Generation and functional characterization of fluorescent, N-terminally tagged CB 1 receptor chimeras for live-cell imaging

Abstract

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 4Cl. 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.