J. Hagen, N. Hopp, M. Grahek, J. Humphrey, G. Du, P. Murtha, M. Cooper, C. Autin, B. Aggeler, A. Kalyuzhny
ABSTRACT
Trafficking of the µ Opioid Receptor (OPRM1) from the cell membrane into the cytoplasm is one of the key mechanisms underlying receptor desensitization, which is implicated in playing a role in the development of tolerance and dependence to narcotic opiates. Endocytosis of OPRM1 is usually analyzed qualitatively by utilizing cells transfected with a receptor that has been tagged with a hapten, such as HA and c-myc, at its N-terminus. The receptor is then detected by immunocytochemistry using tag-specific secondary antibodies conjugated to fluorescent dyes. We have developed a more efficient protocol to monitor receptor endocytosis by employing an Alexa Fluor® 594-conjugated rabbit monoclonal antibody that recognizes and binds the N-terminal part of OPRM1. HEK293 EBNA cells, transfected with rat OPRM1 tagged with Green Fluorescent Protein (GFP) at its C-terminus, were treated with the synthetic opioid peptide DAMGO. DAMGO produced a strong internalization of receptors, which was blocked by the antagonist Naloxone. The specificity of internalization was confirmed by detecting complete overlap of green (GFP) and red (Alexa Fluor® 594) fluorescence using confocal microscopy. The extent of OPRM1 internalization was further analyzed using the High Content Screening (HCS) platform, which allows for the automated examination of hundreds to thousands of drug-treated cells. The advantage of this new technique is that it can be applied to native neuronal cells as the transfection of hapten-containing Oprm1 constructs is not needed. By measuring the fluorescence intensity of OPRM1 localized on the cell membrane.
