N-linked glycosylation is essential for the functional expression of the recombinant P2X2 receptor

被引:56
作者
Torres, GE [1 ]
Egan, TM [1 ]
Voigt, MM [1 ]
机构
[1] St Louis Univ, Ctr Hlth Sci, Dept Pharmacol & Physiol Sci, St Louis, MO 63104 USA
关键词
D O I
10.1021/bi981209g
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
P2X receptors are integral membrane proteins that belong to the growing family of transmitter-gated ion channels. The extracellular domain of these receptors contains several consensus sequences for N-Linked glycosylation that may contribute to the functional expression of the channel. We have previously reported the extracellular orientation of asparagine residues 182, 239, and 298 of the P2X(2) receptor subunit by showing that the protein is glycosylated at each site [Torres, G. E., et al. (1998) FEES Lett. 425, 19-23 (1)]. In this study, we focused on the consequences of removing N-linked glycosylation from the P2X(2) receptor by using two different approaches, tunicamycin treatment or site-directed mutagenesis. HEK-293 cells stably transfected with the P2X(2) receptor subunit showed little or no response to ATP after tunicamycin treatment. In addition, loss of function was observed with the elimination of all three N-Linked glycosylation sites from P2X(2). Cell surface labeling with biotin or indirect immunofluorescence revealed that the expression of the nonglycosylated receptors produced by either tunicamycin or site-directed mutagenesis is greatly reduced at the cell surface, indicating that the nonglycosylated P2X(2) receptors are retained inside the cell. These data provide the first direct evidence for a critical role of N-linked glycosylation in the cell surface expression of a P2X receptor subunit.
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页码:14845 / 14851
页数:7
相关论文
共 32 条
  • [1] NEW STRUCTURAL MOTIF FOR LIGAND-GATED ION CHANNELS DEFINED BY AN IONOTROPIC ATP RECEPTOR
    BRAKE, AJ
    WAGENBACH, MJ
    JULIUS, D
    [J]. NATURE, 1994, 371 (6497) : 519 - 523
  • [2] An antagonist-insensitive P-2X receptor expressed in epithelia and brain
    Buell, G
    Lewis, C
    Collo, G
    North, RA
    Surprenant, A
    [J]. EMBO JOURNAL, 1996, 15 (01) : 55 - 62
  • [3] CANNESA CM, 1994, AM J PHYSIOL, V267, P1682
  • [4] CHEN C, 1995, NATURE, V367, P428
  • [5] Chen DN, 1998, J NEUROCHEM, V70, P349
  • [6] Collo G, 1996, J NEUROSCI, V16, P2495
  • [7] Egan TM, 1998, J NEUROSCI, V18, P2350
  • [8] N-glycosylation is not a prerequisite for glutamate receptor function but is essential for lectin modulation
    Everts, I
    Villmann, C
    Hollmann, M
    [J]. MOLECULAR PHARMACOLOGY, 1997, 52 (05) : 861 - 873
  • [9] FREDHOLM BB, 1994, PHARMACOL REV, V46, P143
  • [10] Characterization of recombinant human P2X(4) receptor reveals pharmacological differences to the rat homologue
    GarciaGuzman, M
    Soto, F
    GomezHernandez, JM
    Lund, PE
    Stuhmer, W
    [J]. MOLECULAR PHARMACOLOGY, 1997, 51 (01) : 109 - 118