Generation and functional characterization of fluorescent, N-terminally tagged CB1 receptor chimeras for live-cell imaging

Neil A. McDonald, Christopher M. Henstridge, Christopher N. Connolly, Andrew J. Irving

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

N-terminally tagged CB1 receptor fusion proteins, incorporating enhanced green fluorescent protein (GFP) or super-ecliptic pHluorin (SEP), were generated to study CB1 receptor trafficking and cell surface receptor expression in live COS7 and HEK293 cells and hippocampal neurons. An artificial signal sequence (SS) was required for efficient surface expression of CB, receptor chimeras, which behaved like wild-type CB1 receptors in functional assays. Treatment with cannabinoid ligands led to a rapid down-regulation of SS-GFP-CB1 from the plasma membrane in COS7 and HEK293 cells, associated with trafficking into cytosolic vesicles. Activation, of CB1 receptors was also linked with a time-dependent reduction in cell surface SEP-CB, fluorescence and incorporation of the construct into acidic endosomes, revealed following exposure to NH4Cl. In live hippocampal neurons, SEP-CB1 fluorescence was largely restricted to the axon, consistent with its polarised surface expression. Thus, these new molecular tools are well suited for studying CB1 receptor trafficking and a new generation of GPCR chimeras incorporating SEP at the N-terminus will be especially useful for monitoring dynamic changes in cell surface receptor expression in living cells. (c) 2007 Elsevier Inc. All rights reserved.

Original languageEnglish
Pages (from-to)237-248
Number of pages12
JournalMolecular and Cellular Neuroscience
Volume35
Issue number2
DOIs
Publication statusPublished - Jun 2007

Fingerprint

Cannabinoid Receptor CB1
HEK293 Cells
Cell Surface Receptors
Protein Sorting Signals
Fluorescence
Neurons
Cannabinoids
Endosomes
Green Fluorescent Proteins
Axons
Down-Regulation
Cell Membrane
Ligands
PHluorin
Proteins

Keywords

  • Endocytosis
  • Cannabinoid
  • GPCR
  • pHluorin
  • Green fluorescent protein (GFP)
  • GFP
  • Imaging
  • G protein-coupled receptors (GPCR)

Cite this

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title = "Generation and functional characterization of fluorescent, N-terminally tagged CB1 receptor chimeras for live-cell imaging",
abstract = "N-terminally tagged CB1 receptor fusion proteins, incorporating enhanced green fluorescent protein (GFP) or super-ecliptic pHluorin (SEP), were generated to study CB1 receptor trafficking and cell surface receptor expression in live COS7 and HEK293 cells and hippocampal neurons. An artificial signal sequence (SS) was required for efficient surface expression of CB, receptor chimeras, which behaved like wild-type CB1 receptors in functional assays. Treatment with cannabinoid ligands led to a rapid down-regulation of SS-GFP-CB1 from the plasma membrane in COS7 and HEK293 cells, associated with trafficking into cytosolic vesicles. Activation, of CB1 receptors was also linked with a time-dependent reduction in cell surface SEP-CB, fluorescence and incorporation of the construct into acidic endosomes, revealed following exposure to NH4Cl. In live hippocampal neurons, SEP-CB1 fluorescence was largely restricted to the axon, consistent with its polarised surface expression. Thus, these new molecular tools are well suited for studying CB1 receptor trafficking and a new generation of GPCR chimeras incorporating SEP at the N-terminus will be especially useful for monitoring dynamic changes in cell surface receptor expression in living cells. (c) 2007 Elsevier Inc. All rights reserved.",
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AU - Henstridge, Christopher M.

AU - Connolly, Christopher N.

AU - Irving, Andrew J.

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AB - N-terminally tagged CB1 receptor fusion proteins, incorporating enhanced green fluorescent protein (GFP) or super-ecliptic pHluorin (SEP), were generated to study CB1 receptor trafficking and cell surface receptor expression in live COS7 and HEK293 cells and hippocampal neurons. An artificial signal sequence (SS) was required for efficient surface expression of CB, receptor chimeras, which behaved like wild-type CB1 receptors in functional assays. Treatment with cannabinoid ligands led to a rapid down-regulation of SS-GFP-CB1 from the plasma membrane in COS7 and HEK293 cells, associated with trafficking into cytosolic vesicles. Activation, of CB1 receptors was also linked with a time-dependent reduction in cell surface SEP-CB, fluorescence and incorporation of the construct into acidic endosomes, revealed following exposure to NH4Cl. In live hippocampal neurons, SEP-CB1 fluorescence was largely restricted to the axon, consistent with its polarised surface expression. Thus, these new molecular tools are well suited for studying CB1 receptor trafficking and a new generation of GPCR chimeras incorporating SEP at the N-terminus will be especially useful for monitoring dynamic changes in cell surface receptor expression in living cells. (c) 2007 Elsevier Inc. All rights reserved.

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KW - pHluorin

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KW - GFP

KW - Imaging

KW - G protein-coupled receptors (GPCR)

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