Rapid CB1 cannabinoid receptor desensitization defines the time course of ERK1/2 MAP kinase signaling

Molecular mechanisms regulating the development of physiological and behavioral tolerance to cannabinoids are not well understood. Two cellular correlates implicated in the development and maintenance of tolerance are CB1 cannabinoid receptor internalization and uncoupling of receptor signal transduction. Both processes have been proposed as mediators of tolerance because of observations that chronic Delta(9)-tetrahydrocannabinol (THC) treatment causes both region-specific decreases in CB1 receptors and G-protein coupling in the brain. To determine the balance of these two processes in regulating CB1 receptor signaling during sustained receptor stimulation, we evaluated the parameters affecting ERK1/2 MAP kinase activity in HEK293 cells stably expressing CB1 receptors. CB, receptor stimulation by the potent CB1 receptor agonist, CP 55,940 transiently activated ERK 1/2. To determine if CB1 receptor desensitization or internalization was responsible for the transient nature of ERK1/2 activation, we evaluated ERKI/2 phosphorylation in HEK293 cells expressing a desensitization-deficient CB1 receptor (S426A/S430A CB1). Here, the duration of S426A/S430A CB1 receptor-mediated activation of ERKI/2 was markedly prolonged relative to wild-type receptors, and was dynamically reversed by SR141716A. Interestingly, the S426A/S430A CB1 receptor was still able to recruit beta arrestin-2, a key mediator of receptor desensitization, to the cell surface following agonist activation. In contrast to a central role for desensitization, pharmacological and genetic approaches suggested CB1 receptor internalization is dispensable in the transient activation of ERK1/2. This study indicates that the duration of ERK1/2 activation by CB1 receptors is regulated by receptor desensitization and underscores the importance of G-protein uncoupling in the regulation of CB1 receptor signaling. (c) 2007 Elsevier Ltd. All rights reserved.