Agonist-induced internalization and trafficking of cannabinoid CB1 receptors in hippocampal neurons

Agonist-induced internalization of G-protein-coupled receptors is an import ant mechanism for regulating receptor abundance and availability at the pla sma membrane. in this study we have used immunolabeling techniques and conf ocal microscopy to investigate agonist-induced internalization and traffick ing of CB1 receptors in rat cultured hippocampal neurons. The levels of cel l surface CB1 receptor immunoreactivity associated with presynaptic GABAerg ic terminals decreased markedly (by up to 84%) after exposure to the cannab inoid agonist (+)-WIN55212, in a concentration-dependent (0.1-1 muM) and st ereoselective manner. Inhibition was maximal at 16 hr and abolished in the presence of SR141716A, a selective CB1 receptor antagonist. Methanandamide (an analog of an endogenous cannabinoid, anandamide) also reduced cell surf ace labeling (by 43% at 1 muM). Differential labeling of cell surface and i ntracellular pools of receptor demonstrated that the reduction in cell surf ace immunoreactivity reflects agonist-induced internalization and suggests that the internalized CB1 receptors are translocated toward the soma. The i nternalization process did not require activated G-protein alpha (i) or alp ha (o) subunits. A different pattern of cell surface CB1 receptor expressio n was observed using an undifferentiated F-11 cell line, which had pronounc ed somatic labeling. In these cells substantial CB1 receptor internalizatio n was also observed after exposure to (+)-WIN55212 (1 muM) for relatively s hort periods (30 min) of agonist exposure. In summary, this dynamic modulat ion of CB1 receptor expression may play an important role in the developmen t of cannabinoid tolerance in the CNS. Agonist-induced internalization at p resynaptic terminals has important implications for the modulatory effects of G-protein-coupled receptors on neurotransmitter release.