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Generation and functional characterization of fluorescent, N-terminally tagged CB(1) receptor chimeras for live-cell imaging.
Mol Cell Neurosci. 2007 Mar 3;
Authors: McDonald NA, Henstridge CM, Connolly CN, Irving AJ
N-terminally tagged CB(1) receptor fusion proteins, incorporating enhanced green fluorescent protein (GFP) or super-ecliptic pHluorin (SEP), were generated to study CB(1) 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(1) receptor chimeras, which behaved like wild-type CB(1) receptors in functional assays. Treatment with cannabinoid ligands led to a rapid down-regulation of SS-GFP-CB(1) from the plasma membrane in COS7 and HEK293 cells, associated with trafficking into cytosolic vesicles. Activation of CB(1) receptors was also linked with a time-dependent reduction in cell surface SEP-CB(1) fluorescence and incorporation of the construct into acidic endosomes, revealed following exposure to NH(4)Cl. In live hippocampal neurons, SEP-CB(1) fluorescence was largely restricted to the axon, consistent with its polarised surface expression. Thus, these new molecular tools are well suited for studying CB(1) 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.
PMID: 17467290 [PubMed - as supplied by publisher]
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