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1.
Figure 1

Figure 1. From: Distinct Dynamin-dependent and -independent Mechanisms Target Structurally Homologous Dopamine Receptors to Different Endocytic Membranes .

Localization of D1 and D2 dopamine receptors in neuroblastoma cells. HA-tagged D1 receptors (arrowheads) and FLAG-tagged D2 receptors (arrows) present in the plasma membrane of transfected N2A neuroblastoma cells were specifically labeled at 37°C with HA.11 and M1 mAbs, respectively, as described in Materials and Methods. The localization of antibody-labeled receptors was then visualized by fluorescence microscopy after incubation of cells for 30 min in the absence (A–C) or presence (D–F) of 10 μM dopamine.

Ross G. Vickery, et al. J Cell Biol. 1999 January 11;144(1):31-43.
2.
Figure 6

Figure 6. From: Distinct Dynamin-dependent and -independent Mechanisms Target Structurally Homologous Dopamine Receptors to Different Endocytic Membranes .

Neither D1 nor D2 receptors undergo rapid degradation. The degradation rates of biotinylated D1 or D2 receptors (A and B, respectively) were determined in the absence of agonist (dashed line) or in the continuous presence (solid line) of the chemically stable D1 or D2 receptor agonists 6-chloro-PB (10 μM) or quinpirole (10 μM), respectively, as described in Materials and Methods. Each data point represents the mean fraction of biotinylated receptor remaining at various time points (expressed as a percentage of the total amount of biotinylated surface receptor determined at t = 0), determined from densitometric scanning of streptavidin blots from multiple experiments (n = 3). Error bars represent the standard error of the mean.

Ross G. Vickery, et al. J Cell Biol. 1999 January 11;144(1):31-43.
3.
Figure 5

Figure 5. From: Distinct Dynamin-dependent and -independent Mechanisms Target Structurally Homologous Dopamine Receptors to Different Endocytic Membranes .

Endocytosis of D2 receptors is dynamin independent. Stably transfected HEK293 cells expressing FLAG-tagged D2 receptors were transiently transfected with K44E mutant dynamin and analyzed by fluorescence microscopy after incubation for 30 min in the absence (A and B) or presence (C and D) of 10 μM dopamine. In contrast to the complete blockade of D1 receptor internalization caused by dominant negative mutant dynamin, endocytosis of D2 receptors, both in the absence and presence of agonist, was readily detected (A and C, arrows). Biochemical assay of D2 receptor internalization using the surface biotinylation assay confirmed that K44E mutant dynamin did not inhibit either the constitutive (compare lanes 3 and 5) or agonist-induced components (compare lanes 4 and 6) of D2 receptor endocytosis (E, quantitated in F).

Ross G. Vickery, et al. J Cell Biol. 1999 January 11;144(1):31-43.
4.
Figure 8

Figure 8. From: Distinct Dynamin-dependent and -independent Mechanisms Target Structurally Homologous Dopamine Receptors to Different Endocytic Membranes .

Selective endocytosis of D1 and D2 receptors by distinct membrane vesicles in neuroblastoma cells. Neuro2A neuroblastoma cells coexpressing the HA-tagged D1 and FLAG-tagged D2 receptors were surface labeled at 4°C with specific mAbs in the presence of 10 μM dopamine, as described in Materials and Methods. Cells were then warmed to 37°C for various time periods in the continued presence of dopamine, to allow endocytosis of both receptors to occur, then cells were fixed and processed for dual label fluorescence microscopy to visualize the localization of D1 and D2 receptors in the same cells. At short times after warming cells (e.g., 4 min), internalized D1 and D2 receptors were visualized predominantly in different endocytic vesicles, as apparent in the monochrome image pairs (A and B, respectively) and emphasized in the merged color image (C, D1 immunoreactivity is indicated in green and D2 in red). This differential localization of receptors was most pronounced upon examination of regions close to the plasma membrane at higher magnification (C, inset), where endocytic membranes differentially concentrated in D1 (arrowheads) or D2 (arrows) receptors were observed.

Ross G. Vickery, et al. J Cell Biol. 1999 January 11;144(1):31-43.
5.
Figure 4

Figure 4. From: Distinct Dynamin-dependent and -independent Mechanisms Target Structurally Homologous Dopamine Receptors to Different Endocytic Membranes .

Endocytosis of D1 receptors is dynamin dependent. Stably transfected HEK293 cells expressing FLAG-tagged D1 receptors were transiently transfected with an expression plasmid encoding HA-tagged wild-type or K44E mutant dynamin, as described in Materials and Methods, and receptor localization was visualized by fluorescence microscopy. In cells incubated in the absence of agonist, D1 receptors (A) were localized exclusively in the plasma membrane, whereas mutant dynamin was observed throughout the cytoplasm (B). In the presence of dopamine, D1 receptors failed to endocytose in cells expressing mutant dynamin (C and D, respectively) and instead clustered in small puncta that remained associated with the plasma membrane (C, arrows). Identical experiments performed using wild-type dynamin did not block internalization of D1 receptors (not shown), indicating that the inhibition caused by the K44E dominant negative mutant is biochemically specific. (E) The effect of mutant dynamin on D1 receptor endocytosis was analyzed biochemically using the surface biotinylation assay. Mutant dynamin blocked D1 receptor internalization observed in the presence of dopamine (compare lanes 4 and 6) and also blocked the small amount of residual endocytosis observed in the absence of agonist (compare lanes 3 and 5). (F) The effect of mutant dynamin on D1 receptor internalization was quantitated by densitometric scanning of streptavidin blots. This analysis indicated that K44E mutant dynamin reduced agonist-induced internalization of D1 receptors observed after 30 min in the presence of 10 μM dopamine by ∼85% (compare bars 2 and 4).

Ross G. Vickery, et al. J Cell Biol. 1999 January 11;144(1):31-43.
6.
Figure 3

Figure 3. From: Distinct Dynamin-dependent and -independent Mechanisms Target Structurally Homologous Dopamine Receptors to Different Endocytic Membranes .

Localization and endocytosis of D2 receptors in HEK293 cells. The localization of FLAG-tagged D2 receptors expressed in stably transfected HEK293 cells was examined (as in Fig. 2) in cells incubated for 30 min in the absence (A) or presence (B) of 10 μM dopamine. (C) Constitutive and agonist- induced internalization of D2 receptors was assayed biochemically using cell surface biotinylation. The total amount of biotinylated surface D2 receptor and the efficiency of biotin cleavage are indicated in lanes 1 (Total) and 2 (Strip), respectively. Receptor internalization was determined in cells incubated at 37°C either in the absence or presence of 10 μM dopamine for 5 min (lanes 3 and 4), 15 min (lane 5 and 6), 30 min (lanes 7 and 8), or 60 min (lanes 9 and 10). To test the role of antagonist on endocytosis, internalization of D2 receptors was compared in cells incubated for 30 min in the absence of ligand (inset, lane 1), in the presence of 1 μM haloperidol (inset, lane 2), or in the presence of 1 μM haloperidol combined with 10 μM dopamine (inset, lane 3). The D2 antagonist haloperidol did not by itself induce endocytosis but was unable to inhibit endocytosis of receptors observed in the absence of agonist, confirming that this endocytosis occurs constitutively. (D) Quantitation of these results by densitometric scanning of biotinylation data obtained in the absence (dashed line) or presence (solid line) of 10 μM dopamine. Each data point represents the mean from multiple experiments (n = 3–4), and error bars represent the standard error of the mean.

Ross G. Vickery, et al. J Cell Biol. 1999 January 11;144(1):31-43.
7.
Figure 2

Figure 2. From: Distinct Dynamin-dependent and -independent Mechanisms Target Structurally Homologous Dopamine Receptors to Different Endocytic Membranes .

Localization and endocytosis of D1 receptors in HEK293 cells. The localization of FLAG-tagged D1 receptors expressed in stably transfected HEK293 cells was visualized by fluorescence microscopy in cells incubated for 30 min in the absence (A)   or presence (B) of 10 μM    dopamine. (C) Agonist- induced internalization of D1 receptors was determined biochemically using cell surface biotinylation assay, as described in Materials and Methods. The total amount of D1 receptor protein biotinylated at the cell surface is represented in lane 1 (Total). The efficiency of biotin cleavage from receptors present in the plasma membrane was indicated by the nearly quantitative removal of biotin observed upon incubating biotinylated cells on ice with glutathione, shown in lane 2 (Strip). Internalized receptors were detected by their resistance to cleavage by glutathione at various times after warming cells to 37°C in the absence or presence of 10 μM dopamine for 5 min (lanes 3 and 4), 30 min (lane 5 and 6), or 60 min (lanes 7 and 8). To test the role of antagonist on endocytosis, internalization of D1 receptors was compared in cells incubated for 30 min in the absence of ligand (inset, lane 1), in the presence of 1 μM SCH23390 (inset, lane 2), or in the presence of 1 μM SCH23390 combined with 10 μM dopamine (inset, lane 3). The antagonist SCH23390 by itself had no effect on receptor endocytosis, but it completely inhibited agonist-induced endocytosis caused by dopamine. (D) Receptor internalization was quantitated by densitometric scanning of streptavidin blot data determined in the absence (dashed line) or presence (solid line) of 10 μM dopamine. Each data point represents the mean fraction of endocytosed D1 receptors (expressed as a percentage relative to the total amount of biotinylated receptor present without cleavage, n = 3–4). Error bars represent standard error of the mean.

Ross G. Vickery, et al. J Cell Biol. 1999 January 11;144(1):31-43.
8.
Figure 7

Figure 7. From: Distinct Dynamin-dependent and -independent Mechanisms Target Structurally Homologous Dopamine Receptors to Different Endocytic Membranes .

Both D1 and D2 receptors recycle back to the plasma membrane after endocytosis. A biochemical assay using surface biotinylation was used to determine the recycling of endocytosed receptors (inaccessible to cleavage by glutathione) back to the plasma membrane (where they become susceptible to cleavage by glutathione). HEK293 cells expressing FLAG-tagged D1 receptors were stimulated for 30 min in the presence of 10 μM dopamine to drive endocytosis, chilled to 4°C to arrest membrane trafficking, and then reincubated at 37°C to allow recycling for 5 min (A, lane 2), 15 min (lane 3), or 30 min (lane 4). D1 receptor completely recycled within 15 min when compared with the total pool of endocytosed D1 receptor (compare lanes 1 and 3). The identical experiment was performed using HEK293 cells expressing FLAG-tagged D2 receptors at comparable levels (B). Total endocytosed D2 receptors observed after incubation of cells for 30 min in the presence of 10 μM dopamine were determined (lane 1) and compared with the amount of internalized D2 receptor remaining in identical cells that were washed extensively on ice to remove agonist after stimulating endocytosis, then reincubated at 37°C in the absence of agonist for either 15 min (lane 2) or 30 min (lane 3). This analysis indicated that a significant amount of D2 receptor recycles back to the plasma membrane within 30 min (compare lanes 1 and 3). These biochemical data were quantitated by densitometric scanning of streptavidin blots to compare the recycling of D1 and D2 receptors (C, solid line and dashed line, respectively). Each data point represents the mean fraction of receptors that became accessible to glutathione (expressed as a percentage of the total internalized pool determined at t = 0) from determinations performed in duplicate from samples prepared in two independent experiments. Error bars represent the standard error of the mean. (D–G) Recycling of endocytosed D1 and D2 receptors was visualized by fluorescence microscopy. FLAG-tagged D1 or D2 receptors expressed in HEK293 cells were labeled in the plasma membrane with M1 mAb, and receptor endocytosis was allowed to occur for 30 min in the presence of 10 μM dopamine. Cells were then chilled on ice to arrest membrane trafficking, washed extensively to remove residual dopamine, and antibodies attached to receptors remaining in the plasma membrane were eluted by washing intact cells in the presence of EDTA, thereby specifically labeling only D1 or D2 receptors located in endocytic vesicles (D and F, respectively). Cells were then incubated at 37°C for 30 min in the absence of agonist, and recycling of antibody-labeled D1 and D2 receptors back to the plasma membrane was visualized by fluorescence microscopy (E and G).

Ross G. Vickery, et al. J Cell Biol. 1999 January 11;144(1):31-43.

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