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First published online 19 September 2006
doi: 10.1242/jcs.03217

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The adaptor protein Dab2 sorts LDL receptors into coated pits independently of AP-2 and ARH

¹ Molecular and Cellular Biology Program, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue N, Seattle, WA 98109, USA
² Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue N, Seattle, WA 98109, USA

View larger version (23K): [in a new window]   Fig. 1. Knockdown of both Dab2 and ARH results in accumulation of LDLR at the cell surface. (A) Western blot analysis of HeLa total cell lysates demonstrates that ARH, Dab2 and CHC were efficiently depleted following two rounds of transfection with short RNA duplexes specific for each target. As a control, MAPK levels remained constant in siRNA-treated cells. (B) Surface proteins were biotinylated and precipitated using streptavidin-linked agarose beads followed by western blot analysis for LDLR. Total cell lysates were also analyzed for LDLR on the same gel. (C) Densitometry was used to compare surface (biotinylated) LDLR to total cellular LDLR for each siRNA condition. The steady-state ratio of surface: total LDLR in control cells was set as 1. Depletion of ARH did not significantly change the amount of LDLR at the surface, whereas depletion of Dab2 caused a minor but significant increase (*P<0.01 as determined by t-test analysis). Depletion of both ARH and Dab2, or CHC, caused significant increases in relative surface LDLR (**P<0.001).

View larger version (34K): [in a new window]   Fig. 2. Endocytosis of LDLR, but not the TfnR, is inhibited in the absence of both ARH and Dab2. (A-F) LDLR antibody internalization. siRNA-treated cells on coverslips were incubated at 4°C with anti-LDLR for 1 hour, followed by internalization for 0 or 10 minutes at 25°C. Non-internalized antibody was removed by acid stripping and cells were fixed and permeabilized. Internalized antibody was detected by immunofluorescence using a DeltaVision microscope. A 4 µm flattened Z-projection of a representative cell is shown for each condition. Bar, 15 µm. (G) The internalization experiment was repeated three times and for each experiment approximately 100 cells were assessed for internalization for each condition. Percent of cells internalizing antibody was averaged for each experiment. Depletion of both ARH and Dab2 or CHC caused a significant reduction in LDLR endocytosis (**P<0.001, as determined by t-test). (H) On separate coverslips, knockdown efficiency was evaluated for each target by immunofluorescence. Approximately 100 cells were analyzed for each siRNA. (I-Q) LDLR antibody and Alexa Fluor-488–Tfn internalization. LDLR internalization was analyzed as for A-F with the addition of labeled-Tfn to assess TfnR endocytosis. A 4 µm Z-stack projection is shown for a representative field for control (I-K), Dab2/ARH-depleted (L-N), and CHC-depleted cells (O-Q). LDLR endocytosis was intact in control cells (I), but was inhibited by depletion of ARH/Dab2 (L) and CHC (O). Tfn was internalized in control (J) and ARH/Dab2-depleted cells (M), but not in CHC-depleted cells (P). Bar, 15 µm.

View larger version (50K): [in a new window]   Fig. 3. Depleting Dab2 alone inhibits LDLR endocytosis in arh–/– HDFs. (A) Western blot analysis. arh–/– HDFs were treated with siRNA for Dab2 and CHC, and cell lysates were analyzed by western blot for efficiency of knockdown and total LDLR levels. (B-D) LDLR endocytosis was assessed in arh–/– fibroblasts as in Fig. 2A-F. Bar, 15 µm. (E) The percent of cells internalizing LDLR was determined in two separate experiments by counting approximately 50 cells of each condition, and these were averaged together. Depletion of either Dab2 or CHC caused a significant block in LDLR endocytosis (**P<0.01).

View larger version (17K): [in a new window]   Fig. 4. Dab2-depletion slows the rate of miniLDLR endocytosis. siRNA-treated HeLa cells were detached from dishes with 10 mM EDTA, and antibody-binding and -internalization experiments were performed on cells in suspension. The mean fluorescence intensity for each population was measured by FACS analysis, and average values and standard errors are shown for three independent experiments. (A) Initial steady-state receptor levels in control versus siRNA-treated cells were compared (P<0.05 as determined by t-test). (B) Receptor endocytosis was followed by monitoring the decrease in surface receptor levels following 0, 5 and 10 minutes at 25°C (*P<0.01 and P<0.05 as determined by t-test).

View larger version (72K): [in a new window]   Fig. 5. Dab2-mediated endocytosis does not require AP-2. (A,B) Treatment with siRNA to µ2 adaptin causes loss of adaptin from coated pits. Single 0.2-µm sections at the bottom surfaces of representative cells are shown. (C-F) Steady-state surface levels of TfnR and miniLDLR were measured in control and siRNA-treated cells. Cell surface proteins were biotinylated. For TfnR analysis, lysates were immunoprecipitated using a mouse anti-TfnR antibody, and precipitated proteins were analyzed by western blot analysis first using streptavidin-HRP to detect biotinylated receptor (C, top), and then with rabbit anti-TfnR to detect total cellular TfnR (C, bottom). (E) Ratios of surface to total TfnR were determined for all conditions and compared with control cells. Depletion of AP-2 µ2 in combination with any other protein caused an increase in TfnR at the surface. For the miniLDLR, biotinylated proteins were precipitated with streptavidin-agarose beads and analyzed by western blotting using anti-HA antibody to detect the receptor (D, top). Total cell lysates were also analyzed to determine the total amount of miniLDLR (D, bottom). (F) Ratios of surface to total miniLDLR were determined for each condition and compared with controls. Depletion of AP-2 µ2 caused a large increase in miniLDLR at the surface only if Dab2 was also depleted. Error bars in E and F represent range of duplicate experiments. (G-M) Endocytosis of miniLDLR was measured in siRNA-treated cells. 4 µm Z-stack projections are shown for a representative field from each condition. No internal receptor was detected at 0 minutes (G). Following a 10-minute incubation, miniLDLR was internalized in control cells (H) but not in ARH/Dab2-depleted cells (I). Depletion of AP-2 µ2 alone (J) or in combination with ARH (K) did not affect miniLDLR endocytosis, but in combination with Dab2 (L) or Dab2/ARH (M), endocytosis was completely blocked. Bars, 15 µm.

View larger version (61K): [in a new window]   Fig. 6. Surface distribution of miniLDLR is altered in the absence of Dab2. (A-E) Surface miniLDLR distribution in non-permeabilized siRNA-treated cells. The miniLDLR was detected with anti-HA antibody and visualized using a DeltaVision microscope. Single 0.2-µm sections at the bottom surfaces of representative cells are shown. Dab2 and ARH immunofluorescence (not shown) was used to confirm depletion. (F) To quantify receptor distribution, a 100x100 pixel area was analyzed for 10-14 cells of each condition. The average histogram values for these images were plotted. Punctate staining (as in control and ARH-depleted cells) results in a high number of black pixels (arrowhead) and bright white pixels spread out over a broad range of intensities (arrow, magnified region). Diffuse staining (as in Dab2, Dab2/ARH, and CHC siRNA cells) results in fewer black pixels, more grey pixels (open arrowhead) and fewer white pixels. Based on the histogram for control cells, a threshold (TH) was set. Pixels above this TH indicate clustered receptor and below this threshold indicate diffuse receptor. Similar results were obtained in independent experiments. (G) A plot of the mean pixel intensity above and below the TH shows that in Dab2, ARH/Dab2, and CHC siRNA-treated cells, there is less clustered receptor and more diffuse receptor compared with control cells and ARH siRNA treated cells (**P<0.001). (H-L) Surface TfnR distribution was compared in siRNA-treated cells. Only in CHC-depleted cells is there a change in receptor localization, where there is more diffuse receptor accumulating on the surface. Bars, 15 µm.

View larger version (48K): [in a new window]   Fig. 7. Dab2-mediated receptor traffic requires binding to clathrin, LDLR and phosphoinositides. (A) Drawing of p96 and p67 LDLR constructs, different motifs, binding sites, PtdIns(4,5)P2 (PIP2) and FxNPxY sites are indicated. (B-G') Surface distribution of miniLDLR for cells depleted of both Dab2 and ARH, re-expressing vector alone, wild-type, or mutant T7-tagged mouse Dab2. miniLDLR was detected in non-permeabilized cells, and then cells were permeabilized and stained for T7-Dab2. Shown are 0.2 µm sections at the bottom surfaces of representative cells. Bar, 15 µm. (H,I) As in Fig. 6, a 100x100 pixel area was selected for approximately ten cells of each condition, and the average histogram for miniLDLR intensity was plotted for each siRNA (not shown). A treshhold (TH) was determined based on the distribution of miniLDLR in control cells, and the mean value above (H) this TH and mean value below (I) this TH was calculated for each condition. Overall pixel intensities were decreased compared with those in Fig. 6 owing to use of isotype-specific secondary antibodies that produce a less intense signal. Receptor remained clustered in control cells and double knockdown cells re-expressing T7-p96, as indicated by the higher mean (above TH) and lower mean (below TH), whereas receptor was not clustered in double knockdown cells re-expressing vector, T7-p67, T7-p96S122T or T7-p96K53Q (**P<0.001; *P<0.01; P<0.05). Bar, 15 µm.

View larger version (18K): [in a new window]   Fig. 8. Proposed model. Dab2 binds to clathrin, PtdIns(4,5)P2 (PIP2), LDLR, MyoVI and accessory proteins, independently of AP-2 (left), and actively mediates LDLR movement to, or trapping in, coated pits (step 1), where it recruits clathrin and completes budding (steps 2,3). By contrast, ARH binds clathrin, PtdIns(4,5)P2 and LDLR but only recruits accessory proteins indirectly via AP-2 (right). Recruitment of LDLR to coated pits by ARH may be by passive diffusion (step 4). As clathrin assembles, it may displace ARH from AP-2 and LDLRs may remain trapped without a specific adaptor (steps 5,6). See text for explanation and evidence.