Authors: Shazina S Khan Frank J S Lee
Publish Date: 2013/11/22
Volume: 53, Issue: 1, Pages: 10-21
Abstract
Both the cannabinoid CB1 receptor CB1 and dopamine D2 receptor D2R are G proteincoupled receptors that are linked to inhibitory Gαi/o protein whereby activation of the receptor leads to the inhibition of cAMP production Moreover previous findings have shown evidence of crosstalk between the dopamine and endocannabinoid systems In this report we confirm the interaction of CB1 and D2R with coimmunoprecipitation experiments using human embryonic kidney 293T HEK293T cells coexpressing both receptors We also generated GST and Histagged fusion proteins of the D2R and CB1 and conducted affinity purification assays and in vitro binding experiments to show that the CB1–D2R complex can be formed by a direct protein–protein interaction This interaction is mediated by the carboxyl terminus of the CB1 receptor and the third intracellular loop of the D2 receptor Cotransfection of an inhibitory minigene resulted in decreased levels of the CB1–D2R complex Using a cAMP biosensor we show that activation of D2R or CB1 alone in HEK293T cells coexpressing both receptors leads to an inhibition of forskolinstimulated cAMP accumulation However coactivation of both receptors resulted in a loss of this inhibition on cAMP accumulation Our findings characterize the physical interaction between CB1 and D2R as well as demonstrate the potential functional outcome of the receptor complexWe thank Dr Mary Abood for kindly providing the human CB1 cDNA and Dr Ken Mackie for providing the rat CB1 cDNA We also thank Dr Martin Lohse for providing the epac1camp plasmid We thank Beryl Luk for technical assistance and Dr Tim Beischlag for reviewing the manuscript This study was funded by a Young Investigator Grant from the MIND BC Foundation and from a grant from the William and Ada Isabelle Steel Fund
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