Dopamine responsiveness is regulated by targeted sorting of D2 receptors

Abstract

Aberrant dopaminergic signaling is a critical determinant in multiple psychiatric disorders, and in many disease states, dopamine receptor number is altered. Here we identify a molecular mechanism that selectively targets D2 receptors for degradation after their activation by dopamine. The degradative fate of D2 receptors is determined by an interaction with G protein coupled receptor-associated sorting protein (GASP). As a consequence of this GASP interaction, D2 responses in rat brain fail to resensitize after agonist treatment. Disruption of the D2-GASP interaction facilitates recovery of D2 responses, suggesting that modulation of the D2-GASP interaction is important for the functional down-regulation of D2 receptors.

Fig. 1.

D2Rs degrade and D1s recycle. (A) Immunocytochemical analysis of receptor trafficking. (A Upper) HEK cells stably expressing D2Rs were fed antibody to the extracellular FLAG-tag to label surface receptors and treated with dopamine or left untreated (NT, Left). Treatment with dopamine for 60 min (DA60, Center) promotes endocytosis of D2Rs. After agonist treatment, any remaining surface receptors were stripped of antibody and antagonist haloperidol (H) was added to monitor recycling and prevent further endocytosis. Internalized D2Rs fail to return to the surface in the presence of antagonist (Right). (A Lower) HEK293 cells stably expressing FLAG-tagged D1R were treated as above, except the antagonist SCH23390 (SCH) was used. (B) Antagonist prevents dopamine-induced endoctytosis. HEK cells stably expressing D2Rs (Upper) or D1Rs (Lower) were fed antibody to the extracellular FLAG-tag to label surface receptors. Cells were then treated simultaneously for 60 min with both dopamine and either haloperidol for the D2Rs (H, Upper), or SCH23390 (SCH, Lower). (C) Stability of endocytosed receptor was assessed by using the BPA on the cell lines used for A and in cells stably expressing both receptors. Cells stably expressing D2R (Top and Upper Middle) or D1R (Lower Middle and Bottom) were biotinylated and treated with the agonists dopamine (for both D1R and D2R) or quinpirole (Q, for D2R) for the times indicated. Remaining surface receptors were then stripped of biotin by using a membrane impermeant reducing agent and the “protected” internalized receptor pool was immunoprecipitated and visualized. D2Rs are degraded after internalization with either quinpirole or dopamine (Top, compare DA60 and Q60 with DA180 and Q180), whereas D1Rs showed no apparent degradation after internalization (Upper Middle, compare DA60 and DA180). Cells stably expressing both a FLAG-tagged D2R and an HA-tagged D1R were biotinylated and treated with dopamine for the times indicated. Remaining surface receptors were then stripped of biotin, the lysate divided into two samples and the “protected” internalized receptor pool was immunoprecipitated with either anti-FLAG or -HA antibodies and visualized (Lower Middle and Bottom).

Fig. 2.

GASP modulates D2R trafficking. (A) GASP (Top) coimmunoprecipitates selectively with FLAG-tagged D2Rs (Middle, compare D1R and D2R lanes). (Bottom) Lysate samples immunoblotted for GASP. HEK, HEK293 cells with no additional receptor; IP, immunoprecipitate; IB, immunoblot. (B) Cells were generated that stably overexpress GFP-cGASP. Lysates from cells stably expressing D2R only (left lane, undiluted lysate) or stably expressing both D2R and GFP-cGASP (all other lanes, lysate serially diluted 2-, 4-, 8-, or 80-fold) were immunoblotted for GASP. In the D2R-cGASP cell line, cGASP is >80-fold more abundant than endogenous GASP. (C) Cells stably expressing D2R were transiently transfected with GFP-cGASP. Cells were then treated with dopamine for 30 min (DA30) to monitor endocytosis, or dopamine for 30 min followed by agonist washout and antagonist haloperidol treatment for 30 min (DA30 + H30) to monitor recycling. D2Rs in adjacent cells that did (gray stars) or did not (white arrows) receive GFP-cGASP endocytosed in response to dopamine (Left). D2Rs in cells that overexpressed cGASP were recycled in 30 min (gray stars, Right). (D) Cells stably expressing FLAG-D2R (Upper) and stably overexpressing GFP-cGASP (Lower) were treated with dopamine for 90 min (DA90) to monitor endocytosis (Left) or with dopamine for 90 min, followed by a haloperidol (H) chase to monitor recycling (Right). D2Rs in these cells were recycled (compare to cells with D2R alone Fig. 1 A). (E) Cells from B either stably overexpressing HA-tagged D2R alone (Upper) or overexpressing both GFP-cGASP and HA-tagged D2R (Lower) were monitored for postendocytic trafficking by using the BPA. D2R degradation is attenuated by overexpression of GFP-cGASP (compare Upper and Lower DA120 and Q120 lanes). The D2Rs that were not degraded in the presence of GFP-cGASP were recycled within 30 min (compare DA120 to DA90/H30 and Q120 to Q90/H30 in the GFP-cGASP/D2R cell line, Lower).

Fig. 3.

D2Rs bind to GASP in the rat brain. (A) Rat brain. GASP (Upper) coimmunoprecipitates with the D2R (Lower) from rat brain. Increasing concentrations of D2R antibody (αD2R) immunoprecipitate increasing amounts of both D2R and GASP. L, rat brain lysate. B (beads), protein G agarose alone added. The arrow shows the position of the D2R signal. Upper band is IgG heavy chain. (B) GASP from rat brain interacts with the cytoplasmic tail of the D2R but not the D1R expressed as GST fusion proteins. (Upper) Protein eluted from GST resin that was immunoblotted for GASP. (Lower) The Coomassie stain of the input protein. L, brain lysate; B (beads), empty GST resin; G, empty GST protein; D1, GST-D1R c-tail fusion protein; D2, GST-D2R c-tail fusion protein. (C-H) GASP is localized in dopaminergic neurons of the VTA. (C) Coronal slices were stained by using affinity-purified anti-GASP antibody. (D) The GASP signal in C was blocked by preincubation of the antibody with purified GASP protein (block). (Scale bar, 50 μm.) (E and G) GASP (G) colocalizes with TH (E). GASP is distributed in both cell bodies and dendrites (white arrow). (F and H) D2Rs (H) colocalize with TH (F). D2Rs, like GASP, are also distributed on dendrites (white arrow). (Scale bar, 20 μm.) (I) Recovery of D2R responsiveness in the VTA. Slices were pretreated for 25 min with quinpirole (Q), then washed and allowed to recover for 35 min (washout). Quinpirole was then reapplied to monitor recovery of D2R function. D2R function did not recover. The GABA_B agonist baclofen (Bacl) still elicited a response.

Fig. 4.

GASP modulates D2R responsiveness in the rat VTA. (A) Anti-GASP antibody, but not rabbit IgG, inhibits the in vitro interaction between the GST-D2R-c-tail fusion protein and GASP (compare lanes 4 and 5). (B) Intracellular filling of dopaminergic cells of the VTA with rabbit IgG antibody. (a) Confocal image of a representative VTA cell, which was filled with rabbit IgG antibody during electrophysiological recording. Tissue was stained with Cy3-conjugated anti-rabbit antibody (red) and anti-TH antibody (FITC, green). Nuclei are stained with DAPI (blue). Both neuronal perikaryon and dendrites (arrows in a) are filled with antibody (red). (b) The antibody filled cell is dopaminergic TH-positive (arrow in Right). (c) A high-magnification image of antibody filled dendritic fragments showing the spread of antibody into spines (arrows) presumably containing synaptic sites. (Scale bars, 50 μm in a and 10 μm in c.) (C) Anti-GASP antibody facilitates the recovery of D2R responses in VTA dopaminergic neurons. Neurons filled with 0.5 mg/ml control rabbit IgG antibody (n = 4, green triangles) or 0.5 mg/ml anti-GASP antibody (n = 6, filled red circles) showed hyperpolarization upon application of quinpirole. Rechallenge with quinpirole after washing and recovery for 35 min caused hyperpolarization of only the neurons containing anti-GASP antibody (n = 6, solid red circles). This hyperpolarization was blocked by the D2R antagonist raclopride (Racl 5 μM, n = 4, open red circles), which was added at the start of the washout and was present for the remainder of the experiment.