Agonist-induced internalization and trafficking of cannabinoid CB1 receptors in hippocampal neurons

Angela A. Coutts, Sharon Anavi-Goffer, Ruth A. Ross, David J. MacEwan, Ken Mackie, Roger G. Pertwee, Andrew J. Irving

    Research output: Contribution to journalArticle

    129 Citations (Scopus)

    Abstract

    Agonist-induced internalization of G-protein-coupled receptors is an important mechanism for regulating receptor abundance and availability at the plasma membrane. In this study we have used immunolabeling techniques and confocal microscopy to investigate agonist-induced internalization and trafficking of CB1receptors in rat cultured hippocampal neurons. The levels of cell surface CB1 receptor immunoreactivity associated with presynaptic GABAergic terminals decreased markedly (by up to 84%) after exposure to the cannabinoid agonist (+)-WIN55212, in a concentration-dependent (0.1–1 µm) and stereoselective manner. Inhibition was maximal at 16 hr and abolished in the presence of SR141716A, a selective CB1 receptor antagonist. Methanandamide (an analog of an endogenous cannabinoid, anandamide) also reduced cell surface labeling (by 43% at 1 µm). Differential labeling of cell surface and intracellular pools of receptor demonstrated that the reduction in cell surface immunoreactivity reflects agonist-induced internalization and suggests that the internalized CB1 receptors are translocated toward the soma. The internalization process did not require activated G-protein a(i) or a(o) subunits. A different pattern of cell surface CB1 receptor expression was observed using an undifferentiated F-11 cell line, which had pronounced somatic labeling. In these cells substantial CB1 receptor internalization was also observed after exposure to (+)-WIN55212 (1 µm) for relatively short periods (30 min) of agonist exposure. In summary, this dynamic modulation of CB1 receptor expression may play an important role in the development of cannabinoid tolerance in the CNS. Agonist-induced internalization at presynaptic terminals has important implications for the modulatory effects of G-protein-coupled receptors on neurotransmitter release.
    Original languageEnglish
    Pages (from-to)2425-2433
    Number of pages9
    JournalJournal of Neuroscience
    Volume21
    Issue number7
    Publication statusPublished - Apr 2001

    Fingerprint

    Cannabinoid Receptor CB1
    Neurons
    rimonabant
    Cannabinoids
    Presynaptic Terminals
    Cell Surface Receptors
    G-Protein-Coupled Receptors
    Cannabinoid Receptor Agonists
    Carisoprodol
    GTP-Binding Proteins
    Confocal Microscopy
    Neurotransmitter Agents
    Immunohistochemistry
    Cell Membrane
    Cell Line

    Keywords

    • Internalization
    • Cannabinoid
    • Receptor trafficking
    • CB1
    • Hippocampal
    • F-11

    Cite this

    Coutts, A. A., Anavi-Goffer, S., Ross, R. A., MacEwan, D. J., Mackie, K., Pertwee, R. G., & Irving, A. J. (2001). Agonist-induced internalization and trafficking of cannabinoid CB1 receptors in hippocampal neurons. Journal of Neuroscience, 21(7), 2425-2433.
    Coutts, Angela A. ; Anavi-Goffer, Sharon ; Ross, Ruth A. ; MacEwan, David J. ; Mackie, Ken ; Pertwee, Roger G. ; Irving, Andrew J. / Agonist-induced internalization and trafficking of cannabinoid CB1 receptors in hippocampal neurons. In: Journal of Neuroscience. 2001 ; Vol. 21, No. 7. pp. 2425-2433.
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    abstract = "Agonist-induced internalization of G-protein-coupled receptors is an important mechanism for regulating receptor abundance and availability at the plasma membrane. In this study we have used immunolabeling techniques and confocal microscopy to investigate agonist-induced internalization and trafficking of CB1receptors in rat cultured hippocampal neurons. The levels of cell surface CB1 receptor immunoreactivity associated with presynaptic GABAergic terminals decreased markedly (by up to 84{\%}) after exposure to the cannabinoid agonist (+)-WIN55212, in a concentration-dependent (0.1–1 µm) and stereoselective manner. Inhibition was maximal at 16 hr and abolished in the presence of SR141716A, a selective CB1 receptor antagonist. Methanandamide (an analog of an endogenous cannabinoid, anandamide) also reduced cell surface labeling (by 43{\%} at 1 µm). Differential labeling of cell surface and intracellular pools of receptor demonstrated that the reduction in cell surface immunoreactivity reflects agonist-induced internalization and suggests that the internalized CB1 receptors are translocated toward the soma. The internalization process did not require activated G-protein a(i) or a(o) subunits. A different pattern of cell surface CB1 receptor expression was observed using an undifferentiated F-11 cell line, which had pronounced somatic labeling. In these cells substantial CB1 receptor internalization was also observed after exposure to (+)-WIN55212 (1 µm) for relatively short periods (30 min) of agonist exposure. In summary, this dynamic modulation of CB1 receptor expression may play an important role in the development of cannabinoid tolerance in the CNS. Agonist-induced internalization at presynaptic terminals has important implications for the modulatory effects of G-protein-coupled receptors on neurotransmitter release.",
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    Coutts, AA, Anavi-Goffer, S, Ross, RA, MacEwan, DJ, Mackie, K, Pertwee, RG & Irving, AJ 2001, 'Agonist-induced internalization and trafficking of cannabinoid CB1 receptors in hippocampal neurons', Journal of Neuroscience, vol. 21, no. 7, pp. 2425-2433.

    Agonist-induced internalization and trafficking of cannabinoid CB1 receptors in hippocampal neurons. / Coutts, Angela A.; Anavi-Goffer, Sharon; Ross, Ruth A.; MacEwan, David J.; Mackie, Ken; Pertwee, Roger G.; Irving, Andrew J.

    In: Journal of Neuroscience, Vol. 21, No. 7, 04.2001, p. 2425-2433.

    Research output: Contribution to journalArticle

    TY - JOUR

    T1 - Agonist-induced internalization and trafficking of cannabinoid CB1 receptors in hippocampal neurons

    AU - Coutts, Angela A.

    AU - Anavi-Goffer, Sharon

    AU - Ross, Ruth A.

    AU - MacEwan, David J.

    AU - Mackie, Ken

    AU - Pertwee, Roger G.

    AU - Irving, Andrew J.

    N1 - dc.publisher: Society for Neuroscience dc.description.sponsorship: Wellcome Trust (Grants 47368 and 055291) Medical Research Council (Grant G9901500)

    PY - 2001/4

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    N2 - Agonist-induced internalization of G-protein-coupled receptors is an important mechanism for regulating receptor abundance and availability at the plasma membrane. In this study we have used immunolabeling techniques and confocal microscopy to investigate agonist-induced internalization and trafficking of CB1receptors in rat cultured hippocampal neurons. The levels of cell surface CB1 receptor immunoreactivity associated with presynaptic GABAergic terminals decreased markedly (by up to 84%) after exposure to the cannabinoid agonist (+)-WIN55212, in a concentration-dependent (0.1–1 µm) and stereoselective manner. Inhibition was maximal at 16 hr and abolished in the presence of SR141716A, a selective CB1 receptor antagonist. Methanandamide (an analog of an endogenous cannabinoid, anandamide) also reduced cell surface labeling (by 43% at 1 µm). Differential labeling of cell surface and intracellular pools of receptor demonstrated that the reduction in cell surface immunoreactivity reflects agonist-induced internalization and suggests that the internalized CB1 receptors are translocated toward the soma. The internalization process did not require activated G-protein a(i) or a(o) subunits. A different pattern of cell surface CB1 receptor expression was observed using an undifferentiated F-11 cell line, which had pronounced somatic labeling. In these cells substantial CB1 receptor internalization was also observed after exposure to (+)-WIN55212 (1 µm) for relatively short periods (30 min) of agonist exposure. In summary, this dynamic modulation of CB1 receptor expression may play an important role in the development of cannabinoid tolerance in the CNS. Agonist-induced internalization at presynaptic terminals has important implications for the modulatory effects of G-protein-coupled receptors on neurotransmitter release.

    AB - Agonist-induced internalization of G-protein-coupled receptors is an important mechanism for regulating receptor abundance and availability at the plasma membrane. In this study we have used immunolabeling techniques and confocal microscopy to investigate agonist-induced internalization and trafficking of CB1receptors in rat cultured hippocampal neurons. The levels of cell surface CB1 receptor immunoreactivity associated with presynaptic GABAergic terminals decreased markedly (by up to 84%) after exposure to the cannabinoid agonist (+)-WIN55212, in a concentration-dependent (0.1–1 µm) and stereoselective manner. Inhibition was maximal at 16 hr and abolished in the presence of SR141716A, a selective CB1 receptor antagonist. Methanandamide (an analog of an endogenous cannabinoid, anandamide) also reduced cell surface labeling (by 43% at 1 µm). Differential labeling of cell surface and intracellular pools of receptor demonstrated that the reduction in cell surface immunoreactivity reflects agonist-induced internalization and suggests that the internalized CB1 receptors are translocated toward the soma. The internalization process did not require activated G-protein a(i) or a(o) subunits. A different pattern of cell surface CB1 receptor expression was observed using an undifferentiated F-11 cell line, which had pronounced somatic labeling. In these cells substantial CB1 receptor internalization was also observed after exposure to (+)-WIN55212 (1 µm) for relatively short periods (30 min) of agonist exposure. In summary, this dynamic modulation of CB1 receptor expression may play an important role in the development of cannabinoid tolerance in the CNS. Agonist-induced internalization at presynaptic terminals has important implications for the modulatory effects of G-protein-coupled receptors on neurotransmitter release.

    KW - Internalization

    KW - Cannabinoid

    KW - Receptor trafficking

    KW - CB1

    KW - Hippocampal

    KW - F-11

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    Coutts AA, Anavi-Goffer S, Ross RA, MacEwan DJ, Mackie K, Pertwee RG et al. Agonist-induced internalization and trafficking of cannabinoid CB1 receptors in hippocampal neurons. Journal of Neuroscience. 2001 Apr;21(7):2425-2433.