CB1 Cannabinoid Receptors Stimulate Gβγ-GRK2-Mediated FAK Phosphorylation at Tyrosine 925 to Regulate ERK Activation Involving Neuronal Focal Adhesions

Abstract

CB 1 cannabinoid receptors (CB 1) are abundantly expressed in the nervous system where they regulate focal adhesion kinase (FAK) and the mitogen-activated protein kinases (MAPK) extracellular signal-regulated kinase 1 and 2 (ERK1/2). However, the role of CB 1-stimulated FAK 925 tyrosine phosphorylation (Tyr-P) in regulating ERK1/2 activation remains undefined. Here, immunoblotting analyses using antibodies against FAK phospho-Tyr 925 and ERK2 phospho-Tyr 204 demonstrated CB 1-stimulated FAK 925 Tyr-P and ERK2 204 Tyr-P (0-5 min) which was followed by a decline in Tyr-P (5-20 min). CB 1 stimulated FAK-Grb2 association and Ras-mediated ERK2 activation. The FAK inhibitors Y11 and PF 573228 abolished FAK 925 Tyr-P and partially inhibited ERK2 204 Tyr-P. FAK 925 Tyr-P and ERK2 204 Tyr-P were adhesion-dependent, required an intact actin cytoskeleton, and were mediated by integrins, Flk-1 vascular endothelial growth factor receptors, and epidermal growth factor receptors. FAK 925 Tyr-P and ERK2 204 Tyr-P were blocked by the Gβγ inhibitor gallein, a GRK2 inhibitor, and GRK2 siRNA silencing, suggesting Gβγ and GRK2 participate in FAK-mediated ERK2 activation. Together, these studies indicate FAK 925 Tyr-P occurs concurrently with CB 1-stimulated ERK2 activation and requires the actin cytoskeleton and G i/o βγ-GRK2mediated cross-talk between CB 1 , integrins, and receptor tyrosine kinases (RTKs).

In Vitro Characterization of 6-Methyl-3-(2-nitro-1-(thiophen-2-yl)ethyl)-2-phenyl-1H-indole (ZCZ011) at the Type 1 Cannabinoid Receptor: Allosteric Agonist or Allosteric Modulator?

“…Canonical signaling of CB 1 includes phosphorylation of the mitogen-activated protein kinases ERK1/2, attributed to both G α and G βγ signaling. − This signaling paradigm was investigated using the AlphaLISA Surefire pERK kit, where all responses were normalized to pERK response in the presence of vehicle (0%) or 1 μM CP55940 (100%) at 3 min 30 s, the time point for peak pERK activity.…”

“…Negative allosteric modulation or competitive antagonism could explain the negative interaction observed, although more investigation is required. This negative interaction is interesting because ZCZ011 was found to be a positive allosteric modulator of ERK1/2 phosphorylation, a signaling pathway known to be mediated by G proteins. − These contrasting findings could be attributed to a mutual inconsistency in pERK and G protein dissociation assay sensitivities, such that low levels of pERK may not be detectable (and if so, apparent PAM effects observed could be overestimated), or that PAM effects were underestimated in G protein dissociation assays due to limits in detecting high efficacy responses. As Gαi3 was the only G protein investigated in the G protein dissociation assay, it is possible that ZCZ011 potentiates the dissociation of a different Gα subtype thus causing an increase in ERK1/2 phosphorylation.…”

Effects of Gαi2 and Gαz protein knockdown on alpha2A-adrenergic and cannabinoid CB1 receptor regulation of MEK-ERK and FADD pathways in mouse cerebral cortex

“…Thus, WINinduced ERK stimulation may course via Gαi 1/3 proteins, or perhaps via β-arrestin pathway [11]. Furthermore, other cell-based studies also implicated Gβγ, rather than Gα proteins, in CB 1 -R-induced ERK activation [41,42]. Further studies will be needed to fully determine the role of these molecules in transducing CB 1 -R-to-ERK signals in native brain tissue.…”

“…However, in rodent brain, PI3K inhibition had no effect on cannabinoid-induced ERK stimulation, and the role of Gαi/o proteins was unclear [39,40]. Alternatively, CB 1 -R-mediated PI3K stimulation may occur via Gβ/γ, and the subsequent transactivation of receptor tyrosine kinases may ultimately stimulate ERK signaling [41,42].…”

Cell Adhesion Molecules and Protein Synthesis Regulation in Neurons

“…MAPK inhibitors inhibit neurite outgrowth induced by soluble fragments of NEGR1 in embryonic mouse cortical neurons (Pischedda and Piccoli, 2015 ), and block an increase in the expression of MAP2 induced by antibodies against L1 in mouse hippocampal neurons (Poplawski et al, 2012 ). The MAPK pathway is also triggered by other CAM families, such as cadherins (Yasuda et al, 2007 ; Lelievre et al, 2012 ) and integrins (Dalton et al, 2020 ). Interestingly, the loss of N-cadherin-mediated adhesion also results in the activation of the MAPK pathway in cultured cortical neurons (Ando et al, 2011 ).…”