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 CB(1)/dopamine D(2) interactions we sought to generate HEK293 cells expressing FLAG-tagged D(2) for use in antibody-based assays of GPCR localisation and trafficking activity, however observed that stable FLAG-hD(2) expression was particularly challenging to maintain. In contrast, when expressed in cell lines expressing hCB(1) robust and stable FLAG-hD(2) expression was observed. We hypothesised that co-expression of CB(1) might stabilise surface FLAG-hD2 expression, and therefore investigated this further. Here, we describe the observation that co-expression of either cannabinoid CB(1) or CB(2) receptors in HEK293 decreases the sulfation of a FLAG epitope appended at the N-terminus of the dopamine D(2) 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-hD(2) receptor, and importantly, has wider implications for the utilisation and interpretation of receptor studies involving epitope tags.