acid, ceramide 1-phosphate, or lysophosphatidylcholine (Fig. 1 C). Similar results were found using the orthologous mouse TDAG8 DNA (data not shown). The PSY response was not blocked by pretreatment of RH7777 cultures with pertussis toxin (PTX; 100 ng/ml for 24 h), suggesting the involvement of PTX-insensitive G proteins, perhaps Gαz (Kozasa and Gilman 1995). SPC also was active in this assay, but this response, which was PTX sensitive (not shown), probably proceeds through an endogenous receptor in RH7777 cells (Im et al. 2000a). This suspicion was confirmed when SPC failed in further assays (see below). The ability of PSY to activate TDAG8 was confirmed by this lipid evoking Ca2+ transients in TDAG8/HEK293 cells (Fig. 2) and the PSY-driven internalization of a TDAG8/GFP fusion protein in HEK293T cells (Fig. 3). Both actions were mimicked by related lysoglycolipids (e.g., GlcPSY and lysosulfatide), but not by SPC or N-acetyl PSY at concentrations up to 10 μM (data not shown).

Bottom Line: The glycosphingolipid, psychosine (d-galactosyl-beta-1,1' sphingosine), accumulates to micromolar levels in GLD patients who lack the degradative enzyme galactosyl ceramidase.Here we document that an orphan G protein-coupled receptor, T cell death-associated gene 8, is a specific psychosine receptor.Treatment of cultured cells expressing this receptor with psychosine or structurally related glycosphingolipids results in the formation of globoid, multinuclear cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA.

ABSTRACT
Globoid cell leukodystrophy (GLD) is characterized histopathologically by apoptosis of oligodendrocytes, progressive demyelination, and the existence of large, multinuclear (globoid) cells derived from perivascular microglia. The glycosphingolipid, psychosine (d-galactosyl-beta-1,1' sphingosine), accumulates to micromolar levels in GLD patients who lack the degradative enzyme galactosyl ceramidase. Here we document that an orphan G protein-coupled receptor, T cell death-associated gene 8, is a specific psychosine receptor. Treatment of cultured cells expressing this receptor with psychosine or structurally related glycosphingolipids results in the formation of globoid, multinuclear cells. Our discovery of a molecular target for psychosine suggests a mechanism for the globoid cell histology characteristic of GLD, provides a tool with which to explore the disjunction of mitosis and cytokinesis in cell cultures, and provides a platform for developing a medicinal chemistry for psychosine.

Show MeSH
Related in: MedlinePlus