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Intracellular retention of the two isoforms of the D₂ dopamine receptor promotes endoplasmic reticulum disruption

¹ DEPSN, UPR 2197, Institut de Neurobiologie Alfred Fessard, CNRS, Avenue de la Terrasse, F91198 Gif-sur-Yvette Cedex, France
² UMR 144 CNRS Institut Curie, Laboratoire C Burg, 12 Rue Lhomond, 75005 Paris, France
³ Genaxis Biotechnology, Nîmes, France
^* These authors contributed equally to this work

View larger version (80K): [in a new window]   Fig. 1. Immunodetection of the epitope-tagged D2 receptor isoforms in transfected COS-7 and HeLa cell lines. COS-7 cells (A,B) and HeLa cells (C,D), have been transfected by either D2b, revealed by the monoclonal 9E10 anti-myc antibody coupled to Texas-Red (A,C), or D2a, revealed by the monoclonal P5D4 anti VSV-G antibody coupled to Texas-Red (B,D). These confocal microscope images reveal the large predominance of intracellular labeling detected with these antibodies. Bar, 2.5 µm.

View larger version (31K): [in a new window]   Fig. 2. Effect of tunicamycin on the labeling and the transport of the D2 receptors to the plasma membrane. COS-7 cells transfected by the myc-tagged D2b receptor were treated with tunicamycin for 12 hours at 10 ng/ml (B) and 20 ng/ml (C), and compared with control cells (A). The receptors were visualized with the monoclonal 9E10 antibody, coupled to Texas-Red. Note the massive restriction of the receptor localization close to the nuclear membrane with increasing doses of tunicamycin compared with control cells. Bar, 3 µm.

View larger version (53K): [in a new window]   Fig. 3. Comparison of the distribution of the D2b receptor revealed by anti epitope-tag antibody and GFP-tagged fusion in transiently transfected COS-7 cells. The labeling exhibited by the D2b receptor fused to the GFP is seen both intracellularly and at the plasma membrane (A,B) and is very similar to that obtained with anti-D2 receptor polyclonal antibody, coupled to Texas-Red (C). In sharp contrast, the D2b receptor-GFP construct (B) provides a visualization significantly different from that obtained with the monoclonal 9E10 antibody, coupled to Texas-Red (D), which revealed mainly intracellular compartments. Confocal microscope images; bars, 1 µm.

View larger version (21K): [in a new window]   Fig. 4. Relative quantification of the proportion of the D2 receptor isoforms located at the plasma membrane. After membrane biotinylation (see Materials and Methods), the COS-7 cells transfected by either D2a or D2b receptors were lysed and the whole cell lysate was fractionated by centrifugation to provide a nuclear pellet (N; nuclei and cell debris) and a post-nuclear supernatant. This supernatant was submitted to streptavidine chromatography to separate biotinylated membranes from nonbiotinylated membranes. In the three fractions, the amount of D2a (grey bars) and D2b (empty bars) receptors were quantified by [3H]-spiperone binding (A) and compared with the total activity of alkaline phosphatase, a marker of plasma and nuclear membrane (B). The histograms correspond to values ± s.e.m (n=3).

View larger version (47K): [in a new window]   Fig. 5. The D2 receptor isoforms are not found in the transferrin endocytosis-pathway. The intracellular labeling obtained with rhodamine-labeled transferrin either after 10 minutes (C) or 60 minutes (F) incubation at 37°C does not match that of the tagged-D2b receptor revealed by the monoclonal 9E10 antibody coupled to FITC (A,D) at the same incubation time. Superimposition of confocal images of the transferrin and D2 receptor labeling (B,E) clearly shows that the two types of labeling are mutually exclusive. Confocal microscope images; bar, 2 µm.

View larger version (68K): [in a new window]   Fig. 6. The D2 receptor isoforms are poorly co-localized with Rab6, a marker of the Golgi complex, in COS-7 and HeLa cells. As analyzed with a confocal microscope, the labeling for the D2a isoform obtained with the monoclonal P5D4 antibody, coupled to FITC, in HeLa cells (A) and with the monoclonal 9E10 antibody, coupled to FITC, for the D2b isoform (C), overlap only for a very small part with that obtained with an anti-rab6 polyclonal antibody, coupled to Texas-Red, (B,D). The D2a receptor transfected in COS-7 cells displays essentially the same pictures (E,F). This receptor labeling is not significantly modified by cell treatment with BFA (10 µg/ml) for 1 hour (G), whereas that of Rab6 spreads over the cytoplasm (H). Bar, 2.5 µm.

View larger version (45K): [in a new window]   Fig. 7. The intracellular D2 receptor isoform induces a vacuolization of the endoplasmic reticulum, which is overcome by PTX treatment. In HeLa cells, the co-transfection of the Ii invariant chain-coding vector (the Ii chain is used as an ER marker revealed with a specific polyclonal antibody; 1/2000) coupled to Texas-Red; (B,D) with either the D2a (A,B) or the D2b (C,D) receptor-encoding vectors, labeled with the monoclonal P5D4 (A) and 9E10 (C) antibodies, respectively and revealed by FITC, shows a partial colocalization of the receptor with the ER marker. In addition, the presence of the D2 receptors in these intracellular membranes promotes a dramatic vacuolization of the ER (B,D). When the cells co-transfected by the Ii invariant chain and the D2a (E,F,G,H) are treated by PTX (0.1 µg/ml; E,F) at the same time as the transfection procedure, only minor alterations in the ER morphology are seen 12 hours later (F). Conversely, the ER disruption is still obvious (H) when the incubation with PTX begins only 2 hours after cell transfection (G-H). Confocal microscope images; bars, 2.5 µm.

View larger version (30K): [in a new window]   Fig. 8. Localization of the D1A receptor isoform co-transfected with the D2b receptor in COS-7 cells. D1A-GFP fusion construct expressed in COS-7 cells is mainly present at the plasma membrane (A; confocal microscope image). In cells where it is co-expressed with the D2b receptor, it appears retained into altered intracellular compartments (B). The co-expressed myc-tagged D2 receptor is visualized with the monoclonal 9E10 antibody coupled to Texas-Red (C). Bars, 2 µm.

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