Identification of Two Functionally Distinct Endosomal Recycling Pathways for Dopamine D₂Receptor

Abstract

Dopamine D 2 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 activitydependent 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.

The Serotonin Transporter Undergoes Constitutive Internalization and Is Primarily Sorted to Late Endosomes and Lysosomal Degradation

“…It has previously been suggested that constitutively internalized DAT to some extent take advantage of this fast pathway of recycling (17). Furthermore, in a recent study on the dopamine D2 receptor, it was shown that constitutive recycling of dopamine D2 receptor, and hence steady state surface levels, is sensitive to Rab4, whereas agonist-dependent recycling is sensitive to Rab11 (44). Because the transit time in rapid recycling of Rab4-positive endosomes was estimated to occur with a t1 ⁄ 2 of 1-2 min (45), a decrease in TacSERT surface expression would be expected upon treatment with the recycling blocker monensin if the steady state level of surface SERT was dependent on rapid recycling.…”

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

“…Our culture protocol could also be used for other types of neurons in the brain. For example, we recently used this protocol to culture striatal medium spiny neurons in a study to examine dopamine D2 receptor (DRD2) insertion in cultured mouse striatal medium spiny neurons 9 . Figure 2 shows mouse striatal medium spiny neurons cultured using our protocol at DIV 8.…”

“…The use of TIRFM to image superecliptic pHluorin-tagged receptors has been applied to several different types of neuronal receptors [7][8][9][10][11][12][13][14][15][16][17] .…”

“…Attaching a GFP molecule to the extracellular domain of a membrane protein may interfere with the protein's function, and it is therefore critical to verify that this tagging strategy does significantly interfere with the function of the protein under investigation 9 . Moreover, an overexpressed receptor may or may not be subject to the regulatory mechanisms that govern the trafficking of endogenous receptors.…”

“…Standard approaches for examining receptor trafficking include surface biotinylation followed by biochemical detection, which lacks both the necessary spatial and temporal resolutions; and fluorescence microscopy examination of immunolabeled surface receptors, which requires chemical fixation of cells and therefore lacks sufficient temporal resolution [1][2][3][4][5][6] . To overcome these limitations, we and others have developed and employed a new strategy that enables visualization of the dynamic insertion of receptors into the PM with excellent spatial and temporal resolutions [7][8][9][10][11][12][13][14][15][16][17] . The approach includes tagging of a pH-sensitive GFP, the superecliptic pHluorin…”