Supporting Information

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Fig. 5. (A) Quantification of D1 and D2 dopamine receptor (D1R and D2R) internalization. Data from at least three independent experiments in Fig. 1C were quantified and analyzed by using one-way ANOVA, followed by Tukey posttest: dopamine and quinpirole (Q) induced endocytosis of D2Rs: ***, P < 0.001 D2R DA60 and Q60 min versus D2R NT; dopamine induced endocytosis of D1Rs: ***, P < 0.001 D1R 60 min versus D1R NT; postendocytic degradation of D2Rs: ^###, P < 0.001 D2R, DA 180 min versus D2R DA 60 min; ^###, P < 0.001 D2R, Q 180 min versus Q 60 min. (B) Quantification of D2R internalization and recycling in the presence and absence of GFP-cGASP. Data from at least three independent experiments in Fig. 2E were quantified and analyzed by using one-way ANOVAs, followed by Tukey posttest. Dopamine and Q induced endocytosis of D2Rs in the D2R and D2R/GFP-cGASP cell lines: ***, P < 0.001 D2R 60 min versus D2R NT in D2R and D2R/GFP-cGASP cell lines; D2R degradation is attenuated by overexpression of GFP-cGASP, ^££, P < 0.01 D2R, DA120 and Q 120 min versus D2R/GFP-cGASP DA120 and Q 120 min, compare right and left histograms. The D2Rs that were not degraded in the presence of GFP-cGASP were recycled within 30 min: lll, P < 0.001 D2R/GFP- cGASP DA90+H30 and Q90+H30 min versus D2R/GFP-cGASP, DA120 and Q120 min. (C) Immunoblot of VTA lysate probed with anti-GASP antibody.

Fig. 6. Effect of haloperidol on recycling and cell viability. (A and B) Cells stably expressing FLAG-D1R (B) or both FLAG-D1R and hemagglutinin (HA)-D2R (A) were fed antibody to the extracellular tag(s) and treated with 10 mM SKF38393 for 60 min to promote internalization of D1 dopamine receptors (D1R). Remaining surface FLAG antibody was stripped by washing in PBS without calcium and both 20 mM SCH23390 (to prevent additional D1R internalization), and 20 mM haloperidol (to test whether haloperidol blocked recycling) were added. Cells were fixed with 4% formaldehyde in PBS. After fixation, cells were permeabilized in blotto with 0.1% Triton X-100 and stained with fluorescently conjugated subtype-selective secondary antibodies (Alexa Fluor 488-IgG2b for FLAG, Alexa Fluor 594-IgG1 for HA both at 1:500, Molecular Probes). D1Rs efficiently recycled in the presence of 20 mM haloperidol whether D2R was present or not. (C) Cells stably expressing D1R and D2R were treated with haloperidol for 30 or 60 min and cell viability was monitored as described in Supporting Text. Haloperidol did not affect cell viability.

Supporting Text

Cell Viability Test

HEK293 cells stably expressing the SFD1/HAD2 receptors were treated with 20 mM Haloperidol for 30 or 60 min or left untreated. Subsequently, cells were lifted in EDTA and immediately stained with EtBr (nonviable cell count) or lysed and stained with EtBr (total cell count). EtBr-stained nuclei were counted in a Nucleo Counter (New Brunswick Scientific). % viability was calculated as = {([total cell count] – [non-viable cell count])/[total cell count]}´ 100.

Cells stably expressing FLAG-D1R or both FLAG-D1R and HA-D2R were fed antibody to the extracellular tag(s) and treated with 10 m M SKF38393 for 60 min to promote internalization of D1R. Remaining surface FLAG antibody was stripped by washing in PBS without calcium and both 20 mM SCH23390 (to prevent additional D1R internalization) and 20 mM haloperidol (to test whether haloperidol blocked recycling) were added. Cells were fixed with 4% formaldehyde in PBS. After fixation, cells were permeabilized in blotto with 0.1% Triton X-100 and stained with fluorescently conjugated subtype-selective secondary antibodies (Alexa Fluor 488-IgG2b for FLAG, Alexa Fluor 594-IgG1 for hemagglutinin both at 1:500, Molecular Probes). D1Rs efficiently recycled in the presence of 20 mM haloperidol whether D2R was present or not.