The CB1 cannabinoid receptor C-terminus regulates receptor desensitization in autaptic hippocampal neurones

BACKGROUND AND PURPOSE The cannabinoid CB1 receptor is the chief mediator of the CNS effects of cannabinoids. In cell culture model systems, CB1 receptors both desensitize and internalize on activation. Previous work suggests that the extreme carboxy-terminus of this receptor regulates internalization via phosphorylation of residues clustered within this region. Mutational analysis of the carboxy-terminus of CB1 receptors has demonstrated that the last six serine/threonine residues are necessary for agonist-induced internalization. However, the structural determinants of CB1 receptor internalization are also dependent on the local cellular environment. The importance of cell context on CB1 receptor function calls for an investigation of the functional roles of these residues in neurones. EXPERIMENTAL APPROACH To determine the structural requirements of CB1 internalization in neurones, we evaluated the signalling properties of carboxyterminal mutated CB1 receptors expressed in cultured autaptic hippocampal neurones, using electrophysiological methods. KEY RESULTS CB1 receptors transfected into CB1 knockout neurones signalled and desensitized as did wild-type neurones, allowing us to test specific CB1 receptor mutations. Deletion of the last 13 residues yielded a CB1 receptor that inhibited excitatory postsynaptic currents but did not desensitize. Furthermore, mutation of the final six serine and threonine residues to alanines resulted in a non-desensitizing receptor. In contrast, CB1 receptors lacking residues 419-460, leaving the last 14 residues intact, did desensitize. CONCLUSIONS AND IMPLICATIONS The distal thirteen residues of CB1 receptors are crucial for their desensitization in cultured neurones. Furthermore, this desensitization is likely to follow phosphorylation of serines and threonines within this region.