1 Zentrum für Biochemie und Molekulare Zellbiologie, Biochemie II, Universität Göttingen, Heinrich-Düker-Weg 12, D-37073 Göttingen, Germany
2 School of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, UK
*Author for correspondence (e-mail: pschu{at}gwdg.de)
Accepted September 13, 2001
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SUMMARY |
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Key words: AP-1, clathrin, endocytosis, exocytosis, MPR300/IGF-II receptor
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INTRODUCTION |
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The cation-dependent mannose-6-phosphate receptor (MPR46) and the cation-independent mannose-6-phosphate/IGF-II receptor (MPR300) are transported by AP-1 at the TGN and by the homologous AP-2 complex at the plasma membrane. They transport different sets of soluble lysosomal enzymes from the TGN to endosomes from where the enzymes reach the lysosome. The receptors are transported back from endosomes to the TGN for another round of transport. Both receptors also appear at the plasma membrane. The mechanisms controlling endosome-to-TGN versus endosome-to-plasma-membrane transport are not known (Kornfeld, 1992; Hille-Rehfeld, 1995). The MPR300 is phosphorylated upon TGN export, and phosphorylation of MPR46 appears to influence endosomal sorting. The importance of these modifications for intracellular sorting are however controversial (Johnson and Kornfeld, 1992; Chen et al., 1993; Hemer et al., 1993; Méresse and Hoflack, 1993; Breuer et al., 1997). MPR46 accumulates in µ1A-deficient cells in EEA1-positive early endosomes, because MPR46 is not transported back to the TGN (Meyer et al., 2000). In control cells, MPR300 and MPR46 are found in the same endosomes, but they appear to be differentially sorted in the endosomes (Klumperman et al., 1993; Meyer et al., 2000). Moreover, MPR300 and MPR46 have different transport functions. Although both receptors are transported to the plasma membrane, leading to 10% located at the cell surface, only the MPR300 is able to bind mannose-6-phosphate (M6P)-carrying proteins and other ligands at the cell surface and to deliver them to endosomes. The MPR300 function critical for development and cell survival is the removal of the insulin-like growth factor IGF-II from the circulation. MPR300-deficient mice are larger than their control litter mates at birth and die perinataly. Mice deficient for both MPR300 and IGF-II are viable (Ludwig et al., 1996; Dittmer et al., 1998). MPR300 also binds to the growth and differentiation factor LIF, retinoic acid and the urokinase receptor, and it is the death receptor for granzyme B of cytotoxic T-cells (Kang et al., 1997; Nykjaer et al., 1998; Blanchard et al., 1999; Motyka et al., 2000). The luminal domain of MPR300 is composed of 15 homologous repeats, indicating that the receptor is able to endocytose additional, yet unidentified, ligands. The calculated Stokes radius and the ability to bind different ligands simultaneously indicates an elongated conformation of the receptor (York et al., 1999).
In the µ1A-deficient cells, MPR300 mediated endocytosis of ligands is six to seven times higher than in control cells (Meyer et al., 2000). We analyzed the mechanism underlying this strikingly enhanced endocytic capacity by determining MPR300 transport between the plasma membrane and the endosome and vice versa and its distribution within the plasma membrane.
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MATERIALS AND METHODS |
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MPR300 biotinylation experiments
MPR300 biotinylation/surface biotinylation
The cells were grown on 3 cm ø culture dishes overnight to about 90% confluency. They were washed 4 x 5 minutes with ice cold biotinylation buffer (10 mM NaPi pH 8.0; 135 mM NaCl; 10 mM KCl) and incubated on ice with 1 mg/ml sulfo-NHS-SS-biotin (PIERCE) for 3 hours at 4°C in a cold room with temporary agitation. To remove the unbound biotin reagent, the cells were washed 6 x 5 minutes with biotinylation buffer.
Determination of surface MPR300
Surface biotinylated cells were washed, harvested in TIN (0.5% Triton X-100; 50 mM imidazol pH 7; 150 mM NaCl and proteinase inhibitor mix) and homogenized by ultrasonication. Protein concentration was determined with the BIORAD reagent, and 100 µg samples of each clone were frozen until further analysis. Homogenates were centrifuged for 20 minutes at 48,000 g, supernatants were collected and volume equivalents to 300-500 µg total protein were subjected to precepitation with strepavidin-agarose (Pierce) overnight at 4°C. Bound proteins were spun down for 1 minute at 12,000 g, and agarose beads were washed 5 times with PBS/0.1% Triton X-100. Beads and 100 µg of homogenates were boiled in SDS-PAGE sample buffer under non-reducing conditions for 3 minutes and separated on a 5% SDS-gel. After western transfer onto a nitrocellulose membrane (Sartorius) MPR300 was detected with a polyclonal rabbit serum and HRP-coupled secondary antibody. Signals were developed with the chemoluminescent ECL kit (PIERCE), quantified by densitometry (WinCam 2.2 software). Signals obtained from the biotinylated MPR300 were normalized for the protein content.
Internalisation assay
Cells were biotinylated and washed as described. One set of cells was harvested in TIN to measure total cell surface MPR300. Then the cells were incubated for 1 minute at 37°C in DMEM. Cells were cooled by washing with ice cold buffer, and biotin of cell-surface proteins was cleaved off by incubation for 20 minutes with gluthatione solution (600 mg gluthatione; 9 ml 83 mM NaCl, 1 mM MgCl2, 1 mM CaCl2; 300 µl 10 M NaOH; 100 µl 1 M DTT; 1 ml 10% BSA) on ice. As a control, one set of cells was incubated without rewarming. Cells were homogenized in TIN by ultrasonication, incubated for 30 minutes on ice and centrifuged for 20 minutes at 48,000 g. Supernatants were immunoprecipitated with a polyclonal rabbit serum overnight at 4°C. Precipitates were separated on a 5% non-reducing SDS-gel, transfered onto a nitrocellulose membrane and blocked with 1% BSA in PBS/0.2% Tween 20. Biotinylated MPR300 was detected by incubating with streptavidin-HRP (Dianova; 1:5000 in blocking buffer) for 2 hours. Blots were developed with the ECL-kit (PIERCE) and X-ray film exposure. Films were scanned and signals quantified (WinCam 2.2 software).
Recycling assay
Cells were biotinylated and washed as described and loaded with biotinylated receptors by rewarming for 2 minutes in DMEM at 37°C. The biotin of cell-surface proteins was cleaved off as described above, and one set of cells was harvested in TIN. Sequentially, cells were subjected to rewarming/cleaving for 1, 2 or 3 times to measure appearance of internalized receptors at the cell surface. From the cells, MPR300 was extracted and immunoprecipitated as described above.
MPR300 surface labelling and immunoelectron microscopy
Cells were grown on 35 mm culture dishes to subconfluency. The cells were rinsed twice with PBS and then incubated with 3% BSA on ice for 10 minutes. The cell surface was then labelled with rabbit anti-MPR300 (1:200 in 3% BSA/PBS) on ice for 1 hour. The monolayer was washed on ice four times with 1% BSA, and then twice with PBS, before being fixed in 4% para-formaldehyde on ice for 15 minutes. After washing in PBS and quenching in 0.12% glycine, the bound anti-MPR300 was detected with goat F(ab)2 anti-rabbit IgG 10 nm gold (British BioCell, Cardiff). Finally, the cells were washed in PBS-BSA and PBS, fixed in 1% glutaraldehyde, scraped off the dish, pelleted, postfixed in 1% osmium tetroxide and embedded in Epon. Cell-surface labelling was quantitated from 80 nm thin sections by counting gold particles under the electron microscope. The length of membrane on section was measured by intersection counting (Griffiths, 1993). Statistical significance was tested by applying the Mann-Whitney U-test. It is a non-parametric test, which does not request a normal distribution of the measurement results. p-values below 0.05 indicate statistical significance, and this increases with values smaller then 0.01 and 0.001.
LDL and EGF-R degradation
LDL endocytosis and degradation
Cells were grown in DMEM with 5% FCS in 3 cm ø dishes. The medium was replaced by DMEM with 5% LDL free human serum and cultures were incubated for an additional 24 hours. Purified huLDL was labelled with 125I by the iodine monochloride method to 300 cpm/ng. 300.000 cpm/dish of 125I-labeled huLDL was added to the cultures, and cells were incubated for an additional 24 hours. The accumulated non-TCA-precipitable radioactivity in the cells and media was determined by liquid scintillation counting. Background LDL decay was determined by incubating labelled huLDL-containing medium in the absence of cells (Goldstein et al., 1983).
EGF-R endocytosis and degradation
Cells were grown to confluency in 6 cm dishes, serum starved for 1 hour in DMEM, 1% BSA, 20 mM HEPES pH 7.4, and downregulation was induced by the addition of 500 ng/ml EGF (Santa Cruz Biotechnology Inc.). Cells were washed with PBS, scraped off the plates and protein extracts were prepared. 30 µg of proteins were resolved on 5% SDS-PAGE and blotted onto nitrocellulose membranes. Western blots were developed with rabbit anti-EGF-R serum (Santa Cruz Biotechnology Inc.) and mouse monoclonal anti--adaptin antibodies (BD Transduction Laboratories) as an internal control (Wiley et al., 1991). Quantification was performed as described above.
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RESULTS |
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To determine the fraction of MPR300 present at the cell surface at steady state, the cells were rapidly cooled to 0°C and cell-surface proteins were biotinylated. The biotinylated proteins were collected with streptavidin-agarose. MPR300 was quantified in the cell extracts and in the streptavidin-bound fraction by western-blot (Fig. 1). To our surprise, despite a seven-fold increase in the internalization rate for MPR300 ligands, we did not detect an increase in the number of receptors at the cell surface. In control cells, 11.5% (±2.3; n=3) of total MPR300 was located at the plasma membrane, a value corresponding to data in the literature (Klumperman et al., 1993). In µ1A-deficient cells, the fraction of MPR300 accessible to biotinylation at the plasma membrane was 9.2% (±0.5; n=3).
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MPR300 recycling endosomes
To characterize the endosomes containing MPR300, we performed double-immunofluorescence microscopy for MPR300 and various endosomal markers.
The early endosomal antigen EEA1 interacts with Rab5 and phosphatidylinositol-3-phosphate, facilitating early endosome fusion (Christoforidis et al., 1999). In control cells, we find MPR300/EEA1 colocalization restricted to a few EEA1-positive endosomes (Fig. 4). In µ1A-deficient cells, a large number of EEA1-positive endosomes also contain MPR300. As a measure of colocalization, the yellow spectra of the merged images were quantified. This revealed a 2.3 fold more intense staining in µ1A/ cells compared to control cells. We also performed double immunofluorescence with lysobisphosphatidic acid (LBPA), a marker for late endosomes (Kobayashi et al., 1998). We did not observe colocalization of MPR300 with LBPA in control or µ1A-deficient cells (Fig. 4). We also could not detect colocalization with LAMP-1, another marker for late endosomes and lysosomes (Meyer et al., 2000). This demonstrates a redistribution of MPR300 from the TGN to early endosomes.
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The prohormone convertase furin recycles between the TGN and endosomes, and endosomes and the plasma membrane, as MPR300. Transport out of the TGN occurs via AP-1 clathrin-coated vesicles, and the fraction of furin appearing at the cell surface is endocytosed by AP-2 clathrin-coated vesicles (Schäfer et al., 1995; Molloy et al., 1999; Stroh et al., 1999; Teuchert et al., 1999). Available antibodies did not permit us to study colocalization of MPR300 and furin. To detect a redistribution of furin, we performed double-immunofluorescence microscopy for EEA1 and furin. Similar to MPR300, furin is concentrated in the perinuclear region. In µ1A-deficient cells, it is redistributed into peripheral vesicles. In contrast to the MPR300 colocalization with EEA1, furin is not detectable in either cell line (Fig. 4), indicating that the transport pathways of furin and MPR300 are differentially affected by µ1A deficiency.
Distribution of MPR300 in the plasma membrane
To obtain insight into the mechanisms underlying the enhanced internalization rate of MPR300, we compared MPR300 distribution in the plasma membrane of µ1A-deficient and control cells by immunogold staining. Cells were labelled with anti-MPR300 antibodies at 4°C, fixed and processed for immunoelectron microscopy with goat anti-rabbit IgG gold.
In control cells, only 3.95% of coated pits were labelled with gold particles, whereas in µ1A-deficient cells, 17.28% were labelled (Table 2; Fig. 5). Coated areas were labelled in both cell lines by 1.4 gold particles on average per cell. When clathrin-coated pits and deeply invaginated coated pits were counted, 22.5% fewer clathrin-coated pits were detected in µ1A-deficient cells, but statistical analysis indicates that this is not significantly different. Taken together, these data indicate that in µ1A-deficient cells, a higher fraction of clathrin-coated pits contain MPR300.
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DISCUSSION |
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The fraction of MPR300 present at the plasma membrane has been determined in this and other studies to be 10% (Klumperman et al., 1993). This fraction is not increased in µ1A-deficient cells. The endocytosis rate of the MPR300 receptor however is about five times faster. The increase can account for most, and probably even all, of the seven times higher endocytosis rate observed for MPR300 ligands. The fraction of previously endocytosed receptors that is exocytosed per unit of time was significantly smaller in µ1A-deficient cells. This indicates that the endocytosed receptors mix with a much larger pool of endosomal receptors (Fig. 1; Fig. 2; Fig. 3).
The size of the endosomal pool of MPR300 that recycles between the plasma membrane was calculated from the trafficking rates and the steady-state distributions to be 4% of the total cellular MPR300. In NRK and HepG2 cells expressing human MPR300, the fraction of MPR300 in early endosomes has previously been determined to be 10%±2.3 by immunogold labelling (Klumperman et al., 1993). In µ1A-deficient cells, 90% of the cellular receptors are localized to endosomes from which they recycle to the plasma membrane. Thus, MPR300 endosome-to-TGN retrograde transport is blocked in the µ1A-deficient cells.
We characterized this endosomal MPR300 pool in µ1A/ cells by double-immunofluorescence microscopy for MPR300 and endosomal markers. We found extensive colocalization with EEA1, but none with LBPA or LAMP-1. Colocalization with transferrin-containing endosomes is not higher than in control cells, indicating that MPR300 and transferrin receptors are separated along the recycling pathway (Stoorvogel et al., 1989). Future experiments will have to characterize the MPR300-containing endosomes and the recycling pathway of MPR300.
How can the enhanced internalization rate of MPR300 be achieved? It is apparent from the normal internalization rate observed for LDL-R-mediated uptake of LDL-and EGF-triggered delivery of surface EGF-R to lysosomes that the enhanced internalization rate is specific for MPR300, and possibly a few other receptors, but does not reflect a general increase in AP-2 mediated internalization. More MPR300 were found to be clustered outside clathrin-coated pits in µ1A/ cells compared with control cells. MPR300 itself does not form dimers or homooligomers unlike MPR46. MPR300, however, can be crosslinked by multivalent ligands (York et al., 1999). We can exclude the possibility that ligand-mediated crosslinking contributes to the clustering of MPR300 and its enhanced internalization rate. Treatment of the cells for 2 hours with cycloheximide to clear the biosynthetic pathway from endogenous MPR300 ligands and washing of the cells with M6P did not affect subsequent endocytosis of arylsulfatase A in the presence of cycloheximide (not shown). Therefore only a minor fraction of cell-surface MPR300 is occupied by endogenous ligands.
At the plasma membrane of µ1A-deficient cells, more MPR300 are concentrated in clusters than in control cells. We propose that a larger fraction of MPR300 arrive at the plasma membrane in a more clustered form. These clusters are likely to be more rapidly integrated into clathrin-coated pits, either due to their diffusion as a raft impairing their rapid lateral diffusion in the plane and/or because they serve directly as nucleation centers for rapid formation of clathrin-coated pits. In µ1A-deficient cells, we found 40% more MPR300 clustered outside coated-pits and 4.4-fold more coated pits and vesicles were labelled in µ1A-deficient cells compared with control cells. In conjunction with the faster internalization rate, this indicates that clustering of MPR300 enhances significantly the rate sy which MPR300 is recruited into coated pits and vesicles.
In the absence of AP-1, MPR300 accumulates in endosomes owing to its impaired return to the TGN. We propose that the higher concentration of MPR300 in endosomes favours clustering of the receptor and increases its exocytosis to the plasma membrane. As discussed above, arrival of clustered receptors at the plasma membrane is thought to accelerate its incorporation into clathrin-coated vesicles to an extent that the steady-state concentration at the plasma membrane is not increased inspite of an increased exocytosis rate of MPR300. Thus AP-1-mediated transport of the MPR300 from endosomes to the TGN indirectly controls, in a negative manner, the trafficking rate of the receptor between the plasma membrane and endosomes and thereby controls MPR300-mediated endocytosis of growth and differentiation factors and lysosomal enzymes.
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ACKNOWLEDGMENTS |
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