Polyubiquitination of the Epidermal Growth Factor Receptor Occurs at the Plasma Membrane upon Ligand-induced Activation
2000; Elsevier BV; Volume: 275; Issue: 18 Linguagem: Inglês
10.1074/jbc.275.18.13940
ISSN1083-351X
AutoresEspen Stang, Lene E. Johannessen, Sigrun L. Knardal, Inger Helene Madshus,
Tópico(s)Glycosylation and Glycoproteins Research
ResumoWe have previously shown that, although overexpression of mutant dynamin inhibits clathrin-dependent endocytosis and disrupts high affinity binding of epidermal growth factor (EGF) to the EGF receptor (EGFR), it does not inhibit ligand-induced translocation of the EGFR into clathrin-coated pits. In the present study, we demonstrate that, upon ligand binding and incubation at 37 °C, the EGFR was polyubiquitinated regardless of overexpression of mutant dynamin. In cells not overexpressing mutant dynamin, the EGFR was rapidly internalized and deubiquitinated. In cells being endocytosis-deficient, due to overexpression of mutant dynamin, however, the EGFR was upon prolonged chase first found in deeply invaginated coated pits, and then eventually moved out of the coated pits and back onto the smooth plasma membrane. Polyubiquitination occurred equally efficiently in cells with or without intact clathrin-dependent endocytosis, while the kinetics of ubiquitination and deubiquitination was somewhat different. We further found that the EGF-induced ubiquitination of Eps15 occurred both in the absence and presence of endocytosis with the same kinetics as polyubiquitination of the EGFR, but that the EGF-induced monoubiquitination of Eps15 was somewhat reduced upon overexpression of mutant dynamin. Our data show that EGF-induced polyubiquitination of the EGFR occurs at the plasma membrane. We have previously shown that, although overexpression of mutant dynamin inhibits clathrin-dependent endocytosis and disrupts high affinity binding of epidermal growth factor (EGF) to the EGF receptor (EGFR), it does not inhibit ligand-induced translocation of the EGFR into clathrin-coated pits. In the present study, we demonstrate that, upon ligand binding and incubation at 37 °C, the EGFR was polyubiquitinated regardless of overexpression of mutant dynamin. In cells not overexpressing mutant dynamin, the EGFR was rapidly internalized and deubiquitinated. In cells being endocytosis-deficient, due to overexpression of mutant dynamin, however, the EGFR was upon prolonged chase first found in deeply invaginated coated pits, and then eventually moved out of the coated pits and back onto the smooth plasma membrane. Polyubiquitination occurred equally efficiently in cells with or without intact clathrin-dependent endocytosis, while the kinetics of ubiquitination and deubiquitination was somewhat different. We further found that the EGF-induced ubiquitination of Eps15 occurred both in the absence and presence of endocytosis with the same kinetics as polyubiquitination of the EGFR, but that the EGF-induced monoubiquitination of Eps15 was somewhat reduced upon overexpression of mutant dynamin. Our data show that EGF-induced polyubiquitination of the EGFR occurs at the plasma membrane. epidermal growth factor epidermal growth factor receptor growth hormone growth hormone receptor phosphate-buffered saline phenylmethylsulfonyl fluoride sodium acetate buffer with 0.5 m NaCl polyacrylamide gel electrophoresis Binding of epidermal growth factor (EGF)1 to its receptor at the plasma membrane results in activation and autophosphorylation of the EGF receptor (EGFR), as well as phosphorylation and activation of other molecules (1.Carpenter G. Annu. Rev. Biochem. 1987; 56: 881-894Crossref PubMed Scopus (1053) Google Scholar). Activation of EGFR thereby initiates signal transduction cascades important for cellular growth and differentiation. Furthermore, binding of EGF mediates translocation of the EGFR to clathrin-coated pits, internalization of the EGFR via clathrin-coated vesicles, transport through endosomes, and eventually degradation in lysosomes (2.Renfrew C.A. Hubbard A.L. J. Biol. Chem. 1991; 266: 21265-21273Abstract Full Text PDF PubMed Google Scholar, 3.Felder S. Miller K. Moehren G. Ullrich A. Schlessinger J. Hopkins C.R. Cell. 1990; 61: 623-634Abstract Full Text PDF PubMed Scopus (352) Google Scholar, 4.Haigler H.T. McKanna J.A. Cohen S. J. Cell Biol. 1979; 81: 382-395Crossref PubMed Scopus (338) Google Scholar). Activation of the EGFR by ligand binding at 37 °C was also demonstrated to induce polyubiquitination of the EGFR (5.Galcheva-Gargova Z. Theroux S.J. Davis R.J. Oncogene. 1995; 11: 2649-2655PubMed Google Scholar). No EGF-stimulated EGFR ubiquitination was found in experiments using a mutant kinase-negative EGFR or in experiments where clathrin-dependent endocytosis was prevented by low temperature or K+ depletion (5.Galcheva-Gargova Z. Theroux S.J. Davis R.J. Oncogene. 1995; 11: 2649-2655PubMed Google Scholar). This suggested a functional coupling between clathrin-dependent endocytosis and ubiquitination of the EGFR. Ubiquitination has been shown to be essential for endocytosis of the yeast α-factor receptor (6.Hicke L. Riezman H. Cell. 1996; 84: 277-287Abstract Full Text Full Text PDF PubMed Scopus (670) Google Scholar) and for the growth hormone receptor (GHR) (7.Strous G.J. van Kerkhof P. Govers R. Ciechanover A. Schwartz A.L. EMBO J. 1996; 15: 3806-3812Crossref PubMed Scopus (265) Google Scholar). In cells with a temperature-sensitive defect in ubiquitin conjugation, neither growth hormone (GH)-dependent internalization nor GH-dependent degradation of the GHR was observed at the non-permissive temperature (7.Strous G.J. van Kerkhof P. Govers R. Ciechanover A. Schwartz A.L. EMBO J. 1996; 15: 3806-3812Crossref PubMed Scopus (265) Google Scholar). Further studies have demonstrated that when the internalization of the GHR was inhibited, GHR ubiquitination was also inhibited (8.Govers R. van Kerkhof P. Schwartz A.L. Strous G.J. EMBO J. 1997; 16: 4851-4858Crossref PubMed Scopus (91) Google Scholar). These results also imply a connection between ubiquitin conjugation and endocytosis. It has so far been unclear whether ubiquitination occurs prior to or following the budding of clathrin-coated vesicles. In a recent study, Levkowitz et al. (9.Levkowitz G. Waterman H. Zamir E. Kam Z. Oved S. Langdon W.Y. Beguinot L. Geiger B. Yarden Y. Genes Dev. 1998; 12: 3663-3674Crossref PubMed Scopus (716) Google Scholar) proposed that polyubiquitination of the EGFR occurs in endosomes. This was, however, not directly demonstrated. Instead, the authors demonstrated increased EGFR polyubiquitination and increased EGFR down-regulation by the overexpression of c-Cbl in Chinese hamster ovary cells. They furthermore demonstrated complex formation between c-Cbl and the EGFR upon incubation with EGF in cells overexpressing both c-Cbl and EGFR. Additionally, they showed that c-Cbl and the EGFR colocalized in endosomes upon addition of EGF. These data strongly support the recently suggested role for c-Cbl in the process of EGFR polyubiquitination (10.Waterman H. Levkowitz G. Alroy I. Yarden Y. J. Biol. Chem. 1999; 274: 22151-22154Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar, 11.Yokouchi M. Kondo T. Houghton A. Bartkiewicz M. Horne W.C. Zhang H. Yoshimura A. Baron R. J. Biol. Chem. 1999; 274: 31707-31712Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar), but do not clarify where in the cell ubiquitination occurs. It has been proposed by others that ubiquitination caused endocytosis of GHR by directing the GHR into clathrin-coated pits (8.Govers R. van Kerkhof P. Schwartz A.L. Strous G.J. EMBO J. 1997; 16: 4851-4858Crossref PubMed Scopus (91) Google Scholar). We have previously shown that EGF stimulation of the EGFR induced a rapid relocalization of EGFR from the smooth plasma membrane into clathrin-coated pits in cells being endocytosis-deficient due to overexpression of the GTPase-deficient mutant K44A form of dynamin (12.Ringerike T. Stang E. Johannessen L.E. Sandnes D. Levy F.O. Madshus I.H. J. Biol. Chem. 1998; 273: 16639-16642Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Clathrin-coated pits do not bud in cells overexpressing this mutant form of dynamin (13.Damke H. Baba T. Warnock D.E. Schmid S.L. J. Cell Biol. 1994; 127: 915-934Crossref PubMed Scopus (1040) Google Scholar), and we took advantage of this system to study the trafficking, tyrosine phosphorylation, and ubiquitination of the EGF-stimulated EGFR in the absence of clathrin-dependent endocytosis. We found that in cells overexpressing K44A dynamin the EGFR transiently localized to coated pits, but that hardly any EGF·EGFR complexes were internalized. Nevertheless, polyubiquitination occurred as efficiently in endocytosis-deficient cells as in cells where clathrin-dependent endocytosis took place in a normal fashion. Our data therefore demonstrate for the first time that ubiquitination of the EGFR occurs at the plasma membrane prior to endocytosis. Human recombinant EGF was from Bachem Feinchemikalien AG (Budendorf, Switzerland). All reagents were from Sigma unless otherwise noted. HeLa cells transfected with K44A mutant dynamin (13.Damke H. Baba T. Warnock D.E. Schmid S.L. J. Cell Biol. 1994; 127: 915-934Crossref PubMed Scopus (1040) Google Scholar) were a kind gift from Sandra L. Schmid (Scripps Research Institute, La Jolla, CA). The cells were grown in Dulbecco's modified Eagle's medium (3.7 g/liter sodium bicarbonate) (BioWhittaker, Walkersville, MD) containing 400 μg/ml Geneticin (Life Technologies, Inc., Paisley, United Kingdom), 200 ng/ml puromycin, 2 mm l-glutamine (BioWhittaker), and 1× penicillin-streptomycin-fungizone mixture (17-745, BioWhittaker) supplemented with 10% (v/v) fetal bovine serum (BioWhittaker). Cells were seeded at a density of 15.000 cells/cm2 and grown for 24 h at 37 °C in the presence (uninduced) or absence (induced) of 1 μg/ml tetracycline as described (14.Damke H. Gossen M. Freundlieb S. Bujard H. Schmid S. Methods Enzymol. 1995; 257: 209-220Crossref PubMed Scopus (100) Google Scholar). Expression of the transgene was routinely controlled by Western blotting as described (14.Damke H. Gossen M. Freundlieb S. Bujard H. Schmid S. Methods Enzymol. 1995; 257: 209-220Crossref PubMed Scopus (100) Google Scholar). After 24 h, cells were serum-starved by incubation for another 24 h in the same medium, but with 0.5% fetal bovine serum. The following antibodies were used: sheep anti-EGFR (Life Technologies, Inc., catalog no. 13287-016); rabbit anti-conjugated ubiquitin (Sigma); rabbit anti-Eps15 (C terminus) (Berkeley Antibody Company, Berkeley, CA); mouse anti-phosphotyrosine (Upstate Biotechnology, Inc., Lake Placid, NY); peroxidase-conjugated donkey anti-mouse IgG (Sigma); peroxidase-conjugated goat anti-rabbit IgG, peroxidase-conjugated donkey anti-sheep-IgG, and alkaline phosphatase-conjugated goat anti-rabbit antibodies (Jackson Immunoresearch Laboratories Inc., West Grove, PA); and phosphatase-conjugated anti-mouse antibodies (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom). Cells were incubated with 10−8m EGF for 15 min on ice, washed with ice-cold phosphate-buffered saline (PBS) (140 mm NaCl, 2.7 mm KCl, 10 mm Na2HPO4,2 mm NaH2PO4, pH 7.4), and chased in prewarmed EGF-free medium at 37 °C. At the end of the chase period, the cells were washed with ice-cold PBS, fixed with 4% paraformaldehyde, 0.1% glutaraldehyde in Soerensen's phosphate buffer, and processed for cryosections and immunogold labeling (15.Griffiths G. McDowall A. Back R. Dubochet J. J. Ultrastruct. Res. 1984; 89: 65-78Crossref PubMed Scopus (342) Google Scholar). The sections were labeled using sheep anti-EGFR antibody (Life Technologies, Inc., catalog no. 13287-016), followed by goat anti-sheep IgG-coated 18 nm colloidal gold (Jackson Immunoresearch Laboratory Inc.). To quantify EGFR distribution, the number of gold particles localized to the plasma membrane were counted and sorted into two groups depending on whether the gold localized to clathrin-coated or to smooth plasma membrane areas. Labeling along the tube connecting coated pits with the rest of the plasma membrane was interpreted as labeling of non-coated plasma membrane. From each experiment micrographs from at least 10 randomly chosen cells were examined, and a minimum of 100 gold particles were counted. The percentage of distribution of gold particles to coated pits were calculated as (no. of gold particles on coated plasma membrane/total no. of gold particles at the plasma membrane) × 100. Labeling for Eps15 was performed using rabbit anti-Eps15 antibodies followed by protein A-coated colloidal gold (purchased from G. Posthuma and J. Slot, Utrecht, Holland). HeLa cells were preincubated with brefeldin A (17.Misumi Y. Misumi Y Miki K. Takatsuki A. Tamura G. Ikehara Y. J. Biol. Chem. 1986; 261: 11398-11403Abstract Full Text PDF PubMed Google Scholar, 18.Lippincott-Schwartz J. Yuan L.C. Bonifacino J.S. Klausner R.D. Cell. 1989; 56: 801-813Abstract Full Text PDF PubMed Scopus (1312) Google Scholar) (5 μg/ml) for 1 h at 37 °C. Further processing was as for the experiments described above, but in the presence of brefeldin A. Chase-dependent redistribution of EGFR was quantified after immunolabeling by calculating EGFR labeling density/length of plasma membrane (16.Griffiths G. Fine Structure Immunology. Springer Verlag, Berlin1993Crossref Google Scholar) on micrographs from randomly chosen cells. EGF (10−8m) was added to cells in 12-well microtiter plates on ice for 15 min before the cells were washed with ice-cold PBS and chased in prewarmed EGF-free medium at 37 °C. After incubation the cells were subjected to Western blot analysis as described (19.Skarpen E. Johannessen L.E. Bjerk K. Fasteng H. Guren T.K. Lindeman B. Thoresen G.H. Christoffersen T. Stang E. Huitfeldt H.S. Madshus I.H. Exp. Cell Res. 1998; 243: 161-172Crossref PubMed Scopus (43) Google Scholar). The cells were lysed in lysis buffer (10 mm Tris-HCl (pH 6.8), 5 mmEDTA, 50 mm NaF, 30 mm sodium pyrophosphate, 2% (w/v) SDS (Bio-Rad), 4% (v/v) β-mercaptoethanol, 1 mm Na3VO4, 1 mmphenylmethylsulfonyl fluoride (PMSF) on ice for 10 min. Then 0.033% (w/v) bromphenol blue and 4% (v/v) glycerol) was added, and the cell lysate was incubated at 95 °C for 10 min, centrifuged at 20.000 × g for 15 min, and the supernatant fraction was subsequently subjected to SDS-PAGE (20.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207233) Google Scholar). The proteins were then electrotransferred to nitrocellulose membranes (21.Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44924) Google Scholar). The reactive proteins were detected using an enhanced chemiluminescence method (Amersham Pharmacia Biotech). To quantify band intensity, and when Western blotting with antibody against ubiquitin, proteins were electrotransferred to polyvinylidene difluoride membranes (Hybond-P, Amersham Pharmacia Biotech) following SDS-PAGE. For quantification, the membrane was incubated with primary antibodies as described above before incubation with alkaline phosphatase-conjugated anti-mouse antibodies or alkaline phosphatase-conjugated anti-rabbit antibodies for 2 h at room temperature. The immunobinding was detected by the enhanced chemifluorescence method (Amersham Pharmacia Biotech), and the chemifluorescence was measured by a phosphorofluoroimager (STORM840, Molecular Dynamics, Sunnyvale, CA). Due to dispersion of the EGFR caused by ubiquitination, both the main band and the smear above the main band were quantified. When membranes were incubated with anti-ubiquitin antibody, 3% (w/v) gelatin in Tris-buffered saline containing 1% (w/v) Tween 20 was used as blocking reagent, and the immunobinding was detected by enhanced chemiluminescence using an ECL Plus kit (Amersham Pharmacia Biotech). When immunoprecipitating the EGFR to determine the EGFR phosphorylation, the cells were lysed in immunoprecipitation buffer (PBS with 10 mm EDTA, 1% (v/v) Triton X-100, 10 mm NaF, 200 units/ml aprotinin, 1 mm PMSF, and 1 mmNa3VO4). Then protein G-agarose (Amersham Pharmacia Biotech) was incubated with sheep anti-EGFR antibody for 1 h at room temperature. The complexed antibody was washed twice in the buffer used for immunoprecipitation, and immunoprecipitation was performed at 4 °C for 60 min. In the experiment demonstrating ubiquitination of the EGFR, the immunoprecipitation was performed essentially as described by Strous et al. (7.Strous G.J. van Kerkhof P. Govers R. Ciechanover A. Schwartz A.L. EMBO J. 1996; 15: 3806-3812Crossref PubMed Scopus (265) Google Scholar). The cells were lysed in boiling buffer, containing 1% (w/v) SDS in PBS. After heating the lysates for 5 min at 100 °C, the cell lysates were homogenized using QIAshredder columns (Qiagen GmbH, Hilden, Germany). Then protein A-agarose and protein G-agarose (Amersham Pharmacia Biotech) were incubated with rabbit anti-ubiquitin antibody and sheep anti-EGFR antibody, respectively, for 1 h at room temperature. The complexed antibody was washed twice in the immunoprecipitation buffer, consisting of 1% (v/v) Triton X-100, 0.5% (w/v) SDS, 0.25% (w/v) sodium deoxycholate, 0.5% (w/v) bovine serum albumin, 1 mmEDTA, 1 mm PMSF, 2 mmNa3VO4, 20 mm NaF, 10 μg/ml leupeptin, and 200 units/ml aprotinin in PBS, and immunoprecipitation was performed at 4 °C for 60 min. The immunoprecipitate was washed and subjected to SDS-PAGE and Western blotting, as described above. 125I-EGF (10−8m; Amersham Pharmacia Biotech) was added to the cells on ice in minimal essential medium (Life Technologies, Inc.) without HCO3−. After 15 min at 4 °C, the cells were washed three times with ice-cold PBS to remove unbound ligand. The cells were then chased in minimal essential medium without HCO3− and with 0.1% (w/v) bovine serum albumin at 37 °C for the indicated time periods. The medium was collected, and the 125I-EGF was precipitated using 5% (w/v) tricloroacetic acid and 1% (w/v) phosphotungstic acid, as described (22.Sorkin A.D. Teslenko L.V. Nikolsky N.N. Exp. Cell Res. 1988; 175: 192-205Crossref PubMed Scopus (62) Google Scholar). Both the trichloroacetic acid-precipitable and the trichloroacetic acid-soluble radioactivity were measured in a γ-counter. Cells were washed three times with PBS, treated with 0.2m sodium acetate buffer (pH 4.5 or 7.4) containing 0.5m NaCl (SAB pH 4.5/SAB pH 7.4) on ice for 10 min, and washed once with the same buffer. Then 125I-EGF was precipitated from the cells with trichloroacetic acid and 1% (w/v) phosphotungstic acid. Finally, the precipitate was dissolved with 1m NaOH, and the radioactivity was measured. Counts/min from cells treated with SAB pH 4.5 represent internalized125I-EGF, while counts/min from cells treated with SAB pH 7.5 represent both internalized and surface-localized125I-EGF. Overexpression of K44A dynamin has been shown to inhibit clathrin-mediated endocytosis (13.Damke H. Baba T. Warnock D.E. Schmid S.L. J. Cell Biol. 1994; 127: 915-934Crossref PubMed Scopus (1040) Google Scholar). We have recently shown that although the overexpression of K44A dynamin disrupts high affinity binding of EGF to the EGFR, the EGFR is efficiently recruited into coated pits upon ligand binding (12.Ringerike T. Stang E. Johannessen L.E. Sandnes D. Levy F.O. Madshus I.H. J. Biol. Chem. 1998; 273: 16639-16642Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Due to overexpression of K44A dynamin, coated pits were often found at the end of long tubular plasma membrane invaginations, and sometimes gathered in groups. In our previous study we only examined cells chased for 10 min at 37 °C. As coated pits do not bud and form coated vesicles in cells overexpressing K44A dynamin, we wanted to examine the trafficking of the EGFR upon extended chase periods at 37 °C. EGF (10−8m) was added to cells on ice for 15 min, the cells were washed free of unbound ligand and chased for increasing time periods at 37 °C. At the end of the chase period, the cells were fixed, sectioned, and immunocytochemically labeled using antibodies recognizing the EGFR. To estimate the EGF-induced redistribution of the EGFR, we quantified the amount of EGFR localizing to coated pits compared with the total amount of EGFR found at the plasma membrane. Immunocytochemical labeling showed that in cells not exposed to EGF, as well as in cells fixed immediately after binding of EGF on ice, the EGFR mainly localized outside coated pits (TableI and Fig.1 A). However, upon chasing at 37 °C, the EGFR was seen to relocalize rapidly from non-coated, uninvaginated plasma membrane areas into areas where the plasma membrane had a clear cytoplasmic coat (Fig. 1 B). At early time points (2–5 min) (Table I and Fig. 1 B), several of these coated areas were flat or only slightly invaginated. After a longer chase period (10–15 min) (Table I and Fig. 1 C), however, most of the coat-associated labeling was in coated pits connected to the plasma membrane by long tubular necks. After 10–15 min of chase, almost 50% of the EGFRs were seen in coated pits (TableI). The labeling was usually found associated with the coated, bulb-shaped blind end of the tubules, and it should be noted that almost no labeling was found along the tubular neck. Upon further chase, however, the EGFR started to appear along the tube while the bulb-shaped end remained coated and the number of EGFR associated with coated areas decreased (Table I and Fig. 1, D andE). Upon 60 min of chase at 37 °C, the distribution of plasma membrane-associated EGFR was almost as in cells not exposed to EGF (Table I).Table IEGF-induced redistribution of EGFR to coated pits in cells overexpressing mutant dynaminControl0 min2 min5 min10 min15 min30 min60 minPulse-chase13 ± 211 ± 234 ± 340 ± 449 ± 247 ± 330 ± 319 ± 1Brefeldin A12 ± 211 ± 211 ± 324 ± 241 ± 340 ± 112 ± 110 ± 3The distribution of the EGFR to coated versus non-coated areas of the plasma membrane in cells overexpressing mutant (K44A) dynamin was quantified as described under “Experimental Procedures.” The distribution of EGFR was determined in cells after binding of EGF on ice and chase at 37 °C in EGF-free medium (Pulse-chase) and in cells exposed to brefeldin A prior to, as well as during, a pulse-chase incubation (Brefeldin A). The table shows the percentage of total plasma membrane-associated EGFR labeling, that under each experimental condition localized to coated membrane areas. The control column shows the percentage amount of EGFR localized to coated pits in cells prior to incubation with EGF. The 0, 2, 5, 10, 15, 30 and 60 min columns indicate the percentage amount of EGFR in coated pits upon these times of incubation at 37 °C. Labeling along the tube connecting the coated pits with the uninvaginated plasma membrane was interpreted as labeling of non-coated plasma membrane. Results are presented as the mean ± S.E. from a minimum of three independent experiments. Open table in a new tab The distribution of the EGFR to coated versus non-coated areas of the plasma membrane in cells overexpressing mutant (K44A) dynamin was quantified as described under “Experimental Procedures.” The distribution of EGFR was determined in cells after binding of EGF on ice and chase at 37 °C in EGF-free medium (Pulse-chase) and in cells exposed to brefeldin A prior to, as well as during, a pulse-chase incubation (Brefeldin A). The table shows the percentage of total plasma membrane-associated EGFR labeling, that under each experimental condition localized to coated membrane areas. The control column shows the percentage amount of EGFR localized to coated pits in cells prior to incubation with EGF. The 0, 2, 5, 10, 15, 30 and 60 min columns indicate the percentage amount of EGFR in coated pits upon these times of incubation at 37 °C. Labeling along the tube connecting the coated pits with the uninvaginated plasma membrane was interpreted as labeling of non-coated plasma membrane. Results are presented as the mean ± S.E. from a minimum of three independent experiments. Although the antibodies used recognize the intracellular part of the EGFR, labeling was in some cases found on the extracellular side of the plasma membrane (Fig. 1). Due to the size of the primary antibody recognizing the EGFR and the secondary antibody coating the colloidal gold, the observed distance from the gold particle to the antigen may be more than the width of a membrane (16.Griffiths G. Fine Structure Immunology. Springer Verlag, Berlin1993Crossref Google Scholar). The specificity of the anti-EGFR antibody was confirmed by Western blotting and was furthermore reflected by the EGF-induced change in labeling distribution. The colloidal gold contained a small amount of doublets (see Fig. 1 D), but when quantifying, eventual clusters were counted as one gold particle. Labeling of sections showed that independent of EGF binding and chase at 37 °C, some EGFR localized to the Golgi apparatus (data not shown). This labeling most likely represents newly synthesized EGFR. As including newly synthesized EGFR appearing at the plasma membrane during the chase period could complicate the study of ligand-induced trafficking of the EGFR, we treated cells with brefeldin A before addition of EGF as well as during the chase period. Brefeldin A prevents the transport of newly synthesized proteins from the endoplasmic reticulum to the plasma membrane by redistributing the cis and medial stacks of the Golgi apparatus back into the endoplasmic reticulum (17.Misumi Y. Misumi Y Miki K. Takatsuki A. Tamura G. Ikehara Y. J. Biol. Chem. 1986; 261: 11398-11403Abstract Full Text PDF PubMed Google Scholar, 18.Lippincott-Schwartz J. Yuan L.C. Bonifacino J.S. Klausner R.D. Cell. 1989; 56: 801-813Abstract Full Text PDF PubMed Scopus (1312) Google Scholar). Quantification of labeling on sections from brefeldin A-treated cells showed basically the same EGF-induced EGFR redistribution and transient localization to coated pits as found in cells not exposed to brefeldin A (see Table I). We conclude that, in cells deficient in endocytosis due to overexpression of K44A dynamin, EGFR moves to coated pits upon binding of EGF. The localization to clathrin-coated pits is transient, and the EGFR seems to loose its association with coat components and move back onto smooth, uninvaginated parts of the plasma membrane. To study the ligand-dependent redistribution of EGFR quantitatively, both with and without overexpression of mutant dynamin, we incubated cells with 10−8m125I-EGF on ice for 15 min before washing away unbound ligand and chasing the cells at 37 °C. At different time points, the fate of the initially bound125I-EGF was analyzed as described under “Experimental Procedures.” The results show that the majority of the initially bound 125I-EGF in cells overexpressing K44A dynamin rapidly dissociated from the EGFR. Of the 125I-EGF that remained cell-associated, only a small amount was endocytosed, and eventually degraded (Fig. 2 A). This is consistent with the findings of Vieira et al. (23.Vieira A. Lamaze C. Schmid S.L. Science. 1996; 274: 2086-2089Crossref PubMed Scopus (828) Google Scholar). The inhibition of EGFR endocytosis was further confirmed by immunocytochemical labeling for the EGFR. Quantification of labeling on sections from cells exposed to brefeldin A showed that the EGFR labeling density at the plasma membrane remained unchanged for up to 60 min of chase at 37 °C (data not shown). In cells grown with tetracycline (not overexpressing mutant dynamin),125I-EGF was rapidly endocytosed, with most ligand internalized upon 10 min of chase at 37 °C (Fig.2 B). Tyrosine-phosphorylated and dimerized EGFR have previously been shown to exist in endosomes (24.Sorkin A. Carpenter G. J. Biol. Chem. 1991; 266: 23453-23460Abstract Full Text PDF PubMed Google Scholar). In order to correlate the tyrosine phosphorylation of the EGFR with its cellular localization, we incubated cells with 10−8mEGF on ice for 15 min before washing away unbound ligand and chasing the cells at 37 °C. Incubation with EGF on ice caused tyrosine phosphorylation of the EGFR both in noninduced cells and in cells overexpressing K44A dynamin (Fig.3 A). However, the EGFR was less efficiently tyrosine-phosphorylated in cells overexpressing K44A dynamin, compared with in cells not overexpressing mutant dynamin (Refs. 12.Ringerike T. Stang E. Johannessen L.E. Sandnes D. Levy F.O. Madshus I.H. J. Biol. Chem. 1998; 273: 16639-16642Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar and 23.Vieira A. Lamaze C. Schmid S.L. Science. 1996; 274: 2086-2089Crossref PubMed Scopus (828) Google Scholar; Fig. 3 B). It should be noted that, upon overexpression of mutant dynamin, more EGFR were found at the plasma membrane (12.Ringerike T. Stang E. Johannessen L.E. Sandnes D. Levy F.O. Madshus I.H. J. Biol. Chem. 1998; 273: 16639-16642Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). We have demonstrated the specific tyrosine phosphorylation of the EGFR (Fig. 3 B) by normalizing the intensity of the phosphotyrosine signal with respect to the EGFR signal both in cells with and without overexpression of mutant dynamin. Chase at 37 °C caused a rapid dephosphorylation of the EGFR in both induced and noninduced cells. Once dephosphorylated, the EGFR in cells overexpressing K44A dynamin remained dephosphorylated. The kinetics of the dephosphorylation coincided with both entry of EGFR into coated pits and dissociation of ligand. In cells not overexpressing mutant dynamin, however, some of the EGFRs were transiently rephosphorylated upon prolonged chase at 37 °C (Fig. 3, A andB; 8, 10, and 12 min). When considering the kinetics of trafficking and phosphorylation, the rephosphorylation observed in cells not overexpressing mutant dynamin seemed to occur upon the EGFR reaching endosomes. When Western blotting with the anti-EGFR antibody, we found that the mobility of the EGFR was shifted as a function of ligand binding and chase at 37 °C, and by overexposing the film, a smear was clearly visible (Fig. 4 A). As the W
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