Published online before print September 2, 2004
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* Department of Dermatology, University Medical Centre Mannheim, Ruprecht-Karls University Heidelberg, Germany;
Department of Medical Biochemistry and Microbiology, University of Uppsala, Sweden;
Oklahoma Medical Research Foundation, Oklahoma City; and
Graduate School of Pharmaceutical Sciences, Kyoto University, Japan
1 Correspondence: Department of Dermatology, University Medical Centre Mannheim, University of Heidelberg, Theodor-Kutzer Ufer 1-3, 68167 Mannheim, Germany. E-mail: julia.kzhyshkowska{at}haut.ma.uni-heidelberg.de
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2). By immunofluorescense and confocal microscopy, we established that stabilin-1 is preferentially localized in early endosome antigen-1-positive early/sorting endosomes and in recycling endosomes identified by transferrin endocytosis. Association of stabilin-1 was infrequently seen with p62 lck ligand-positive late endosomes and with CD63-positive lysosomes but not in lysosome-associated membrane protein-1-positive lysosomes. Stabilin-1 was also found in the trans-Golgi network (TGN) but not in Golgi stack structures. Glutathione S-transferase pull-down assay revealed that the cytoplasmic tail of stabilin-1 but not stabilin-2 binds to recently discovered Golgi-localized, 
-ear-containing, adenosine 5'-diphosphate-ribosylation factor-binding (GGA) adaptors GGA1, GGA2, and GGA3 long, mediating traffic between Golgi and endosomal/lysosomal compartments. Stabilin-1 did not bind to GGA3 short, which lacks a part of the Vps27p/Hrs/STAM domain. Deletion of DDSLL and LL amino acid motifs resulted in decreased binding of stabilin-1 with GGAs. A small portion of stabilin-1 colocalized with GGA2 and GGA3 in the TGN in M2. Treatment with brefeldin A resulted in accumulation of stabilin-1 in the TGN. Our results suggest that stabilin-1 is involved in the GGA-mediated sorting processes at the interface of the biosynthetic and endosomal pathways; similarly to other GGA-interacting proteins, stabilin-1 may thus function in endocytic and secretory processes of human macrophages.
Key Words: receptor • endocytosis • traffic • fasciclin domain • sorting signal
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In addition to endothelial cells, stabilin-1, but not stabilin-2, is expressed in mononuclear phagocytes (macrophages). In vivo stabilin-1-positive macrophages are most prominent in placenta and decidua, but they are also found in skin, gut, pancreas, and in cardiac and skeletal muscle [2 , 3 , 12 ]. Macrophages with strong stabilin-1 expression were also detected in several skin lesions [7 , 8 ], and stabilin-1 is used as a diagnostic marker for cutaneous non-Langerhans cell histiocytoses (reviewed in ref. [13 ]). In vitro stabilin-1 expression can be induced by treatment of peripheral blood-derived monocytes with a combined of interleukin (IL)-4 and dexamethasone (Dex). This type of monocyte stimulation represents a well-established, in vitro model of alternative macrophage activation [1 , 7 , 14 15 16 17 ].
Macrophages are versatile cells that adjust their highly flexible, functional programs to different physiological and pathological situations. Fully polarized macrophages (M1; classically activated) and M2 (alternatively activated) macrophages represent the extremes of a spectrum of functional states. Alternatively, activated macrophages (M2) actively participate in anti-inflammatory and healing processes and are key regulators of induction and maintenance of tolerance toward self-components and environmental antigens. Tumor-infiltrating macrophages acquire a M2-polarized phenotype (reviewed in refs. [15
, 16
, 18
]). The endocytic as well as phagocytic properties of alternatively activated macrophages are enhanced, but they do not exert an increased killing potential toward microbes. A broad range of macrophage receptors executes these functions in M2 [19
]. Stabilin-1 may be counted among these multifunctional receptors involved in protein–protein interactions, and dissection of stabilin-1 intracellular distribution may give a clue to its specific function.
In the present study, we analyzed the localization of stabilin-1 in primary human type 2 macrophages. We show here that stabilin-1-positive macrophages expressed a low amount of the receptor on their surface. Intracellular stabilin-1 is mainly present in early/recycling endosomes and in the trans-Golgi network (TGN), and colocalization in late endosomes and in CD63-positive lysosomes was rarely seen. In addition, we observed that stabilin-1 directly interacts and colocalizes with Golgi-localizing, -ear-containing, adenosine 5'-diphosphate-ribosylation factor (ARF)-binding proteins (GGAs), which are recently identified adaptors mediating the traffic of sorting receptors between Golgi and endosomal/lysosomal compartments. We suggest that stabilin-1 is involved in GGA-mediated sorting between early endosomes and Golgi and similarly to other GGA-interacting receptors, potentially functions in endocytosis and secretion.
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was purchased from Becton Dickinson (Heidelberg, Germany). Generation of F4 rabbit polyclonal antibody directed against the stabilin-1 cytoplasmic tail has been described [1
]. Other primary antibodies were mouse monoclonal against early endosome antigen (EEA)1, p62 lck ligand, lysosomal-associated membrane protein (Lamp)-1, GM130, GGA2, and GGA3 (Becton Dickinson); mouse monoclonal anti-CD63 and sheep polyclonal anti-TGN46 (Serotec, Oxford, UK); and mouse monoclonal against cation-independent mannose 6-phosphate receptor (CI-MPR; Alexis Deutschland, Gruenberg, Germany). Secondary antibodies used for immunofluorescence were Cy2-labeled donkey anti-mouse, Cy3-labeled donkey anti-rabbit, and Cy5-labeled donkey anti-sheep (Dianova, Hamburg, Germany). Secondary antibodies used for fluorescein-activated cell sorter (FACS) analysis were fluorescein isothiocyanate (FITC)-conjugated anti-mouse (Becton Dickinson), FITC-conjugated anti-rabbit (DakoCytomation, Hamburg, Germany), biotinylated anti-mouse (Becton Dicksinson), and phycoerythrin (PE)-streptavidin (Becton Dickinson).
To generate glutathione S-transferase (GST)-fusion cytoplasmic tails of stabilins, the cDNA fragments encoding amino acids 2525–2595 of stabilin-1 and amino acids 2482–2552 of stabilin-2 were cloned into a pGEX-3X vector (Amersham, Freiburg, Germany). pGEX4T1-St1-ED was constructed by polymerase chain reaction (PCR) amplification of an extracellular stabilin-1 fragment corresponding to amino acids 1357–1410. pGEX4T1-St1-C
DDSLL and pGEX4T1-St1-CLL were generated by PCR splicing.
The following plasmids were used for in vitro translation: pcDNA3-HA-GGA1, encoding full-length GGA1; pcDNA3-HA-GGA3 long (L), encoding full-length GGA3L; and pcDNA3-HA-GGA3 short (S), encoding full-length GGA3S. Generation of these plasmids was described [20 ]. The expression construct for full-length GGA2 was pcDNA6-V5-GGA2 [21 ].
Macrophages
Isolation and cultivation of human monocytes/macrophages were done as described [17
] with modifications. The cells were purified from individual buffy coats, which were diluted with Ca2+- and Mg2+-free phosphate-buffered saline (PBS; Biochrom, Berlin, Germany) at a ratio of 1:1. Diluted buffy coats (35 ml) were layered on top of 15 ml Ficoll-Paque (Biochrom) in a 50-ml Leucosep tube (Greiner, Fickenhausen, Germany). After 30 min of centrifugation in a swing-out rotor (Beckman Coulter, Krefeld, Germany) at 650 g, peripheral blood mononuclear cells (PBMC) were collected from the Ficoll serum interphase and were washed three times with PBS (Biochrom). The Percoll gradient was preformed by centrifugation of freshly prepared Percoll [13.5 ml Percoll (Amersham), 1.5 ml 10x Earle’s salts solution, 15 ml Spinner’s medium] at 12000 g for 10 min at 20°C without brakes in an F34-6-38 rotor (Eppendorf, Hamburg, Germany). PBMC (5–8x108) were layered on top of the Percoll gradient and centrifuged at 650 g for 30 min at 20°C without brakes. The upper layer, containing 60–80% monocytes, collected and subjected to negative or positive CD14+ magnetic cell sorting using a monocyte isolation kit or CD14 magnetic beads, respectively (all from Miltenyi Biotech, Bergisch Gladbach, Germany), resulted in 90–98% monocyte purity, confirmed by flow cytometry.
For the culture, monocytes were resuspended in X-vivo 10 serum-free medium (Cambrex, Verviers, Belgium) at a concentration of 5 x 105 cells/ml. The cell suspension was supplemented with the combination of IL-4 10 ng/ml (Tebu Bio, Frankfurt, Germany) and 1 x 10–7 M Dex (Sigma, Munich, Germany) and was transferred to cell-culture dishes. The cells were incubated in the presence of 7.5% CO2 for 6 days and subjected to further analysis.
FACS analysis
For FACS analysis, 5 x 105 cells were taken per sample. All incubations except fixation and permeabilization were performed on ice and all centrifugations, at 4°C.
For the analysis of the surface expression of stabilin-1, macrophages were blocked with 2% bovine serum albumin (BSA) in PBS, incubated for 1 h with MS-1 antibody or IgG1 isotype control (Becton Dickinson), and washed twice with 0.2% BSA in PBS. Biotinylated secondary antibody was added to the samples at 1:100 dilution, incubated 30 min, and washed twice with 0.2% BSA in PBS. For detection, PE-streptavidin was added to the samples at a 1:100 dilution, incubated 30 min, washed with 0.2% BSA in PBS, and analyzed by FACS.
For the intracellular staining, blocked cells were fixed and permeabilized as follows: Cells were fixed in 4% paraformaldehyde (PFA) in PBS for 10 min at room temperature (RT), washed in 0.2% BSA in PBS, and permeabilized in PBS containing 0.25% saponin and 4% BSA. Cells were then stained with primary rabbit polyclonal F4 antibody or control preimmune serum (1:500) for 30 min, washed with 0.1% saponin in PBS, incubated with secondary FITC-labeled, anti-rabbit antibody (1:20) for 20 min, washed with 0.1% saponin in PBS, and analyzed by FACS.
The data were acquired with FACSCalibur (Becton Dickinson) and analyzed with WinMDI 2.8 and WinList (Verity Software House, Topsham, ME) software. To quantify the proportion of positive cells, a histogram subtraction algorithm Super-enhanced DMax (SED) was used.
Transferrin uptake
AlexaFluor488-conjugated transferrin was purchased from Molecular Probes (Leiden, The Netherlands). Transferrin was added directly to the macrophage culture medium in a final concentration, 50 µg/ml. Internalization was performed at 37°C for 5 min, 30 min, and 1 h. After this, cells were replaced on ice and used for cytospin preparation and immunofluorescent analysis.
Berefeldin A treatment
Brefeldin A was purchased from MP Biomedicals (Aurora, OH); stock solution, 5 mg/ml, in ethanol was prepared and stored in aliquots at –20°C. Brefeldin was added directly to cell-culture medium at a concentration of 5 µg/ml, and cells were incubated at 37°C for time-points indicated in Results. Following treatment, cells were used for cytospin preparation and immunofluorescent analysis.
Immunofluorescence and confocal microscopy
Culture plates with macrophages were placed on ice for 20 min. Cells were harvested and used for cytospin preparation (4x104 cells per one cytospin). All fixation and staining procedures were performed at RT. Cells were fixed for 10 min in 2% PFA in PBS, permeabilized for 15 min in 0.5% Triton X-100 in PBS, and fixed for 10 min with 4% PFA in PBS. After extensive washing in PBS, cytospins were dried and stored at –80°C. Staining was performed as described with modifications [22
]. Briefly, samples were blocked with 3% BSA in PBS, incubated with a combination of primary antibodies for 1 h, 15 min, washed, and incubated with a combination of appropriate secondary antibodies. Anti-stabilin-1 F4 and preimmune serum were used at a 1:800 dilution, anti-mouse Cy2 labeled at 1:100, anti-rabbit Cy3 labeled at 1:400, and anti-sheep Cy5, at 1:400. Dilutions of other primary antibodies are available upon request. Samples were mounted with immunofluorescence mounting medium (DakoCytomation, Carpinteria, CA) and analyzed by confocal microscopy.
Confocal laser-scanning microscopy was performed using a Leica TCS SP2 laser-scanning spectral confocal microscope, equipped with a 63 x 1.32 objective. Excitation was with an argon laser, emitting 488 nm; a krypton laser, emitting at 568 nm; and a helium/neon laser, emitting at 633 nm. Data were acquired and analyzed with Leica confocal software. All two- or three-color images were acquired using a sequential scan mode.
In vitro protein–protein interactions
GST pull-down assays were performed in general as described [23
]. GST-fused proteins were expressed in Escherichia coli strain BL21-CodonPlus-RIL (Stratagene, La Jolla, CA). Protein expression and purification were performed under nondenaturing conditions as described; aliquots of proteins immobilized on sepharose 4B (Amersham) were stored at –80°C. Amounts of GST-purified proteins were analyzed by comparison with BSA standards using gel electrophoresis followed by protein staining with Gelcode blue stain reagent (Pierce Biotechnology, Rockford, IL).
35S-Methionine-labeled proteins were generated by in vitro translation according to the standard protocol (Promega, Mannheim, Germany), using 2 µl RedivueL [35S]methionine (Amersham). In vitro translation efficiency for all GGAs was on a similar level, and 5 µl each was used for one binding reaction. Binding was performed in 400 µl ELB (125 mM NaCl, 50 mM HEPES, pH 7.0, 0.1% Nonidet P-40, 0.5 mM dithiothreitol, 0.5 mM EDTA). All binding reactions were rotated at 4°C for 1.5 h, washed five times in 1 ml ELB buffer, and analyzed by polyacrylamide gel electrophoresis (PAGE). Gels were incubated in enhancer solution, dried, and exposed to Kodak BioMax MR film at –80°C.
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2IL-4/Dex). Using rabbit polyclonal F4 antibody generated against the cytoplasmic tail of stabilin-1, FACS analysis of PFA-fixed and saponin-permeabilized cells revealed that 92% of all M2 in the culture expressed stabilin-1 (Fig. 1A
). To analyze the surface expression of stabilin-1 separately in intact cells, M2IL-4/Dex were stained with mouse monoclonal MS-1 antibody [2
]. Unfortunately, MS-1 antibody does not recognize PFA-fixed antigen and therefore, could not be used for intracellular staining. Staining of M2IL-4/Dex with a combination of MS-1 antibody and FITC-labeled secondary antibody did not result in a clear, positive FACS signal (data not shown). To increase the signal intensity, we used the MS-1 antibody in combination with a biotinylated secondary antibody and PE-streptavidin. With this more sensitive method, stabilin-1 was detected on the surface of the same percentage of M2IL-4/Dex (Fig. 1B)
as had been found for permeabilized cells.
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Figure 1. FACS analysis of stabilin-1 expression in M2IL-4/Dex. (A) Staining of permeabilized cells with rabbit polyclonal F4 antibody and preimmune serum as a control, showing 92% positive cells. (B) Surface staining with mouse monoclonal MS-1 antibody and IgG1 as a control, showing 91% positive cells. Dashed line, Isotype control histogram; solid line, positive histogram; filled area, result of positive versus isotype histogram subtraction, performed using the SED method.
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2IL-4/Dex derived from 12 healthy donors. In all macrophage preparations, colocalization of stabilin-1 with EEA1 was observed as depicted in Figure 2
and was especially strong in large endosomes. However, single cells without detectable EEA1 expression containing large stabilin-1-positive vesicles with the shape of standard EEA1-positive endosomes were observed in each macrophage preparation, suggesting that the presence of stabilin-1 in the endosome does not require EEA1 (data not shown). To extend the characterization of stabilin-1-containing endosomes, we performed a time-course experiment of AlexaFluor-488-labeled transferrin uptake. Transferrin is a monomeric serum glycoprotein that binds to the recycling transferrin receptor and therefore, is a marker of recycling endosomes. Macrophages were incubated with transferrin for 5 min, 30 min, and 1 h. At all time-points, high levels of colocalization were observed (Fig. 3
) with more that 90% of the transferrin detected in stabilin-1-positive endosomes.
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Figure 2. Stabilin-1 is localized in EEA1-positive early/sorting endosomes. PFA-fixed macrophages were stained with rabbit polyclonal F4 and mouse monoclonal anti-EEA1 antibody. Secondary antibody combination was Cy2 anti-mouse and Cy3 anti-rabbit. Green corresponds to EEA1 staining, red corresponds to stabilin-1 staining, and yellow corresponds to the area of colocalization (Merge). h, Human.
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Figure 3. Stabilin-1 is present in recycling endosomes. AlexaFluor 488-labeled transferrin (green) was internalized by macrophages for indicated times. Stabilin-1 was visualized in PFA-fixed cells by staining with F4 and anti-rabbit Cy3-labeled antibody (red). Most transferrin colocalized with stabilin-1 (yellow).
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-interacting protein is a ubiquitously expressed protein originally identified as a binding partner of T cell src tyrosine kinase. Its mouse homologue A170 is induced by oxidative stress in mouse macrophages [25
]. Intracellularly, p62 lck ligand was shown to localize to lysosome-targeted, late endosomes; it colocalizes with Rab7 and partially with Lamp-1 and lysosomal integral membrane protein-II but not with Rab5 or the transferrin receptor [26
]. In M2IL-4/Dex, p62 lck ligand was well-expressed and localized in cytoplasmic vesicles (Fig. 4
). Immunofluorescent analysis revealed that stabilin-1 and p62 lck ligand mostly do not colocalize; however, rare sites of clear colocalization were also observed. Double-positive vesicles were, in general, smaller than stabilin-1-positive but p62 lck ligand-negative endosomes.
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Figure 4. Stabilin-1 in late endosomes and lysosomes. Immunofluorescence and confocal microscopy were performed as described in Figure 2
. Limited vesicular colocalization is observed for p62 lck ligand and CD63. Overlapping localization is observed with Lamp-1 only in the perinuclear area but not in cytoplasmic vesicles.
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2IL-4/Dex, we observed CD63-positive vesicles of diverse sizes. In less than 10% of the stabilin-1-positive macrophages, occasional colocalization of stabilin-1 with CD63 was detected (Fig. 4)
.
To investigate the association of stabilin-1 with the lysosomal compartment further, we analyzed its colocalization with Lamp-1, a ubiquitously expressed major lysosomal membrane protein. In the steady state, Lamp-1 is localized on the membrane of lysosomes and late endosomes, but small amounts of Lamp-1 are also found in early endosomal membranes and at the plasma membrane. Newly synthesized Lamp-1 is transported from the TGN to endosomes/lysosomes, mainly via an intracellular route (reviewed in ref. [30
]). In contrast to CD63, Lamp-1 is not enriched in exosomes [31
]. In M2IL-4/Dex, small Lamp-1-positive vesicles, evenly distributed in the cytoplasm, were stabilin-1-negative. In contrast, Lamp-1 staining in the perinuclear space coincided with stabilin-1 staining (Fig. 4)
, suggesting their colocalization in Golgi-associated compartments.
Stabilin-1 and Golgi compartments
With respect to the Golgi apparatus, stabilin-1 was found in extremely close proximity but not colocalized with the Golgi stack compartment, as shown by double-staining for stabilin-1 and for GM130 (Fig. 5
). Next, we analyzed whether stabilin-1 was localized within the TGN, i.e., interconnected tubules and vesicles, at the trans face of the Golgi stack [32
]. For this purpose, we used TGN46, a putative, cargo-binding protein that maintains a steady-state level in the dynamic TGN structure by active retention and recycling. Colocalization of stabilin-1 and TGN46 was observed in the TGN (Fig. 5)
. Taking these data together, we concluded that stabilin-1 may traffic between the endosomal system and the TGN, sparing the lysosomal compartment.
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Figure 5. Stabilin-1 in Golgi compartments. Double-staining with the Golgi marker GM130 (green) and stabilin-1 (red) and double-staining with the TGN marker TGN46 (blue) and stabilin-1 (red).
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Upon searching for a specific motif responsible for the intracellular traffic of stabilin-1, we identified a DDSLL sequence in the middle of its cytosolic tail. The DDSLL motif belongs to the dileucine-based sorting signals of DXXLL type [34 ] (Fig. 6 ). DXXLL signals are described for several receptors and proteins, which cycle between the TGN and endosomes. In addition to the highly conserved residues D and LL, which are strictly required for the sorting signal, the presence of one or more serine residues upstream or within the acidic cluster is a highly specific feature of DXXLL sorting signals. In the cytoplasmic tail of stabilin-1, two serine residues are present that are related to the sorting signal: one located eight amino acids upstream of the motif and one within the acidic cluster (Fig. 6) . Unlike other dileucine-based signals, DXXLL signals do not bind AP complexes; instead, they specifically interact with the Vps27p/Hrs/STAM (VHS) domains of the GGAs [34 35 36 ].
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Figure 6. DXXLL-type sorting signals in cytoplasmic tails of receptors. Bold/shaded, Transmembrane domain (predicted by SOSUI http://sosui.proteome.bio.tuat.ac.jp/sosui_submit.html); boxed/shaded, essential serines; boxed, DXXLL sorting signal. Stabilin-1, but not stabilin-2, contains DXXLL sorting signal.
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Figure 7. Stabilin-1 interaction with GGAs. GST-fused cytoplasmic domains of stabilin-1 and stabilin-2, extracellular EGF-like domain of stabilin-1, and GST alone were immobilized on gluthatione-sepharose and incubated with in vitro-translated 35S-labeled GGA. (A) The binding reaction was analyzed by sodium dodecyl sulfate-PAGE and autoradiography. GGA1, GGA2, and GGA3L bind to stabilin-1, but not to stabilin-2, or to control proteins. (C) GGA2 binding to stabilin-1 cytoplasmic tail with deleted amino acids DDSLL and LL is decreased in comparison with full-length form. (B and D) Control of GST-fused, immobilized protein amounts used for binding experiments (A and C, respectively).
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2IL-4/Dex and exhibited two types of localization. The main area of GGA localization, observed in the majority of macrophages, was in the peri-Golgi-like areas. Partial colocalization of stabilin-1 was observed with GGA2 and GGA3 (Fig. 8
). GGAs function in receptor-mediated cargo transport from the Golgi to the endosomal/lysosomal system. Processing and sorting of receptor/cargo protein complexes by the GGAs occur in the highly dynamic and complex TGN. Indeed, triple immunofluorescent staining revealed that a small portion of stabilin-1 is colocalized with GGA2 and GGA3 in the TGN (Fig. 9
).
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Figure 8. Colocalization of stabilin-1 (red) with GGA2 and GGA3 (green) in human macrophages. Stabilin-1 partially colocalizes with GGA2 and to a lesser extent, with GGA3, preferentially in the TGN areas.
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Figure 9. Triple immunofluorescence for stabilin-1 (red), GGA (green), and TGN46 (blue). Stabilin-1 partially colocalizes with GGA2 and GGA3 in the TGN (white indicates triple-positive structures).
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Brefeldin A affects stabilin-1 localization
It was demonstrated recently that GGAs are recruited to the TGN by virtue of the interaction with guanosine 5'-triphosphate-bound ARF, and ARF regulates GGA-mediated trafficking events from TGN to endosomal/lysosomal compartments [37
]. Brefeldin A is a fungal metabolite, which inhibits guanine exchange factors for ARFs, thereby interfering with membrane association of ARFs. Brefeldin A treatment results in GGA cytoplasmic redistribution within minutes [37
]. We hypothesized that brefeldin A interference with GGA-mediated traffic will result in stabilin-1 redistribution, i.e., in increased localization in TGN. Indeed, treatment of M2IL-4/Dex with brefeldin A resulted in relocalization of a large portion of stabilin-1 from endosomal vesicles to TGN (Fig. 10
). The effect was concentration- and time-dependent. Although single cells showed relocalization of stabilin-1 already after 30 min, clear redistribution was observed after 2 h treatment. Brefeldin A activity was controlled by a complete absence of GGA2 and GGA3 in TGN structures after 30 min (data not shown). These data indicate that a block of TGN–endosome traffic results in stabilin-1 accumulation in TGN.
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Figure 10. Influence of brefeldin A on stabilin-1 localization. In nontreated macrophages, only a minor portion of stabilin-1 (red) colocalizes with TGN46 (blue). Most of stabilin-1 is relocalized in TGN after 2 h treatment with 5 µg/ml brefeldin A. Areas of colocalization are shown in pink.
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2), exhibit an increased endocytic potential and express a broad range of multifunctional receptors (reviewed in refs. [16
, 19
]). Macrophage receptors combine scavenging functions, such as uptake and clearance of modified host molecules, apoptotic cells, microorganisms, and their products, with the regulation of adhesion and signaling events. Cell type-specific receptor functions are based not solely on expression levels but also depend on the intracellular environment including the compartmentalization and the interaction with trafficking machinery.
In the present study, we analyzed the intracellular distribution of stabilin-1 in primary human M2IL-4/Dex. A minor portion of the receptor is expressed on the cell surface. Predominant intracellular localization was observed in early/sorting and recycling endosomal compartments as well as in the TGN. It was rare that stabilin-1 was detected in late endosomes and CD63-positive lysosomes. In a search for APs mediating this specific intracellular distribution, we found that stabilin-1 binds in vitro with GGAs. Within the limits of light microscopy, we demonstrated that a small portion of stabilin-1 colocalizes with GGAs in the TGN. A brefeldin A-induced block of ARF-dependent TGN-endosomal/lysosomal traffic resulted in accumulation of stabilin-1 in TGN.
Several independent groups [38 39 40 41 42 ] reported recently the discovery of GGAs, which are ubiquitously expressed, monomeric APs that interact with the cytosolic tails of certain receptors and regulate the assembly of intracellular, clathrin-coated, transport vesicles. The main function of GGAs is to mediate receptor shuttling and cargo delivery from the TGN to endosomal/lysosomal compartments (reviewed in refs. [43 , 44 ]), and GGA shuttling is regulated by the ubiquitin system [45 46 47 ]. In contrast to AP-adaptor complexes, GGA-mediated intracellular traffic has as yet been described only for a few receptors, including the cation-dependent (CD)-MPR and CI-MPR [35 , 48 49 50 ], ß-secretase [51 ], sortilin [52 ], SorLA [53 ], and low density lipoprotein receptor-related protein 3 (LRP3) [35 ]. GGA-interacting receptors are multifunctional, type-1, transmembrane-sorting receptors involved in two processes: endocytosis and delivery of cargo from the TGN to the endosome/lysosome system (Table 1 ). At least two of them, i.e., CI-MPR and sortilin, possess endocytic and intracellular sorting functions. The most distant in function is ß-secretase (BACE1), a membrane-associated, aspartic protease that initiates cleavage of the ß-APP, leading to the production of ß-amyloid peptide, the central event in Alzheimer’s disease (reviewed in refs. [60 , 62 ]). ß-Secretase and APP are internalized from the plasma membrane to endosomes for cleavage, and GGA1 and/or GGA2 were suggested to mediate ß-secretase endocytosis [21 , 51 ].
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Table 1. GGA-Interacting Mammalian Receptors: Endocytosis and Intracellular Sorting
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The intracellular distribution of stabilin-1 resembles that of CI-MPR. In contrast to CD-MPR, CI-MPR has been implicated in several additional physiological processes including endocytosis of IGF-II, activation of latent transforming growth factor-ß1 precursor, and uptake of granzyme B [54
]. Several research groups analyzed CI-MPR intracellular localization. CI-MPR appears briefly on the surface and is rapidly internalized. Its major portion is localized in intracellular compartments [54
]. Stabilin-1 and CI-MPR can be found in early and recycling endosomes, late endosomes, and the TGN area and are absent from classical lysosomal compartments. However, occasional colocalization of stabilin-1 with CD63-positive vesicles was observed. CD63 was found as a transmembrane component of exosomes in dendritic cells, B cells, platelets, and other cell types [28
]. As stabilin-1 can be found in CD63-positive vesicles but not in Lamp1-positive lysosomes, we presume that stabilin-1 does not enter the major lysosomal compartments, thus escaping degradation, as shown for CI-MPR [64
]. In M2IL-4/Dex, we also observed partial colocalization of stabilin-1 and CI-MPR in the TGN area (data not shown).
In summary, we have demonstrated here that stabilin-1 is distributed subcellularly in the endosomal system and the TGN of human macrophages and that stabilin-1 traffic between these compartments may be mediated by GGA adaptors. We hypothesize that stabilin-1, similarly to other GGA-interacting, multifunctional receptors, can be involved in endocytosis as well as in delivery of newly synthesized material from the biosynthetic compartment to the secretory system.
Received May 20, 2004; revised July 19, 2004; accepted August 2, 2004.
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-adaptin ear homology domain, ADP-ribosylation factor-binding (GGA) proteins interact with acidic dileucine sequences within the cytoplasmic domains of sorting receptors through their Vps27p/Hrs/STAM (VHS) domains J. Biol. Chem. 276,28541-28545-adaptin and VHS domains that facilitate trafficking between the trans-Golgi network and the vacuole/lysosome J. Cell Biol. 149,67-80-adaptin "ear" domains J. Biol. Chem. 275,7176-7183 ear homology domain conserved in -adaptin and GGA proteins that interact with -synergin Biochem. Biophys. Res. Commun. 271,719-725[CrossRef][Medline]-secretase quartet conduct Alzheimer’s amyloid ß-peptide generation EMBO J. 23,483-488[CrossRef][Medline]This article has been cited by other articles:
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J. Kzhyshkowska, G. Workman, M. Cardo-Vila, W. Arap, R. Pasqualini, A. Gratchev, L. Krusell, S. Goerdt, and E. H. Sage Novel Function of Alternatively Activated Macrophages: Stabilin-1-Mediated Clearance of SPARC J. Immunol., May 15, 2006; 176(10): 5825 - 5832. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Kzhyshkowska, S. Mamidi, A. Gratchev, E. Kremmer, C. Schmuttermaier, L. Krusell, G. Haus, J. Utikal, K. Schledzewski, J. Scholtze, et al. Novel stabilin-1 interacting chitinase-like protein (SI-CLP) is up-regulated in alternatively activated macrophages and secreted via lysosomal pathway Blood, April 15, 2006; 107(8): 3221 - 3228. [Abstract] [Full Text] [PDF]
|
|
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M. S. Arredouani, A. Palecanda, H. Koziel, Y.-C. Huang, A. Imrich, T. H. Sulahian, Y. Y. Ning, Z. Yang, T. Pikkarainen, M. Sankala, et al. MARCO Is the Major Binding Receptor for Unopsonized Particles and Bacteria on Human Alveolar Macrophages J. Immunol., November 1, 2005; 175(9): 6058 - 6064. [Abstract] [Full Text] [PDF]
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