Abstract
Syntaxin 1C is an alternative splice variant lacking the transmembrane domain of HPC-1/syntaxin 1A. We found previously that syntaxin 1C is expressed as a soluble protein in human astroglioma (T98G) cells, and syntaxin 1C expression is enhanced by stimulation with phorbol 12-myristate 13-acetate (PMA). However, the physiological function of syntaxin 1C is not known. In this study, we examined the relationship between syntaxin 1C and glucose transport. First, we discovered that glucose transporter-1 (GLUT-1) was the primary isoform in T98G cells. Second, we demonstrated that glucose uptake in T98G cells was suppressed following an increase in endogenous syntaxin 1C after stimulation with PMA, which did not alter the expression levels of other plasma membrane syntaxins. We further examined glucose uptake and intracellular localization of GLUT-1 in cells that overexpressed exogenous syntaxin 1C; glucose uptake via GLUT-1 was inhibited without affecting sodium-dependent glucose transport. The value of Vmax for the dose-dependent uptake of glucose was reduced in syntaxin 1C-expressing cells, whereas there was no change in Km. Immunofluorescence studies revealed a reduction in the amount of GLUT-1 in the plasma membrane in cells that expressed syntaxin 1C. Based on these results, we postulate that syntaxin 1C regulates glucose transport in astroglioma cells by changing the intracellular trafficking of GLUT-1. This is the first report to indicate that a syntaxin isoform that lacks a transmembrane domain can regulate the intracellular transport of a plasma membrane protein.
Highlights
Found previously that syntaxin 1C is expressed as a sol- attachment protein receptor) constitutes a widely accepted uble protein in human astroglioma (T98G) cells, and model in which dynamic interactions among proteins within syntaxin 1C expression is enhanced by stimulation with the acceptor (t-SNARE: syntaxin and SNAP-25) and donor phorbol 12-myristate 13-acetate (PMA)
We demonstrated that glucose transport and the amount of glucose transporters (GLUTs)-1 in the plasma membrane were suppressed in astroglioma cells by stimulation with phorbol 12-myristate 13acetate (PMA)
It is likely that the suppression of glucose transport by PMA was caused by a decrease in the number of GLUT-1 molecules that is present in the plasma membrane, and that this was caused by an increase in the syntaxin 1C expression because: 1) PMA increased the endogenous expression of syntaxin 1C without changing the total expression of syntaxin 2– 4, or GLUT-1 in astroglioma cells and 2) overexpression of exogenous syntaxin 1C caused the similar phenomenon to that by PMA
Summary
Reagents—All tissue culture reagents were purchased from Invitrogen, Life Technologies (Carlsbad, CA) with the exception of fetal calf serum (FCS), which was purchased from Sigma. The cDNA template (5 ng) that was synthesized from total RNA in cells was used for semiquantitative PCR, with primer pairs that were specific for hGLUT-1, hGLUT-2, hGLUT-3, and hGLUT-4 Measurement of 2-DG uptake in T98G cells, cultured with low and high concentrations of glucose, is shown in the white and black bar on the left, respectively. Measurement of 2-DG uptake value in T98G cells, cultured with low and high concentrations of insulin, is shown in the white and black bar on the right, respectively. To study basal glucose uptake via sodium-dependent glucose transporters (SGLTs), cells were treated with Naϩ-free HBSS buffer containing 0.03 g/100 ml bovine serum albumin, 138 mM N-methyl-D-(Ϫ)glucamine (NMDG), 5.6 mM KC1, 0.34 mM KHPO4, 0.44 mM KH2PO4, 1.27 mM CaCl2, and 20 mM HEPES (pH 7.4). A p value of Յ 0.05 was considered to be statistically significant
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