An essential role for constitutive endocytosis, but not activity, in the axonal targeting of the CB1 cannabinoid receptor

In central neurons, the cell-surface distribution of cannabinoid receptor subtype-1 (CB(1)) is highly polarized toward axons and is associated with synaptic terminals, in which it is well-positioned to modulate neurotransmitter release. It has been suggested that high levels of constitutive activity mediate CB(1) receptor axonal targeting, leading to domain-specific endocytosis. We have investigated further the mechanisms that underlie CB(1) receptor axonal polarization in hippocampal neurons and found that constitutive activity is not an essential requirement for this process. We demonstrate that the cell-surface distribution of an N-terminally tagged, fluorescent CB(1) receptor fusion-protein is almost exclusively localized to the axon when expressed in cultured hippocampal neurons. Inhibition of endocytosis by cotransfection with a dominant-negative dynamin-1 (K44A) mutant traps both recombinant and endogenous CB(1) receptors at the somatodendritic cell surface. However, this effect could not be mimicked by inhibiting constitutive activity or receptor activation, either by expressing mutant receptors that lack these properties or by treatment with CB(1) receptor antagonists possessing inverse agonist activity. These data are consistent with a revised model in which domain-specific endocytosis regulates the functional polarization of CB(1) receptors, but this process is distinct from constitutive activity.