Selective inhibition of cannabinoid CB₁ receptor-evoked signalling by the interacting protein GAP43

Cannabinoids exert pleiotropic effects on the brain by engaging the cannabinoid CB₁ receptor (CB₁R), a presynaptic metabotropic receptor that regulates key neuronal functions in a highly context-dependent manner. We have previously shown that CB₁R interacts with growth-associated protein of 43 kDa (GAP43) and that this interaction inhibits CB₁R function on hippocampal excitatory synaptic transmission, thereby impairing the therapeutic effect of cannabinoids on epileptic seizures in vivo. However, the underlying molecular features of this interaction remain unexplored. Here, we conducted mechanistic experiments on HEK293T cells co-expressing CB₁R and GAP43 and show that GAP43 modulates CB₁R signalling in a strikingly selective manner. Specifically, GAP43 did not affect the archetypical agonist-evoked (i) CB₁R/G_i/o protein-coupled signalling pathways, such as cAMP/PKA and ERK, or (ii) CB₁R internalization and intracellular trafficking. In contrast, GAP43 blocked an alternative agonist-evoked CB₁R-mediated activation of the cytoskeleton-associated ROCK signalling pathway, which relied on the GAP43-mediated impairment of CB₁R/G_q/11 protein coupling. GAP43 also abrogated CB₁R-mediated ROCK activation in mouse hippocampal neurons, and this process led in turn to a blockade of cannabinoid-evoked neurite collapse. An NMR-based characterization of the CB₁R-GAP43 interaction supported that GAP43 binds directly and specifically through multiple amino acid stretches to the C-terminal domain of the receptor. Taken together, our findings unveil a CB₁R-G_q/11-ROCK signalling axis that is selectively impaired by GAP43 and may ultimately control neurite outgrowth.