ARTICLE |
Correspondence to: Brigitte Krisch, Anatomisches Institut der Universität Kiel, Olshausenstr. 40, D-24098 Kiel, Germany..
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Summary |
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We analyzed the internalization of the receptor subtype 2 (sst2) for the neuropeptide somatostatin in glioma cells at the ultrastructural level using an antibody against an extracellular amino acid sequence. Intact cells derived from solid human gliomas or those of the human glioma cell line U343 were receptor-labeled (a) by classical gold immunocytochemistry using a 15-nm gold-labeled second antibody, (b) directly with the sst2 antibody adsorbed to 5-nm colloidal gold, and (c) with the physiological ligand somatostatin conjugated to 5-nm colloidal gold. The receptor was predominantly internalized via uncoated vesicles budding from the cell membrane but only rarely via coated pits, which has been mostly reported for G-protein-coupled, seven transmembrane-domain receptors. In the presence of ligand and sst2 antibody vesicles, tubule-like structures, and multivesicular bodies were labeled in superficial and in perinuclear portions of the cells within the first 30 min. Lysosomal labeling was observed after 30 min and especially after an hour of internalization time. This internalization route is also used to study the directly labeled sst2 antibody or the labeled ligand. However, the late endosomal compartment appears to be reached more rapidly in these latter experiments. (J Histochem Cytochem 46:12331242, 1998)
Key Words: somatostatin, receptor, internalization, glioma cells, endosomes
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Introduction |
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Endocytosis of cell surface receptors is a crucial step in the initiation of the intracellular signal transduction cascade after binding of extracellular ligands such as neurotransmitters or hormones. Many receptorligand complexes assemble at clathrin-coated pits budding off the membrane to form coated vesicles. After shedding their coats, these vesicles fuse with early endosomes (
Despite the complexity of these internalization processes most investigations were performed with subcellular fractionation techniques or by confocal laser microscopy and used mostly transfected cells that were not the physiological target for the ligand (
The internalization of the sst2 receptor is described at the ultrastructural level by use of an antibody against an amino acid sequence in the extracellular part of the receptor molecule and with an electron-dense conjugate of the ligand. Visualization of the dynamics of the receptor internalization on intact cells was accomplished by (a) classical gold immunocytochemistry using the sst2 antiserum as first antibody and a gold-labeled anti-rabbit immunoglobulin as second antibody, (b) labeling the sst2 antibody directly with colloidal gold, and (c) adsorption of the physiological ligand somatostatin to colloidal gold and affinity-labeling of the receptor.
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Materials and Methods |
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Antisera
The sst2 receptor antibody was raised in rabbits against a synthetic peptide corresponding to residues 3343 (QTEPYYDLTSN) plus a C-terminal cysteine that was conjugated via SH groups to keyhole limpet hemocyanin (Eurogentec; Seraing, Belgium). This sequence does not occur in any of the other sst subtypes, but is common to the splice variants A and B of the human sst2. The antibody was affinity-purified (affinity gelpeptide coupled to EAH Sepharose; Pharmacia, Uppsala, Sweden) and analyzed by SDS-PAGE and ELISA. A further anti-peptide antibody against the C-terminal sequence of sst2A (ETQRTLLNGDLQTSI) was obtained from Gramsch Laboratories (Schwabhausen, Germany); compare
Membrane Fractionation and Western Blotting
A total of 12 x 106 cells were lysed in 10 ml 5 mM Hepes buffer, pH 7.4, with 1 mM phenylmethanesulfonyl fluoride, 0.1 mM N-ethylmaleimide, and 0.1 mg/ml bacitracin and were disrupted by sonication (1 min). After addition of 1 ml 1.4 M NaCl in 200 mM Hepes, pH 7.4, nuclei and cell debris were removed by centrifugation (10 min, 800 x g at 4C) and a membrane fraction obtained (2 hr, 48,000 x g) that was suspended in 1 ml 0.14 M NaCl, 20 mM Hepes, pH 7.4. An aliquot (5-10 µg protein) was reduced and denatured in sample buffer, separated by SDS-PAGE (10% gel), transferred by semidry blotting (90 min, 65 mA) onto a polyvinylidene difluoride membrane (PVDF; Millipore, Bedford, MA) that was blocked with 5% casein, 0.1% Tween, 1 mM EDTA in PBS for 60 min, incubated overnight at 4C with anti-sst2 (1:20 in TweenEDTAPBS), washed (three times for 15 min in EDTAPBS), incubated (30 min) with peroxidase-labeled goat anti-rabbit IgG (1:40,000; Sigma, St Louis, MO), washed (three times for 15 min in EDTAPBS), and visualized by enhanced chemiluminescence (ECL system; Amersham, Poole, UK). Rainbow-colored as well as biotinylated proteins (Amersham, plus streptavidinperoxidase with the second antibody) served as molecular mass markers. The sst2 antibody was inactivated by co-incubation with 50 µM of the peptide QTEPYYDLTSN.
Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and cAMP Assay
RT-PCR for detection of sst2A and sst2B was performed (
Labeling of Antibody and Ligand with Colloidal Gold
A total of 100 µg affinity-purified sst2 antibody dialyzed vs 2 mM borate buffer, pH 9.0, was stirred for 1 hr with 20 ml colloidal gold sol (EM GC 15 or EM GC 5; British Biocell, Cardiff, UK) previously adjusted with 0.2 M K2CO3 to pH 9.0. Then 1 ml of 10% bovine serum albumin (BSA) in bidistilled water adjusted to pH 9.0 was added. After stirring for another 10 min the conjugate was concentrated to 1 ml by ultrafiltration (YM 10 membrane; Amicon, Temecula, CA), purified by gel filtration (Superdex S-300 1.6 x 60-cm column equilibrated and eluted with 0.14 M NaCl, 0.2% BSA, 10 mM Hepes, pH 8.0), and concentrated again by ultrafiltration. Labeling of somatostatin has been previously described (
Cell Culture
Astrocytes were obtained from dissociated cerebral cortices of 2-day-old Fisher F344 rats, purified, and subcultivated (
Receptor Labeling and Internalization
Subconfluent cells (105 U343 or glioma cells) grown on poly-D-lysine-coated coverslips in DMEM plus 10% fetal calf serum for 3 days were equilibrated to room temperature, washed in Gey's balanced salt solution plus 0.6% glucose (GBSS+G), and cooled to 8C in the refrigerator for 60 min. After preincubation with 10 nM somatostatin in GBSS+G (8C, 60 min), cells were exposed to the sst2 antibody (1:500 in GBSS+G, 150 min, 8C) and after a washing step to goat anti-rabbit IgG conjugated to 15-nm colloidal gold (British Biocell; 45 min, 8C), or, alternatively, only to the gold-labeled anti-sst2, all in the presence of 10 nM somatostatin. For light microscopy, surface-labeled cells (8C) were washed briefly in GBSS+G and PBS, fixed with 3% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, for 25 min, washed in PBS (3 min), 0.1 M lysine in PBS (10 min) and twice again in PBS, and then incubated overnight at 8C with goat anti-rabbit IgG coupled to gold particles of less than 0.8 nm (1:30 in PBS plus 0.2% gelatin; Aurion, Wageningen, The Netherlands). After thorough washing in PBS plus 0.1% gelatin and bidistilled water, specimens were silver-intensified with R-Gent (Aurion), washed in bidistilled water, counterstained in Mayer's hemalum, rinsed in tapwater, and embedded. For electron microscopy, cells were washed in cold GBSS+G without somatostatin and immediately fixed in cold 3% glutaraldehyde in PBS (30 min, 8C) or exposed to 37C for different times (5, 10, 15, 20, 30, 45, and 60 min) in GBSS+G. After fixation with 3% glutaraldehyde in PBS (5 min at 20C and 25 min at 8C), the specimens were embedded in Araldite, sectioned, double-contrasted (
Controls
Cells were incubated only with gold-labeled second antibody (anti-rabbit IgGAu), or without anti-sst2, with anti-sst2 replaced by preimmune serum, or with antigen-inactivated anti-sst2 (1 µM peptide, 30 min, 37C) and fixed after 10 min at 37C. Internalization was performed without preincubation with somatostatin and after washing in 20 mM citrate, 140 mM NaCl, pH 5.5. Under hyperosmolar conditions (0.4 M sucrose in GBSS) or in potassium-free buffer (140 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 1 mg/ml glucose, 20 mM Hepes, pH 7.4), internalization times of 5 and 10 min were used.
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Results |
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Glioma Cells Express the sst2A Somatostatin Receptor
Expression of the sst2 receptor in human glioma cells could be verified by Western blotting and RT-PCR experiments. The affinity-purified sst2 antiserum directed to the N-terminal extracellular portion of the receptor strongly reacted with a 75-kD band in U343 human glioma cells (Figure 1). The immunoreactivity was abolished completely in the presence of the immunization peptide or by omitting the primary antibody. A corresponding 75-kD band was also stained by a different sst2A-directed antiserum raised against the C-terminal 15 intracellular residues of this splice variant of the receptor. The mRNA for sst2A could be detected in U343 glioma cells by RT-PCR with two different primer pairs, whereas mRNA for the splice product sst2B was undetectable (not shown).
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Binding of the antibody to sst2 had no influence on its signal transduction. The somatostatin analogue octreotide (sst2-specific in glioma cells at 1 nM due to the absence of sst3 and sst5) reduced intracellular cAMP levels in U343 cells elevated by the adenylyl cyclase activator forskolin (50 µM at 37C for 10 min; compare
The sst2 Receptor Can be Visualized on the Surface of Glioma Cells
After cooling to 8C subconfluent cultures either of the glioma cell line U343 or from solid human gliomas, the somatostatin receptor could be always stained with the sst2 antibody at the light or electron microscopic level on the cell surface, but with different intensity on single cells (Figure 2). Unstained and heavily stained cells were in direct juxtaposition. When the antibody was inactivated with the immunization peptide, no labeling was observed. At the electron microscopic level, the labeling on U343 cells was rather uniformly distributed mainly on the cell somata, whereas the delicate processes of human glioma cells were labeled up to their finest tips (Figure 3).
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The Antibody-labeled Somatostatin Receptor is Internalized via Endosomal Vesicles and Not via Coated Pits
Although U343 cells and cells of human glioblastoma differ in their surface morphology (Figure 3), there were no fundamental differences in their internalization pattern. Cells fixed without warming carried singly dispersed gold particles exclusively on the surface of short, coarse processes in U343 cells or on the filiform network of glioblastoma cells (Figure 3). After warming at 37C for 5 min, assembly of gold particles was observed on the cell somata between the processes, and a few of them were found in inconspicuous vesicles opening at or positioned directly beneath the cell surface (Figure 4A). On tangential sections through the superficial parts of the cell, a high regional amount of gold assembly on limited areas of the cell surface was even more evident (not shown).
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The labeling intensity of these vesicles increased with increasing internalization times; the vesicles sometimes became elongated to little tubules (Figure 4B). They still were most often found in the superficial parts of the cells. Coated pits were only rarely labeled, at most in about 1% of labeled vesicles. After 15 min at 37C a few gold particles were included in multivesicular bodies (Figure 4C). Their number and the number of engulfed gold particles enlarged with increasing internalization times. In addition, after 30 min at 37C, labeled multivesicular bodies were located in the vicinity of Golgi areas and lysosome-like bodies containing gold particles were first observed (Figure 4D). Thereafter, their number and labeling intensity increased, but even after 60 min of internalization labeled lysosome-like bodies were clearly less common than labeled multivesicular bodies. Binding and internalization continued even for longer times at 37C, however, with decreasing labelling intensity at the cell membrane after 45 min and still less intensity after 60 min at 37C. Then, heavily labeled lysosomal bodies were conspicuous although staining was dense in superficial cytoplasmic vesicles, tubules, and multivesicular bodies.
With 5-nm gold-labeled sst2 antibodies, fundamentally the same internalization pattern was observed as with unlabeled primary and 15-nm gold-labeled secondary antibodies. Gold particles were mostly engulfed in uncoated vesicles, moving slowly with longer incubation times into the perinuclear region. Multivesicular bodies took the same route with a slight shift in time. After 5 min the first labeled multivesicular bodies were observed, which is clearly earlier than in experiments using 15-nm goldIgG as indicator for the bound sst2 antibody. However, no differences were evident regarding the appearance of gold-containing lysosome-like bodies.
Incubation Controls. When somatostatin was omitted from the incubation, no differences in the surface labeling could be observed up to 30 min. Gold particles were localized at the cell surface and were included in vesicles immediately beneath the cell membrane. However, they appeared as singlets and their numbers were clearly lower compared with preparations in which the receptor had been activated. Even after 30 min of incubation, late endosomes were hardly labeled. Only after 45 min were a few multivesicular bodies and even fewer lysosome-like bodies seen in perinuclear regions. Under potassium-deficient conditions, labeling was confined to the cell surface; intracellular gold particles were a rare exception (Figure 5A). Under hyperosmolar conditions, the cells exhibited a strongly condensed cytoplasm and internalization could not be observed, the profiles of coated and uncoated vesicles being greatly diminished (Figure 5B). When peptide-inactivated sst2 antiserum or anti-IgGAu was used instead of gold-labeled anti-sst2, surface and cytoplasmic labeling was hardly observable and was presumed to represent background staining (Figure 5C).
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Labeled Somatostatin Is Internalized via Endosomal Vesicles and Not via Coated Pits
The gold-labeled ligand was internalized in the same way as the antibody-labeled receptor. In previous studies we have shown that this electron-dense conjugate of somatostatin to 59-nm colloidal gold is still biologically active in displacing radiolabeled peptide from pituitary or cortical binding sites and in reducing basal or growth hormone-releasing hormone-induced somatotropin release from adenohypophyseal cells (
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Finally, we attempted to double label the receptor with the 5-nm gold-labeled ligand and the 15-nm gold-labeled antibody (either directly or via the second antibody). However, in these combination studies only the 5-nm gold-tagged ligand could be demonstrated. The preparations were completely devoid of 15-nm gold particles, irrespective of whether ligand and receptor antibody were applied together or in sequence.
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Discussion |
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With a triple electron microscopic approach (classical immunocytochemistry with gold-labeled second antibodies, directly gold-labeled antibody, and affinity-labeling with gold-tagged ligand), the internalization pathway of ligand-activated sst2 was studied in glioblastomas cultivated after neurosurgery and in a human glioblastoma cell line (U343). The results achieved in these three approaches are congruent. The predominant internalization route is via uncoated vesicles budding from the cell membrane. Under permanent internalization conditions for half an hour, labeled vesicles and tubule-like structures as well as labeled multivesicular bodies (MVB) are observed in superficial and perinuclear parts of the cells. Lysosomal labeling begins after 30 min and is fully expressed after an hour of internalization time. Only then is labeling intensity at the cell surface decreased. Slight differences between the results achieved with labeled receptor antibodies and labeled ligand appear to concern internalization dynamics. The ligand appears more rapidly in the late endosomal compartment (MVB) than the labeled antibodies. It does not, however, enter the lysosomal compartment more rapidly.
Neither the gold-labeled ligand nor the sst2 antibody is suitable to discriminate between the splice variants sst2A and sst2B which differ in the intracellular C-terminal residues. However, in the U343 cell line and in gliomas, only the subtype 2A is detectable, as has been reported for other tumors, e.g., carcinomas and melanomas (
Internalization processes terminate after very short periods, with sorting in early endosomes being completed after 23 min (
Internalization of receptor-selective ligands (
The alternative internalization route via caveolae or other endosomal vesicles has been suggested only for a few of the other seven transmembrane domain receptors (-subunits has been described (
-subunits (Go
and particularly Gi
2), thus preventing activation of the G
-subunit (
1, Gi
2, and Gi
3. In astrocytes, caveolae have been demonstrated by immunofluorescence microscopy of caveolin-1 and by conventional electron microscopy (
In the present electron microscopic study we did not observe the receptor directly but only indirectly via one or two antibodies or via the labeled ligand. However, given a pH of 6.36.8 in early endosomal vesicles (
The expression of somatostatin receptors on gliomas cannot be associated only with malignancy. In addition to neurons (
In conclusion, in glioma cells the sst2A receptor is an example of a further G-protein-coupled seven transmembrane-domain receptor that after ligand binding is not internalized via coated pits but via uncoated endosomal vesicles.
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Acknowledgments |
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Supported by grants from the Deutsche Forschungsgemeinschaft (Kr 569/7-1) and the Hensel Foundation.
We are greatly indebted to Ms Martina Burmester, Ms Dagmar Freier, and Ms Helga Prien for skillful technical assistance.
This work is dedicated to Prof Dr Helmut Leonhardt on the occasion of his eightieth birthday.
Received for publication June 16, 1998; accepted June 22, 1998.
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