[Frontiers in Bioscience E1, 220-227, June 1, 2009]

Detecting the mu opioid receptor in brain following SDS-PAGE with multiple approaches

Peng Huang, Lee-Yuan Liu-Chen

Department of Pharmacology and Center for Substance Abuse Research, Temple University School of Medicine, Philadelphia, PA19140

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Ligand affinity-labeling: the (3H)beta-FNA-labeled mu opioid receptor (MOPR) is heterogeneous in molecular masses (Mr's) due to differential N-linked glycosylation, depending on species and expression systems
4. Agonist-promoted MOPR phosphorylation: phosphorylated MOPR was shown as bands with median Mr's above 65 kDa
5. Western blot: MOPR has brain region-specific heterogeneity in N-glycosylation revealed by an anti-MOPR antibody and control brain tissues from MOPR-knockout mice
6. Conclusion
7. Acknowledgements
8. References

1. ABSTRACT

In general, it has been difficult to obtain antibodies which are useful for immunoblotting of endogenous seven-transmembrane receptors (7TMRs) despite the claims made by many companies on commercially available antibodies. In this review, we will use the mu opioid receptor (MOPR) in brain as an example to underscore the importance of using knock-out (K/O) mice and multiple independent approaches (ligand affinity-labeling, receptor phosphorylation and immunoblotting) in identifying 7TMRs following sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE). The rigor and convergence of pharmacological and biochemical data provide confidence on the unequivocal identification of MOPR. The distinct relative molecular masses (Mr's) and band patterns are largely due to variations in the extent of N-glycosylation in different cell lines, brain regions and species.