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Results: 9

1.
Figure 6

Figure 6. Analysis of the proteolytic fragmentation of lysine-mutant DORs show delayed destruction specifically of the receptor endodomain. From: The role of ubiquitination in lysosomal trafficking of ?-opioid receptors.

HEK293 cells stably expressing F-DOR-0cK-HA were incubated with 10μM DADLE for the indicated time period before lysis. HA-linked fragments were immunoprecipitated, treated with PNGaseF, and separated via SDS-PAGE. Shown is a representative anti-HA blot (A). Proteolytic fragments resolved to the indicated sizes, corresponding topologically as diagrammed in (B).

Anastasia G Henry, et al. Traffic. ;12(2):170-184.
2.
Figure 7

Figure 7. Persistence of proteolytic cleavage products is mimicked by treatment with Wortmannin. From: The role of ubiquitination in lysosomal trafficking of ?-opioid receptors.

HEK293 cells stably expressing F-DOR-HA were pretreated with vehicle control (DMSO) or 500nM Wortmannin before incubation with 10μM DADLE for the indicated time period before lysis and division into two identical samples. Shown are representative anti-FLAG (A) or anti-HA (C) Western blots. Arrows denote major proteolytic cleavage products, and the highlighted box shows a darker exposure of the proteolytic product at ~30kDa. Blots generated across multiple experiments were scanned and densitometry performed to estimate the amount of FLAG-tagged receptors remaining at each time point relative to agonist naïve cells (C), and the relative abundance of one anti-HA immunoreactive proteolytic product (denoted *) expressed as a percentage of density at 3 hrs agonist treatment (D). Shown are the mean and SEM of multiple experiments (n=6); closed symbols indicate densitometry in control, DMSO treated cells, and open symbols indicate cells treated with 500nM Wortmannin.

Anastasia G Henry, et al. Traffic. ;12(2):170-184.
3.
Figure 9

Figure 9. Model for sequential ubiquitin-independent –and dependent regulation of DOR degradation. From: The role of ubiquitination in lysosomal trafficking of ?-opioid receptors.

The wild type DOR, as well as the lysine mutant DOR0cK that cannot be ubiquitinated, undergo regulated endocytosis following ligand-induced activation (1). Receptors are prevented from traversing the default recycling pathway by 'Sorting step I', which does not require receptor ubiquitination and is sensitive to ubiquitination-independent interaction of receptors with GASPs (2). Receptors undergo topological sorting from the limiting membrane to ILVs; this represents a discrete operation that we call 'Sorting step II'. Sorting step II resembles canonical ubiquitin-dependent sorting of other cargo and requires the ESCRT. The difference is that DORs can still undergo transfer to ILVs, albeit with moderately reduced rate or efficiency, when receptor ubiquitination is prevented (3). This sequential organization of discrete sorting operations, together with (partial) ubiquitination-dependence specifically of the downstream step, explains the ability of lysine-mutant DORs to down-regulate effectively via the canonical pathway. Accordingly, lysine mutation causes a selective and partial inhibition of later proteolytic events that require protease access to the receptor’s cytoplasmic surface (4).

Anastasia G Henry, et al. Traffic. ;12(2):170-184.
4.
Figure 1

Figure 1. Both DOR and DOR-0cK are efficiently excluded from the recycling pathway and can undergo transfer in ILVs of endosomes. From: The role of ubiquitination in lysosomal trafficking of ?-opioid receptors.

A) Schematic representation of the N and C terminally tagged DOR, indicating the positions of the respective tags and lysine residues (K) B) Recycling time course of FLAG-DOR-HA and FLAG-DOR-0cK-HA receptors relative to transferrin receptors. For the opioid receptors, internalization of antibody-labeled receptors was carried out by 30-min pre-incubation with 10μM DADLE. Cells were washed, incubated in the presence of 10μM naloxone, and antibody efflux was assayed at the indicated time points. Recycling of transferrin receptors was estimated by efflux of Alexa488-conjugated transferrin. Points represent mean determinations calculated from three independent experiments. Error bars represent the SEM calculated across experiments (n = 3). HEK293 cells stably expressing F-DOR-HA (A) or F-DOR-0cK-HA (C and D) were treated with 10μM DADLE for 90 minutes before fixation and preparation for cryosectioning, immunolabeling and electron microscopy as described in Materials and Methods. Shown are representative micrographs using anti-HA labeled with 10nm gold particles. Both F-DOR-HA and F-DOR-0cK-HA were resolved in association with intralumenal vesicle membranes of multivesicular bodies. Scale bars indicate 200 nm.

Anastasia G Henry, et al. Traffic. ;12(2):170-184.
5.
Figure 5

Figure 5. Differential proteolysis of N and C-terminal fragments of DOR and DOR-0cK. From: The role of ubiquitination in lysosomal trafficking of ?-opioid receptors.

A) Schematic representation of doubly-tagged receptor constructs of F-DOR-HA and F-DOR-0cK-HA indicating the N-terminal and C-terminal locations of the FLAG and HA epitope tags, respectively. HEK293 cells stably expressing F-DOR-HA (C and F) or F-DOR-0cK-HA (D and G) were incubated with 10μM DADLE for the indicated time period before lysis and division into two identical samples. Shown are representative anti-FLAG blots (C and D) and anti-HA blots (F and G) as indicated. Bracket indicates position of the full-length receptor species. Arrows denote major proteolytic cleavage products. E) Blots generated in multiple experiments were scanned to estimate the amount of FLAG-tagged receptor remaining at each time point after incubation in the presence of 10μM DADLE, expressed as a percentage of that in cells not exposed to agonist, results were pooled and averaged across multiple experiments (shown are mean and SEM, n=5). H) Anti-HA blots were scanned and the relative immunoreactivity of one proteolytic product (denoted *) was measured and expressed as a percentage of density at 3 hrs, where the band for DOR was most intense. Closed symbols indicate the degradation curve measured for F-DOR-HA, open symbols indicate the degradation curve measured in cells transfected with F-DOR-0cK-HA (mean and SEM, n=5)

Anastasia G Henry, et al. Traffic. ;12(2):170-184.
6.
Figure 2

Figure 2. Enlargement of endosomes by Rab5 manipulation illustrates lysyl-mutant DORs differ in their extent of transfer to ILVs. From: The role of ubiquitination in lysosomal trafficking of ?-opioid receptors.

A and B) HEK293 cells were transiently transfected with CFP-Rab5Q79L and either F-DOR-GFP (A) or F-DOR-0cK-GFP (B), and replated onto to coverslips before treatment for 90 minutes with 10μM DADLE. Cells were then imaged by spinning disc confocal microscopy as described in Materials and Methods. Shown are representative still images of the representative acquired image series, scale bars indicate 5μm. C) Line scan analysis to quantify receptor localization to the intralumenal compartment. Normalized diameter represents the diameter of the endosome shown, where 0 and 100 correspond to the pixel distances with the first and second maximum pixel intensities measured across the dashed line, respectively (see inset image). Blue and red traces represent the normalized pixel intensity measured across the dashed line in the blue and red boxes in A and B respectively, where the maximum pixel intensity across the line is normalized to 100. The black box highlights the normalized fluorescence values of pixels from 40 to 60% of the normalized diameter. D) Compiled results of line scan analysis (mean and SEM, *** p<0.001, Student's t-test, n= 88 endosomes, ≥12 cells).

Anastasia G Henry, et al. Traffic. ;12(2):170-184.
7.
Figure 3

Figure 3. Differences in the extent of DOR transfer to ILVs can be detected by live-cell imaging of non-enlarged endosomes. From: The role of ubiquitination in lysosomal trafficking of ?-opioid receptors.

A and B) Live cell imaging of non-enlarged endosomes in HEK293 cells expressing either F-DOR-GFP (A) or F-DOR-0cK-GFP (B) imaged live by spinning disc confocal microscopy after exposure for 90 minutes to 10μM DADLE. Shown are representative still images of the representative acquired image series (see Supplemental Movie 1 and 2. Scale bars are 2μm and 1μm for the insets. C) Quantification of the middle fluorescence, or percentage of endosomal membrane, measured from individual endosomes. Mean and SEM of middle fluorescence values are shown for F-DOR-GFP transfected cells (DOR-GFP, n= 12 cells, 50 endosomes) and F-DOR0cK-GFP transfected cells (** p<0.01, Student's t-test, n=59 endosomes, 13 cells). D-F) The same experiment in cells transfected with F-DOR-GFP and pretreated with 500nM wortmannin (D) or transfected 48 hours before DADLE addition with mCherry-HRS (E). Scale bar on overall image = 2μm and on inset = 1 μm. F) Fluorescence intensity was measured through the center of the endosome as in (A-C) and the mean internal fluorescence is expressed as a percentage of that of the membrane of wortmannin pre-treated F-DOR-GFP transfected cells (Wortmannin, unpaired t-test; ***, p<0.0001, n= 11 cells, 50 endosomes), and F-DOR-GFP and mCherry-HRS transfected cells (HRS, unpaired t-test; ***, p<0.0001, n= 10 cells, 64 endosomes). D) The same experiment in cells transfected with F-DOR0cK-GFP and pretreated with 500nM wortmannin (G) or transfected 48 hours before DADLE addition with mCherry-HRS (H). Scale bar on overall image = 2μm and on inset = 1 μm. I) Quantification of the middle fluorescence. Mean and SEM of middle fluorescence values are shown for wortmannin pre-treated F-DOR0cK-GFP transfected cells (Wortmannin, unpaired t-test; ***, p<0.001, n= 11 cells, 92 endosomes), and F-DOR0cK-GFP and mCherry-HRS transfected cells (HRS, unpaired t-test; **, p<0.01, n= 10 cells, 116 endosomes).

Anastasia G Henry, et al. Traffic. ;12(2):170-184.
8.
Figure 4

Figure 4. Intralumenal wild -type and lysyl-mutant receptors visualized using a pH-sensitive GFP variant. From: The role of ubiquitination in lysosomal trafficking of ?-opioid receptors.

A) Schematic of experimental setup following receptors fused to the GFP variant (ecliptic pHluorin) which fluoresce when located in the cytoplasm but whose fluorescence is efficiently quenched when exposed to the acidic environment of the endosome lumen. Addition of the weak base chloroquine neutralizes endosomal pH and reveals any tagged receptors present in the endosome lumen. B and C) HEK293 cells were transiently transfected with CFP-Rab5Q79L and either F-DOR-SpH (B) or F-DOR-0cK-SpH (C), and replated onto to coverslips before treatment for 90 minutes with 10μM DADLE. Cells were then imaged at a rate of 5 frames per second by spinning disc confocal microscopy and treated with 1mM chloroquine after 50 frames. Image sequences are shown for cells expressing F-DOR-SpH (B) or F-DOR-0cK-SpH (C), see Supplemental Movies 3 and 4 for full sequence. D) Quantification of the chloroquine-induced increase in fluorescence intensity. Mean and SEM are shown for F-DOR-SpH (Endosomal DOR-SpH, n= 8 cells, 24 endosomes) and F-DOR0cK-SpH containing endosomes (Endosomal DOR0cK-SpH, unpaired t-test; ***, p<0.01, n= 7 cells, 22 endosomes), and F-DOR-SpH expressed on the plasma membrane (Plasma Membrane, n= 11 cells). Scale bar = 2 μm.

Anastasia G Henry, et al. Traffic. ;12(2):170-184.
9.
Figure 8

Figure 8. The E3 ubiquitin ligase AIP4 regulates transfer of DORs to ILVs and subsequent C-terminal proteolysis of receptors. From: The role of ubiquitination in lysosomal trafficking of ?-opioid receptors.

A and B) HEK293 cells stably expressing F-DOR-HA were transfected with either control plasmid (pcDNA) or one expressing mycAIP4-C/A. Cells were then incubated with 10μM DADLE for the indicated time period before lysis and division into two identical samples. Blots generated across multiple experiments were scanned and densitometry performed to estimate the amount of FLAG-tagged receptors remaining at each time point relative to agonist naïve cells (A), and the relative abundance of one anti-HA immunoreactive proteolytic product expressed as a percentage of density at 3 hrs agonist treatment (B). Shown are the mean and SEM of multiple experiments (n=6); closed symbols indicate results from control-transfected cells, and open symbols from cells expressing mycAIP4-C/A. C) HEK 293 cells were transfected with F-DOR-GFP and mCherry-AIP4-C/A or mCherry-Nedd4-1-C/A, replated onto coverslips, and incubated for 90 min with 10μM DADLE before imaging. Quantification of the middle fluorescence measured from individual endosomes is shown. Mean and SEM of middle fluorescence values are shown for F-DOR-GFP transfected cells (Control, n= 12 cells, 50 endosomes), F-DOR-GFP and mCherry AIP4-C/A transfected cells (AIP4-C/A, unpaired t-test; ***, p<0.0001, n= 16 cells, 51 endosomes), and F-DOR-GFP and mCherry-Nedd 4-1-C/A transfected cells (Nedd4-1-C/A, n= 10 cells, 82 endosomes). D) HEK 293 cells were transfected with F-DOR0cK-GFP and mCherry-AIP4-C/A, replated onto coverslips, and incubated for 90 min with 10μM DADLE before imaging. the quantification of the mean middle fluorescence, measured from individual endosomes of Control (n= 14 cells, 59 endosomes) and AIP4-C/A (n= 11 cells, 53 endosomes) is shown.

Anastasia G Henry, et al. Traffic. ;12(2):170-184.

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