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  • 2000-2004  (3)
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  • 1
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Science Ltd
    Journal of neurochemistry 76 (2001), S. 0 
    ISSN: 1471-4159
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Medizin
    Notizen: Metabotropic glutamate receptors (mGlu receptors) are coupled to G-protein second messenger pathways and modulate glutamate neurotransmission in the brain, where they are targeted to specific synaptic locations. Very recently, we identified tubulin as an interacting partner of the mGlu1α receptor in rat brain. Using BHK-570 cells permanently expressing the receptor we have shown that this interaction occurs predominantly with soluble tubulin, following its translocation to the plasma membrane. In addition, treatment of the cells with the agonist quisqualic acid induce tubulin depolimerization and its translocation to the plasma membrane. Immunofluorescence detection of both the receptor and tubulin in agonist-treated cells reveals a disruption of the microtubule network and an increased clustering of the receptor. Collectively these data demonstrate that the mGlu1α receptor interacts with soluble tubulin and that this association can take place at the plasma membrane.
    Materialart: Digitale Medien
    Bibliothek Standort Signatur Band/Heft/Jahr Verfügbarkeit
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  • 2
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Science Ltd
    Journal of neurochemistry 79 (2001), S. 0 
    ISSN: 1471-4159
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Medizin
    Notizen: Recent evidence suggests that specialized microdomains, called lipid rafts, exist within plasma membranes. These domains are enriched in cholesterol and sphingolipids and are resistant to non-ionic detergent-extraction at 4°C. They contain specific populations of membrane proteins, and can change their size and composition in response to cellular signals, resulting in activation of signalling cascades. Here, we demonstrate that both the metabotropic γ-aminobutyric acid receptor B (GABAB receptor) and the metabotropic glutamate receptor-1 from rat cerebellum are insoluble in the non-ionic detergent Triton X-100. However, only the GABAB receptor associates with raft fractions isolated from rat brain by sucrose gradient centrifugation. Moreover, increasing the stringency of isolation by decreasing the protein : detergent ratio caused an enrichment of the GABAB receptor in raft fractions. In contrast, depletion of cholesterol from cerebellar membranes by either saponin or methyl-β-cyclodextrin treatment, which solubilize known raft markers, also increased the solubility of the GABAB receptor. These properties are all consistent with an association of the GABAB receptor with lipid raft microdomains.
    Materialart: Digitale Medien
    Bibliothek Standort Signatur Band/Heft/Jahr Verfügbarkeit
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  • 3
    ISSN: 1471-4159
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Medizin
    Notizen: A Chinese hamster ovary cell line has been established which secretes the N-terminal domain of human mGlu1 receptor. The secreted protein has been modified to contain a C-terminal hexa-histidine tag and can be purified by metal-chelate chromatography to yield a protein with an apparent molecular weight of 130 kDa. Following treatment with dithiothreitol the apparent molecular weight is reduced to 75 kDa showing that the protein is a disulphide-bonded dimer. N-terminal protein sequencing of both the reduced and unreduced forms of the protein yielded identical sequences, confirming that they were derived from the same protein, and identifying the site of signal-peptide cleavage of the receptor as residue 32 in the predicted amino acid sequence. Endoglycosidase treatment of the secreted and intracellular forms of the protein showed that the latter was present as an endoglycosidase H-sensitive dimer, indicating that dimerization is taking place in the endoplasmic reticulum. Characterization of the binding of [3H]quisqualic acid showed that the protein was secreted at levels of up to 2.4 pmol/mL and the secreted protein has a Kd of 5.6 ± 1.8 nm compared with 10 ± 1 nm for baby hamster kidney (BHK)-mGlu1α receptor-expressing cell membranes. The secreted protein maintained a pharmacological profile similar to that of the native receptor and the binding of glutamate and quisqualate were unaffected by changes in Ca2+ concentration.
    Materialart: Digitale Medien
    Bibliothek Standort Signatur Band/Heft/Jahr Verfügbarkeit
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