CB1 Cannabinoid Receptors Signal Through Focal Adhesion Kinase To Activate ERK In Neuronal Cells

Abstract

Focal adhesion kinase (FAK) phosphorylated at tyrosine residue 925 (Tyr 925) binds Grb2/Sos which stimulates the tyrosine phosphorylation (Tyr‐P)/activation of the mitogen‐activated protein kinase ERK. We investigated the role that FAK 925 Tyr‐P plays in regulating CB1 cannabinoid receptor (CB1R)‐stimulated ERK Tyr‐P in murine N18TG2 neuroblastoma cells that express endogenous CB1Rs. Immunoblotting experiments revealed the time‐course of CB1R‐stimulated FAK 925 Tyr‐P and ERK Tyr‐P are similar in N18TG2 cells. The synthetic CB1R agonist WIN55212‐2 (10 nM) produced three distinct phases of FAK 925 Tyr‐P and ERK Tyr‐P: Phase I (0–5 min) involved maximal Tyr‐P, Phase II (5–20 min) involved a decline in Tyr‐P, and Phase III (>; 20 min) involved a plateau in Tyr‐P at submaximal levels. Phase I was blocked by the CB1R antagonist SR141716A (1 μM) and pertussis toxin (100 ng/mL) which suggests maximal FAK 925 Tyr‐P/ERK Tyr‐P are mediated by CB1Rs and Gi/o proteins. The Src inhibitor PP2 (2 μM) abolished Phase I FAK 925 Tyr‐P indicating an absolute requirement for Src, while PP2 partially inhibited Phase I ERK Tyr‐P. Phase I FAK 925 Tyr‐P and ERK Tyr‐P also involved transactivation of Flk‐1 vascular endothelial growth factor receptors and epidermal growth factor receptors. The FAK inhibitors Y11 (100 nM) and PF573228 (100 nM) abolished Phase I FAK 925 Tyr‐P, and partially inhibited Phase I ERK Tyr‐ P. These studies demonstrate CB1R‐stimulated maximal ERK activation requires FAK 925 Tyr‐P in neuronal cells. Supported by NIDA grants R01DA003690 (ACH), F32DA026295 (GDD).