(A) Formation of D1/PSD-95/NMDA receptor complex in HEK293T cells. Cells were transfected with cDNA constructs encoding HA-D1, PSD-95-GFP, and/or NR1/NR2B. Coimmunoprecipitation was performed by incubation of cell lysates with the indicated antibodies followed by immunoblotting. (B) Formation of D1, PSD-95, and NMDA receptor complex in the mouse brain. DOC extracts of mouse forebrain tissues were immunoprecipitated with an anti-D1 antibody and the blots were revealed by antibodies against PSD-95, NR1, NR2A, and NR2B, respectively. (C) Colocalization of D1, PSD-95, and NR1 in subsets of spines or puncta in a dendritic process of a cultured mouse hippocampal neuron. Arrowheads indicate representative puncta where D1, PSD-95 and NR1 colocalize. Scale bar, 2 µm. (D, E) Schematics showing D1 domain structures (D) and GST fusion protein constructions (E). (F) PSD-95 binds distal D1 CT domains. GST-D1 CT2 or GST-D1 CT3, but not GST-D1 CT1 or GST alone precipitated PSD-95 from HEK293 cells overexpressing PSD-95-GFP or mouse striatal cultures. Equal amounts of GST fusion proteins were used in the pull-down assays. IgG was used as a control in all immunoprecipitation experiments. (G) Model depicting molecular interactions (thick bars connecting various proteins) that assemble the D1/PSD-95/NMDA receptor complex. PSD-95 and NR1 interact with distinct but a partially overlapping region on the D1 CT. All experiments were repeated at least three times with similar results.

(A) Coimmunoprecipitation of D1 and NR1 in PSD-95 WT and KO mice. Coimmunoprecipitation was performed on forebrain extracts with an anti-D1 antibody and blotted with an anti-NR1 antibody. Total D1, NR1, and PSD-95 levels were measured by western blots. (B) Densitometric analyses of total D1 and NR1 levels in the forebrain of PSD-95 WT and KO littermates. n = 7. (C) Densitometric analysis of NR1 coprecipitated with an anti-D1 antibody. n = 6. (D) shRNA silencing of PSD-95 in primary hippocampal cultures. Cultured hippocampal neurons were transfected by electroporation with sh-PSD-95 or sh-control. Protein levels of PSD-95 and actin were analyzed by western blots. Experiments were repeated 3 times with essentially identical results. (E) Confocal images of hippocampal neurons transfected with sh-control or sh-PSD-95 shRNAs (left; scale bar, 20 µm). Merged GFP, D1 (red), and NR1 (blue) florescence are shown. Representative endogenous D1 and NR1 clusters on dendritic processes of these neurons are shown on the right (scale bar, 2 µm). (F) Quantification of D1 colocalization with NR1. n = 13 – 30 neurons; all data are expressed as mean ± s.e.m. **, P < 0.01, ***, P < 0.001; two-tailed Student’s t-tests.

(A–D) SKF81297-induced HA-D1 internalization in HEK293 cells expressing HA-D1, PSD-95-WT, and NR1/NR2B (A), HA-D1, PSD-95-ΔNT and NR1/NR2B (B), HA-D1, PSD-95-Δ1&2 and NR1/NR2B (C), or HA-D1, PSD-95-ΔNT,1&2 and NR1/NR2B (D). Note that only the PSD-95 mutant lacking both NT and PDZ1&2 domains failed to block the NMDA receptor-dependent inhibition of D1 internalization (D). Receptor internalization assay was performed as in Figure 4. (E) Summary graph showing the effects of overexpressing the various PSD-95 constructs on NMDA receptor inhibition of D1 internalization. Internalization index was defined as the ratio of internalized to total fluorescence intensities. n = 15–17 cells for each group. Data are expressed as mean ± s.e.m. *, P < 0.05; ***, P < 0.0001; n.s., not significant; Two-tailed Student’s t-tests.

(A) Dose response curves of cAMP production in NR1/NR2B-transfected D1 stable cells. Cells were stimulated with increasing concentrations of SKF81297 for 30 min. Stimulation of NMDA receptors by NMDA (50 µM) and glycine (10 µM) increased the maximum cAMP accumulation. cAMP accumulation was measured with an enzyme immunoassay. (B) Dose response curves of cAMP production in D1 stable cells transfected with NR1/NR2B and PSD-95. Stimulation of NMDA receptors failed to increase the maximum cAMP accumulation. (C) Summary of D1-mediated cAMP production induced by 10 µM SKF81297 from independent experiments (n = 3 – 5). Results are presented in arbitrary units normalized to cAMP levels in cells not stimulated by NMDA/glycine. *, P < 0.01; n.s., not significant; Student’s t-tests. (D) Dose response curves of cAMP production in cells transfected with NMDA receptor, or both NMDA receptors and PSD-95, in the absence of NMDA/glycine stimulation. (E) Unaltered competition binding curves for D1 stable cells transfected with NR1/NR2B in the presence or absence of PSD-95 co-transfection. (F) PSD-95 coexpression did not affect IC₅₀ or the total binding determined at saturating doses. The data represents the mean ± s.e.m. of three independent experiments. Competition binding experiments were carried out using 400 pM [³H]SCH23390 and 9 concentrations of competing SKF81297 ranging from 1 pM to 100 µM. Each data point in A, B, D, E represents the mean ± s.e.m. of three replications. All experiments were repeated at least three times with essentially identical results.

(A) Upon activation by DA, stand-alone D1 receptor stimulates adenylate cyclase, increases the production of cAMP, and signals through the PKA/DARPP-32/PP1 cascade to modulate various downstream effectors and substrates, including NMDA receptors. D1 receptors also undergo agonist-induced internalization via the classical dynamin-dependent, vesicle-mediated endocytic pathway (not shown), to regulate the surface availability and responsiveness of the receptor. (B) Association with NMDA receptors can enhance D1 surface availability and signaling through multiple mechanisms, e.g. inhibition of D1 endocytosis, facilitation of D1 exocytosis (not shown), or allosteric trapping by NMDA receptors (not shown), leading to a reciprocal facilitation of the NMDA receptor through the PKA/DARPP-32/PP1 cascade. (C) Presence of PSD-95 in the D1-NMDA receptor complex places a physical barrier between these receptors, weakening their interaction. This physical interference could in theory abolish NMDA receptor-dependent D1 surface recruitment, regardless of the mechanisms involved. As a result, D1-mediated cAMP signaling is dampened, NMDA receptor potentiation is suppressed, and the positive D1-NMDA receptor feedback is ultimately antagonized. PSD-95 also promotes constitutive D1 endocytosis, adding another level of inhibition on D1 signaling. Line thickness and arrowhead size indicate signaling strength.

(A) Coimmunoprecipitation of HA-D1 and NR1 in the presence or absence of PSD-95 overexpression in HEK293T cells. (B, C) Densitometric analyses of total (B) and coprecipitated (C) NR1 and D1 receptors. n = 3–4; data represent mean ± s.e.m. *, P < 0.05, two-tailed Student’s t test. (D) Schematic drawing of PSD-95 truncation constructs. (E) Effects of PSD-95 mutants on D1-NR1 interaction, as measured by coimmunoprecipitation in the presence or absence of overexpression of different PSD-95 constructs. HEK293T cells were transfected with HA-D1, NR1/NR2B, and different PSD-95-GFP constructs or a mock vector. Coimmunoprecipitation was performed by incubating HEK293T cell lysates with an anti-NR1 antibody followed by immunoblotting with an anti-HA or an anti-NR1 antibody. Total levels of D1 and NR1 were measured by western blots. Experiments were repeated 4 times with similar results.

HEK293 cells were transfected with HA-D1 alone (A), HA-D1 and PSD-95-GFP (B), HA-D1 and NR1/NR2B (C), or HA-D1, PSD-95-GFP, and NR1/NR2B (D). Surface receptors were live-conjugated with an anti-HA antibody, and were allowed for endocytosis (37°C, 30 min) under the indicated stimulation conditions. Following internalization, cells were processed for differential staining of remaining surface (before permeabilization) and internalized (after permeabilization) receptors.

(A) Agonist-induced D1 internalization in hippocampal neurons. Neurons were cotransfected with HA-D1 and sh-PSD-95 or sh-control. HA-D1 internalization was measured by an immunofluorescence-based antibody uptake assay. Representative images of the surface (left column) and internalized (middle column) HA-D1 receptors in sh-control- or sh-PSD-95-transfected neurons (identified by GFP expression, right column) under different stimulation conditions are shown. Surface HA-D1 receptors were live-conjugated with an anti-HA antibody and were allowed for endocytosis (37°C, 30 min) in normal culture medium or medium containing SKF81297 (10 µM), NMDA (50 µM)/glycine (10 µM), or both. (B) Quantification of HA-D1 internalization in neurons. n = 10–15 neurons per group. Data are expressed as mean ± s.e.m. *, P < 0.05; n.s., not significant; Two-tailed Student’s t-tests.

(A) shRNA silencing of PSD-95 in striatal cultures. Dissociated striatal neurons were infected with sh-PSD-95 or sh-control lentivirus. Protein levels of PSD-95, D1, and actin were analyzed by western blots. (B, C) D1-mediated cAMP production in striatal neurons infected with sh-PSD-95 (C) or sh-control (B) lentiviruses. Neurons were stimulated (30 min) with combinations of SKF81297 (10 µM), NMDA (50 µM)/glycine (10 µM), and MK-801 (10 µM) as indicated. Data represent mean ± s.e.m. of three replications. *, P < 0.05; **, P < 0.01; ***, P < 0.001; Two-tailed Student’s t test. Experiments were repeated at least four times with similar results.