Generation and functional characterization of fluorescent, N-terminally tagged CB1 receptor chimeras for live-cell imaging.
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 CB1 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-CB1 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.
McDonald
Neil A
NA
Neurosciences Institute, Division of Pathology and Neuroscience, Ninewells Hospital and Medical School, University of Dundee, Dundee, DD1 9SY, UK.
Henstridge
Christopher M
CM
Connolly
Christopher N
CN
Irving
Andrew J
AJ
D000818:Animals; D000831:Animals, Newborn; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D003952:Diagnostic Imaging / Q000379:methods; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006624:Hippocampus / Q000166:cytology; D008164:Luminescent Proteins / Q000378:metabolism*; D016296:Mutagenesis / Q000502:physiology; D050506:Mutant Chimeric Proteins / Q000378:metabolism*; D009474:Neurons / Q000502:physiology*; D051381:Rats; D043884:Receptor, Cannabinoid, CB1 / Q000737:chemistry / Q000378:metabolism*; D014162:Transfection / Q000379:methods
D008164:Luminescent Proteins; D050506:Mutant Chimeric Proteins; D043884:Receptor, Cannabinoid, CB1; D000242:Cyclic AMP