Abstract
Context: Beta-arrestins are known to couple to some G-protein-coupled receptors (GPCRs) to regulate receptor internalization, G-protein coupling and signal transduction, but have not been investigated for most receptors, and for very few receptors in vivo. Previous studies have shown that beta-arrestin2 deletion enhances the efficacy of specific cannabinoid agonists. Objective: The present study hypothesized that brain cannabinoid CB1 receptors are regulated by beta-arrestin2. Methods: Beta-arrestin2+/+ and −/− mice were used. Western blotting was used to determine the relative levels of each beta-arrestin subtype in mouse brain. Receptor binding was measured to determine whether deletion of beta-arrestin2 influences agonist binding to brain CB1 receptors, or the subcellular localization of CB1 in brain membranes subjected to differential centrifugation. A variety of cannabinoid agonists from different chemical classes were investigated for their ability to activate G-proteins in the presence and absence of beta-arrestin2 in cerebellum, hippocampus and cortex. Results: No differences were found in the density of beta-arrestin1 or cannabinoid CB1 receptors in several brains of beta-arrestin2+/+ versus −/− mice. Differences between genotypes were found in the proportion of high- and low-affinity agonist binding sites in brain areas that naturally express higher levels of beta-arrestin2. Cortex from beta-arrestin2−/− mice contained less CB1 in the P1 fraction and more CB1 in the P2 fraction compared to beta-arrestin2+/+. Of the agonists assayed for activity, only Δ9-tetrahydrocannabinol (THC) exhibited a difference between genotypes, in that it was less efficacious in beta-arrestin2−/− than +/+ mouse membranes. Conclusion: Beta-arrestin2 regulates cannabinoid CB1 receptors in brain.
Acknowledgements
The authors thank Dr. Robert J. Lefkowitz, Nobel Laureate and James B. Duke, Professor of Biochemistry, Immunology and Medicine at Duke University Medical Center, for generously supplying beta-arrestin2+/+ and −/− breeder mice, beta-arrestin2 antibodies and purified beta-arrestin1 and 2 proteins. The authors also thank Dr. Steven R. Childers, Professor of Physiology and Pharmacology at Wake Forest University School of Medicine for assistance with the preparation of this article.