Sulfation of the FLAG epitope is affected by co-expression of G protein-coupled receptors in a mammalian cell model.
G protein-coupled receptors (GPCRs) are important therapeutic targets and therefore extensively studied. Like most transmembrane proteins, there has been considerable difficulty in developing reliable specific antibodies for them. To overcome this, epitope tags are often used to facilitate antibody recognition in studies on fundamental receptor signalling and trafficking. In our study of cannabinoid CB1/dopamine D2 interactions we sought to generate HEK293 cells expressing FLAG-tagged D2 for use in antibody-based assays of GPCR localisation and trafficking activity, however observed that stable FLAG-hD2 expression was particularly challenging to maintain. In contrast, when expressed in cell lines expressing hCB1 robust and stable FLAG-hD2 expression was observed. We hypothesised that co-expression of CB1 might stabilise surface FLAG-hD2 expression, and therefore investigated this further. Here, we describe the observation that co-expression of either cannabinoid CB1 or CB2 receptors in HEK293 decreases the sulfation of a FLAG epitope appended at the N-terminus of the dopamine D2 receptor. Sulfation alters epitope recognition by some anti-FLAG antibodies, leading to the detection of fewer receptors, even though expression is maintained. This demonstrates that cannabinoid receptor expression modifies posttranslational processing of the FLAG-hD2 receptor, and importantly, has wider implications for the utilisation and interpretation of receptor studies involving epitope tags.
Department of Pharmacology and Clinical Pharmacology, University of Auckland, Auckland, New Zealand.
Morag Rose
MR
Hunter
Department of Pharmacology and Clinical Pharmacology, University of Auckland, Auckland, New Zealand.
Natasha Lillia
NL
Grimsey
Department of Pharmacology and Clinical Pharmacology, University of Auckland, Auckland, New Zealand.
Michelle
M
Glass
D000939:Epitopes / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D011499:Protein Processing, Post-Translational; D043884:Receptor, Cannabinoid, CB1 / Q000378:metabolism*; D043885:Receptor, Cannabinoid, CB2 / Q000378:metabolism; D017448:Receptors, Dopamine D2 / Q000378:metabolism*; D013431:Sulfates / Q000378:metabolism*
D000939:Epitopes; D043884:Receptor, Cannabinoid, CB1; D043885:Receptor, Cannabinoid, CB2; D017448:Receptors, Dopamine D2; D013431:Sulfates