Identification of two functionally distinct endosomal recycling pathways for dopamine D₂ receptor

Dopamine D₂ receptor (DRD2) is important for normal function of the brain reward circuit. Lower DRD2 function in the brain increases the risk for substance abuse, obesity, attention deficit/hyperactivity disorder, and depression. Moreover, DRD2 is the target of most antipsychotics currently in use. It is well known that dopamine-induced DRD2 endocytosis is important for its desensitization. However, it remains controversial whether DRD2 is recycled back to the plasma membrane or targeted for degradation following dopamine stimulation. Here, we used total internal reflection fluorescent microscopy (TIRFM) to image DRD2 with a superecliptic pHluorin tagged to its N terminus. With these technical advances, we were able to directly visualize vesicular insertion events of DRD2 in cultured mouse striatal medium spiny neurons. We showed that insertion of DRD2 occurs on neuronal somatic and dendritic surfaces. Lateral diffusion of DRD2 was observed following its insertion. Most importantly, using our new approach, we uncovered two functionally distinct recycling pathways for DRD2: a constitutive recycling pathway and a dopamine activity-dependent recycling pathway. We further demonstrated that Rab4 plays an important role in constitutive DRD2 recycling, while Rab11 is required for dopamine activity-dependent DRD2 recycling. Finally, we demonstrated that the two DRD2 recycling pathways play distinct roles in determining DRD2 function: the Rab4-sensitive constitutive DRD2 recycling pathway determines steady-state surface expression levels of DRD2, whereas the Rab11-sensitive dopamine activity-dependent DRD2 recycling pathway is important for functional resensitization of DRD2. Our findings underscore the significance of endosomal recycling in regulation of DRD2 function.