Transactivation of epidermal growth factor receptor EGFR by CB1 cannabinoid receptor agonists

Cannabinoids have long been used as therapeutic agents. The recent identification of the endogenous cannabinoid system in the brain, namely the abundance of the CB1 receptor (CB1R) in the CNS and the discovery of the endogenous ligands, has suggested that the cannabinoid neuromodulatory system plays important roles in many physiological processes, such as in memory, as well as holds therapeutic promise in a wide range of diseases ranging from mood and anxiety disorders to movement disorders and spinal cord injury. The CB1 receptor exerts its effects through several signalling events, which include the activation of the extracellular signal-regulated kinase, or ERKs. The mechanism by which this G-protein coupled receptor triggers ERK activation is not, however, well understood. To investigate the molecular events coupling CB1 activation to PKC and ERK activation, we generated clonal SHSY-5Y neuroblastoma cell lines which stably overexpress EGFP-CB1R and assessed whether this coupling could be mediated via transactivation of growth factor receptors with intrinsic tyrosine kinase activity. We first established using both immunoprecipitation and immunocytochemistry and GFP fluorescent imaging that the EGFP-CB1R was properly glycosylated and targeted to plasma membranes. We now report that the CB1R agonist methanandamide specifically stimulated, in a time-dependent manner, tyrosine phosphorylation of several cellular proteins, including phosphorylation of the EGF receptor and Src kinase on tyrosine residues 1148 and 416, respectively, and of ERK2. These effects were abolished by the specific CB1 antagonist AM251 and by the EGFR antagonist AG1478. These data provide first evidence that in a neuronal cellular background, the CB1R intracellular signalling engages the specific transactivation of the tyrosine kinase EGF receptor to regulate ERK activation and downstream transcriptional events. ©ΦApmakon-Túttoς