Phosphorylation Mechanisms of CB1 Cannabinoid Receptor Desensitization

Ken Mackie, Psychological and Brain Sciences, Indiana University

CB1 cannabinoid receptors are G protein-coupled receptors (GPCR’s) that are widely expressed throughout the CNS where they mediate the effects of exogenous and endogenous cannabinoids, such as tetrahydrocannabinol (THC), anandamide, and 2-AG.  Like most GPCR’s, continued activation of CB1 receptors leads to behavioral tolerance.  In vitro studies using site-directed mutagenesis suggest that C-terminal phosphorylation of CB1 is involved in desensitization of CB1 signaling as well as CB1 receptor internalization.  Recent results with a knockin mouse where two of these residues (serines 426 and 430) have been mutated to alanine further suggest that phosphorylation of these residues is involved in the development of behavioral tolerance to cannabinoids.

To date all studies investigating the regulation of CB1 receptors by phosphorylation have used site-directed mutagenesis:  a residue is hypothesized to be phosphorylated, it is mutated to alanine, aspartate, or glutamate, and function is assessed.  While this approach has been useful, it suffers from two serious drawbacks.  The first is that it gives a binary answer—the residue is important or not.  Important information such as the kinetics, extent, and order of phosphorylation cannot be obtained.  (Strictly speaking, it also does not tell us that the residue is important for phosphorylation, just that it is important.)  The second is that it is inherently a biased approach—we start with the assumption that a residue might be phosphorylated, and there are too many potentially phosphorylation sites for it to be practical to mutate each one.  To overcome these limitations we will systematically determine which residues are phosphorylated in CB1 as it is acutely activated (in cell lines and mice) and as tolerance develops (mice).  The results of these studies will allow us to characterize CB1 phosphorylation as it is activated and assist us in the design of new experiments to determine the role of this phosphorylation in CB1 receptor regulation.