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Primary amines do not prevent the endocytosis of epidermal growth factor into 3T3 fibroblasts
188
Biochimica et Biophysica Acta, 6 7 4 ( 1 9 8 1 ) 1 8 8 - - 2 0 3 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press
BBA 29585
PRIMARY AMINES DO NOT PREVENT THE ENDOCYTOSIS OF EPIDERMAL GROWTH FACTOR INTO 3T3 FIBROBLASTS
Y. Y A R D E N , M. G A B B A Y a n d J. S C H L E S S I N G E R
Department of Chemical Immunology, The Weizmann Institute of Science, P.O. 26. Rehovot (Israel) (Received October 21th, 1980)
Key words: Primary amine; Endocytosis; Epidermal growth factor; (3T3 fibroblast)
Summary Various amines block the degradation of endocytosed epidermal growth factor (EGF) without affecting the binding of the hormone to its surface receptors. However, studies based on fluorescence microscopy demonstrate that amines block the internalization of a~-macroglobulin and EGF by preventing it from clustering in clathrin coated pits. In order to resolve this controversy we have studied in detail the effect of various amines on the localization and processing of fluorescent and radiolabelled EGF. We have explored the effect of amines on EGF binding and localization, receptor mobility, membrane fluidity, receptor down regulation, hormone degradation and release of degradative products as a function of time and temperature. Our conclusions are as follows. 1. Primary amines prevent the formation of visible patches of fluorescent EGF and a2-macroglobulin on the cell surface at least for 15 min, thus increasing the diffusion coefficients and the mobile fraction of EGF-receptor complexes on the cell surface. 2. Amines do not block the endocytosis of EGF and a2-macroglobulin. On most cells fluorescent EGF and a2-macroglobulin are clustered and endocytosed within 30--45 min at 37°C. 3. Amines do not effect the internalization of 12SI-labelled-EGF and the down regulation of EGF receptors. 4. Amines block the degradation of the endocytosed EGF.
Abbreviations: EGF, epidermal growth factor; R-EGF, EGF labelled at the free carboxyl groups with six rhodamine molecules; R-Iact-EGF, EGF conjugated to a-lactalbumin that is labelled with 7--8 rhodamine molecules; R2-CNBr-EGF, rhodamine labelled c y a n o g e n b r o m i d e cleaved E G F ; diI, 3,3'-dioctadecylindocarbocyanine iodide; FPR, f l u o r e s c e n c e p h o t o b l e a c h i n g recovery.
189 Furthermore, our results raise the possibility that EGF molecules become endocytosed prior to the formation of visible patches. We conclude that until the cellular site and mode of action of amines are fully understood it is impossible to use these drugs as a diagnostic tool for studying the mechanism of EGF clustering, endocytosis and mode of action.
Introduction
Epidermal growth factor (EGF) is a potent mitogen of epithelial and epidermal cells and many cells in culture [1]. Cultured fibroblasts, responsive to EGF, have specific, saturable plasma membrane receptors for this molecule [2]. Like EGF, insulin and a2-macroglobulin bind to their respective diffusely distributed, mobile receptors on the membrane of mouse 3T3 fibroblasts [3]. Upon warming the cells to 37°C the hormone receptor complexes cluster into visible, immobile patches above coated regions of the plasma membrane [3--9]. The coated pits are endocytosed and the internalised hormones are degraded by lysosomal hydrolases and their fragments are released into the medium. The internalization of hormone receptor complexes leads to receptor loss i.e., down regulation of receptors. This process is dependent on EGF concentration, time and temperature [2,9]. Various amines block the degradation of the endocytosed EGF without affecting the binding of the hormone to its surface receptors [2]. This leads to a prolonged retention of intracellular intact 12SI-labelled EGF. Several reports presented data indicating that amines inhibit the formation of visible patches of EGF, a2-macroglobulin and 3,3',5-triiodo-L-thyronine and their subsequent internalization [7,11,12]. Amines at doses which inhibit receptor clustering potentiate EGF stimulated DNA synthesis by 3T3 cells [13]. This suggests that receptor clustering and internalization are related to the removal of the hormone from the cell surface rather than to their mode of action [13]. Recently Haigler et al. [14] reported that amines do block the endocytosis of a:-macroglobulin but do n o t block the endocytosis of EGF into 3T3 cells. Previous studies have shown that amines do not inhibit the endocytosis of EGF into human epithelial carcinoma cells (A-431) and fibroblasts [2,5,6] and that the main action of amines is inhibition of hormone degradation [2,5,6,15]. In order to resolve these conflicting reports we have studied in detail the effect of various amines on the localization and processing of fluorescent and radiolabeUed EGF on 3T3 cells. We have studied the effect of various amines on EGF localization, mobility, receptor down regulation, hormone degradation and release as a function of time. We conclude that primary amines block hormone degradation, prevent receptor patching fQr at least 15 rain, but have virtually no effect on EGF binding, internalization and down regulation of EGF receptors. Materials and Methods Materials. EGF was purified from male mouse submaxillary glands by the procedure of Savage and Cohen [16]. l:SI-labelled EGF (100000--120000
190 cpm/ng) was prepared by the chloroamine-T m e t h o d of Hunter and Greenwood [17]. Rhodamine ~-lactalbumin-EGF conjugate was prepared as previously described [18]. Rhodamine conjugate of cyanogen bromide cleaved EGF (R2CNB~-EGF} was prepared as previously reported [19]. Biologically active fluorescent conjugate of EGF labelled with six rhodamine molecules at the free carboxyl groups of EGF was prepared according to a modified procedure (Y. Shechter, unpublished data) developed for labelling nerve growth factor [20]. All sera, growth media, antibiotics and trypsin solutions were from BioLab Laboratories. Na12SI was purchased from Amersham. Bovine serum albumin, protease (type VI, 5 U/mg), becitracin, dansylcadaverine and di-dansylcadaverine were from Sigma. a-Chymotrypsin (49 U/mg) and trypsin were from Worthington. 3,3'-Dioctadecylindocarbocyanine iodide was a generous gift from Dr. A. Waggoner. Cell and cell cultures. The cells studied were mouse Balb 3T3 clone c/3 fibroblasts. All experiments were performed on stationary confluent cells. The cells were plated at (3--5) • 104 cells in 1 ml of Dulbecco's modified Eagle's medium with 10% foetal calf serum i n 1.5 cm diameter disposable multi-dish trays (Costar) and grown under a humidified atmosphere of 95% air/5% CO2 at 37 °C. Binding studies. Binding studies were performed in multi-dish trays. Each 1.5 cm well contained approx. 3 . l 0 s cells. Before experiments the cells were washed with 1.0 ml prewarmed phosphate buffered saline containing 0.1% bovine serum albumin. The binding was performed in 0.4 ml medium with 0.1% bovine serum albumin. Monolayers of 3T3 cells were preincubated with the indicated concentration of drug for 30 min at 37°C prior to the addition of the labelled hormone. Nonspecific binding was measured in the presence of excess unlabelled hormone (usually 500 ng/ml). U n b o u n d radioactivity was removed by washing with 3 ml ice-cold phosphate buffered saline. The cells were solubilized with 1.0 ml of 0.1 N NaOH and the radioactive content was determined. The binding results are the averages of 2--4 measurements for each data point. The variability in all the binding studies was less than 15% of the corresponding values. Visualization o f receptor distribution and measurement o f receptor mobility. Receptor mobility was measured by the fluorescence photobleaching recovery m e t h o d (FPR) [21--25]. We have added to the fluorescence photobleaching recovery apparatus an intensified silicon intensified target or silicon intensified target camera. This camera can detect very low levels of light and is employed to localize the fluorescent reagent on the cells. We also use this camera in order to align and focus the laser beam (Argon, 514 nm, approx. 5 pW) on the cell surface. After the laser beam is focused, a brief (10 ms) intense pulse irreversibly bleaches the fluorescence in a small region (approx. 3/~m 2) on the cell surface. The time course for recovery of fluorescence in the bleached region due to the replenishment of fresh fluorophores from adjacent regions of t h e cell membrane was recorded as previously described [23]. Diffusion coefficients (D) were calculated as previously reported [21--23]. Incomplete fluorescence recovery was interpreted as an indication that a fraction of the fluorophores are immobile on the time scale of the experiments. D < 3 • 10 -~2 cm2/s is considered to indicate immobility [21]. Our fluorescence microscope
191 (Zeiss, Universal) is equipped with three filter sets for selective observation of fluorescein, rhodamine or dansyl fluorescence. Results
In order to understand the effect of amines on EGF clustering, endocytosis and degradation we have performed a detailed study to determine their effect on the localization and processing of fluorescent and radioactively-labelled EGF on 3T3 fibroblasts and other cells. We have compared the effect of various amines, agents which increase intracellular pH, to the effect of either chloroquine, an inhibitor of hormone degradation, or sodium azide and 2-deoxyglucose, metabolic inhibitors, which were claimed to be inhibitors of cellular endocytosis.
The effect of amines on receptor clustering and mobility 3T3 cells were incubated for 30 rain at 37°C with 25 ng/ml R-EGF. As previously reported, the membrane-bound fluorescent hormone appears as visible immobile patches which subsequently become endocytosed [3] (Fig. 1, A,B). When the cells were incubated with 0.1 mM dansylcadaverine (or with either 10 mM ethylamine or methylamine) for 30 rain at 37°C and then with the fluorescent hormone, the hormone was homogeneously distributed over the cell surface (Fig. 1, C,D) [7]. When the buffer was replaced with buffer that did not contain amine, visible patches were seen after 10 min at 23°C. When the cells were pretreated with dansylcadaverine, labelled with R-EGF, washed and then incubated with 0.1 ~M of anti-EGF antibodies, a different result was obtained. Anti-EGF antibodies induced the formation of visible patches of R-EGF after 5--10 rnin at 37°C (Fig. 1, E,F). This result indicates that after 10 rain in the presence of dansylcadaverine, a substantial fraction of the fluorescent EGF molecules is still on the cell surface since the antibodies cannot penetrate into living cells. In order to assess the possibility that the free amine of dansylcadaverine is the molecular moiety responsible for inhibition of receptor clustering, we have compared the effects of monodansylcadaverine and di-dansylcadaverine (two fluorescent amines) on receptor clustering. Dansylcadaverine, which blocks receptor clustering (Fig. 1, C,D), was localised by fluorescence microscopy in phase dense vesicles (presumably lysosomes) and was also diffusely distributed in the cytoplasm (Fig. 2, D,F). However, di-dansylcadaverine, which did not inhibit patch formation (Fig. 2, A,C), was localised in the cytoplasm but did not appear in the phase dense vesicles (Fig. 2A). Clearly a free amino group is required for inhibition of receptor clustering. We measured the diffusion coefficients of R-EGF and the lipid probe 3,3'dioctadecylindocarbocyanine iodide on 3T3 fibroblasts using the fluorescence photobleaching recovery (FPR) method combined with an image intensified video camera. This system allows the correlation between mobility and localization of specific fluorescent markers on a single viable .cell [21]. The results are summarized in Table I. 3T3 cells were incubated for 30 rain at 37°C with 25 ng/ml of R-EGF. As previously reported, most of the hormone-receptor complexes appear as visible,
192
Fig. 1. T h e d i s t r i b u t i o n of R - E G F o n 3 T 3 cells in t h e p r e s e n c e or a b s e n c e o f a m i n e a n d a n t i - E G F antib o d i e s . (a) 3 T 3 cells w e r e i n c u b a t e d f o r 30 rnin at 3 7 ° C w i t h 2 5 n g / m l of R - E G F . T h e cells w e r e w a s h e d a n d v i e w e d as viable cells in D v o r a k c h a m b e r (A p h a s e a n d B f l u o r e s c e n c e m i c r o g r a p h s o f t h e s a m e field). (b) 3 T 3 cells w e r e p r e i n c u b a t e d f o r 30 rain a t 3 7 ° C w i t h 0,1 m M d a n s y l c a d a v e r i n e a n d for an a d d i t i o n a l 30 rain w i t h 2 5 n g / m l R - E G F . T h e cells w e r e w a s h e d a n d v i e w e d as viable cells (C p h a s e a n d D fluoresc e n c e m i c r o g r a p h s o f t h e s a ~ e field). (c) 3 T 3 cells w e r e p r e t r e a t e d w i t h d a n s y l c a d a v e r i n e a n d labelled w i t h R - E G F as d e s c r i b e d in (b), t h e n w a s h e d a n d i n c u b a t e d w i t h 0.1 /~M a n t i - E G F a n t i b o d i e s . P a t c h e s w e r e seen a p p r o x , a f t e r 5 rain at 3 7 ° C (E p h a s e a n d F f l u o r e s c e n c e m i c r o g r a p h s of t h e s a m e field). (Magnification, X1700).
immobile patches [3]. Only 15 -+ 7% of the occupied receptors were mobile with D = (3.2 + 0.7) • 10 -1° cm2/s. When the cells were preincubated with 10 mM m e t h y l a m i n e f or 30 min at 37°C and t hen with the fluorescent h o r m o n e , t h e h o r m o n e was m o r e mobile and in m o s t of the cells h o m o g e n e o u s l y distributed over the cell surface. Approx. 75% of the occupied receptors are mobile with a diffusion coefficient D = (5.1 -+ 0.6) • 10 -1° cm2/s. Clearly, m et hyl am i ne slows th e f o r m a t i o n o f visible patches and the immobilization of E G F - r e c e p t o r complexes. Similar results were obtained when the cells were labeled with
193
Fig. 2. T h e c e l l u l a r l o c a l i z a t i o n o f d a n s y l e a d a v e r i n e a n d d i - d a n s y l c a d a v e r l n e a n d t h e i r e f f e c t o n r e c e p t o r c l u s t e r i n g . 3 T 3 cells w e r e p r e i n c u b a t e d f o r 3 0 r a i n a t 3 7 ° C w i t h 0 . I m M o f e i t h e r d a n s y l e a d a v e r i n e o r d i - d a n s y l c a d a v e r l n e a n d t h e n f o r a n a d d i t i o n a l 3 0 r a i n w i t h 2 5 n g / m l R - E G F . T h e cells w e r e w a s h e d in p h o s p h a t e b u f f e r e d sal/ne a n d v i e w e d viable. P a n e l A s h o w s p h a s e p a t t e r n , p a n e l B s h o w s t h e d i s t r i b u t i o n o f d i - d a n s y l c a d a v e r i n e a n d p a n e l C s h o w s t h e d i s t r / b u t i o n o f R - E G F o f t h e s a m e field, S i m i l a r l y , p a n e l D is a p h a s e m i c r o g r a p h , p a n e l E s h o w s t h e d i s t r i b u t i o n o f d a n s y l c a d a v e r l n e a n d p a n e l F s h o w s t h e d i s t r i b u t i o n o f R - E G F o f t h e s a m e field. ( M a g n i f i c a t i o n , × 1 9 0 0 . )
R2-CNBr-EGF. This analogue binds to EGF receptors b u t does not induce patch formation and it is also devoid of biological activity [19]. After incubation for 1 h at 37°C in the presence of 200 ng/ml R2-CNBr-EGF the hormone. derivative was homogeneously distributed over the cell surface. The diffusion coefficient of this derivative (D = (6.9 ± 1.2) • 10 -1° cm2/s) is larger than the diffusion coefficient o f R-EGF in the presence of amine. The diffusion coefficient of EGF receptors labelled with R2-CNBr-EGF is presumably the closest value to the diffusion coefficient of the unoccupied receptors.
194 TABLE EFFECT PROBE
I
OF METHYLAMINE
ON THE LATERAL
DIFFUSION
OF EGF RECEPTORS
AND
A LIPID
V a l u e s o f l a t e r a l d i f f u s i o n c o e f f i c i e n t m e a s u r e d b y F P R w e r e o b t a i n e d f r o m t h e average o f m e a s u r e m e n t s t a k e n f r o m t e n d i f f e r e n t c e l l s , a t least t e n m e a s u r e m e n t s p e r cell. A l l the i n c u b a t i o n s for labelling o f f l u o r e s c e n t h o r m o n e s w e r e c a r r i e d o u t at 3 7 ° C and all F P R m e a s u r e m e n t s w e r e c a r r i e d o u t at r o o m t e m perature. Marker and binding conditions
D (cm 2/s)
R-EGF R-EGF. methylamine R2-CNBr-EGF diI diI, methylamine
(3.2 (5.1 (6.9 (8.II (9.28
-+ 0 . 7 ) -+ 0 . 8 ) + 1.2) -+ 1 . 5 4 ) -+ 1 . 5 )
• • • • •
10 -10 10 -10 10 -10 10 -9 10 -9
Mobile fraction (% r e c o v e r y )
Cellular distribution
1 5 -+ 7 75 + 11 8 0 -+ 1 5 ~-I00 ~100
patches d i f f u s e o n m o s t cells diffuse diffuse diffuse
In order to examine the possible effect of amines on the viscosity of the lipid matrix we measured the diffusion coefficient of the lipid probe 3,3'-dioctadecylindocarbocyanine iodide in the presence of absence of amine. The cells were pretreated with 10 mM methylamine and then labelled with 3,3'-dioctadecylindocarbocyanine iodide as previously described [26]. The lipid probe was homogeneously distributed on both the amine treated and non-treated cells. Table I summarizes the results. In absence of amine D = (8.11 -+ 1 . 5 4 ) . 10 -9 cm2/s; in presence of amine the diffusion coefficient of 3,3'-dioctadecylindocarbocyanine iodide was D = (9.28 + 1.5) • 10 -9 cm:/s. Under both conditions the fractional recovery was 100%, indicating the continuity of the lipid matrix for the diffusion of this probe. This result indicates that the viscosity of the lipid matrix is n o t significantly altered b y amines and that the change in mobility o f R-EGF in the presence of amine is n o t the consequence of alterations in lipid fluidity.
The effect o f amines on the patching and internalization o f fluorescent conjugates o f EGF We have explored the effect of amines on the patching and internalization of R-EGF, R-lact-EGF and rhodamine~2-macroglobulin on 3T3 and human epithelial carcinoma (A-431) cells. 3T3 cells were incubated for 30 min at 37°C with 10 mM ethylamine (or 0.1 mM dansylcadaverine) and then for another 20 min at 37°C with R-EGF. Then the buffer containing R-EGF was replaced with buffer containing amine and the cellular location of R-EGF was observed as a function of time. Initially the fluorescent conjugate appeared diffusely distributed on most of the cells (Fig. 1 C and D). However, after 5--10 min at 37°C patches of R-EGF were seen on many cells. After an additional 10 min at 37°C patches of R-EGF were seen all over the cells. Fig. 3 (A and B) shows the distribution of R-EGF after 1 h at 37°C in the presence of 0.1 mM of dansylcadaverine. Similar results were obtained when human epithelial carcinoma cells (A-431) were incubated with R-EGF in the presence of 10 mM ethylamine or when 3T3 cells were exposed to either R-EGF, R-lact-EGF or rhodamine-a2-macroglo-
195
Fig. 3. Dansylcadaverine d o e s n o t prevent the e n d o e y t o s i s o f R-EGF. 3T3 cells were pretreated w i t h 0.1 mM dansylcadaverine for 30 min at 37°C and then for an additional 30 ml n with 25 ng/ ml R-EGF. T h e cells were washed and maintained in buffer containing dansylcadaverine, (A) Phase and (B) fluorescence m i c r o g r a p h s o f cells observed after o n e h o u r at 37 ° C. (Magnification, × 1615.)
bulin in the presence of 10 mM ethylamine or other amines such as dansylcadaverine or methylamine. In all our experiments amines slow rather than block the formation of visible patches of fluorescent conjugates of EGF and a2-macroglobulin. Furthermore, amines did n o t inhibit the subsequent endocytosis of EGF and a:-macroglobulin.
Binding of ~2sI-iabelled EGF to 3T3 cells at 4°C We have tested the effect o f various amines on the binding of EGF to 3T3 cells. The cells were first incubated for 30 min at 37°C with buffer containing the amines. The cells were then cooled to 4°C and incubated for 2 h with 5 ng/ml ~2SI-labelled EGF at this temperature, then the specific cell-associated radioactivity was determined. Similar amounts of ~2SI-labelled EGF bind to the cells (53 ± 4 pg EGF/3 • l 0 s cells) in the presence of 5 mM ethylamine, 0.1 mM chloroquine, 5 mM methylamine, 10 mM ammonium chloride or in the absence of amines. We have also tested the effect of these drugs on cell viability and adhesion to the substrate. The same concentrations of amines did n o t alter cell morphology, adhesion to the substrate or the exclusion of trypan blue from the cells for more than 4 h. However, higher concentrations of amines: 30 mM ethylamine, 1 mM chloroquine, and 0.5 mM dansylcadaverine were toxic to the cells. At these concentrations the cells started to detach from the substrate and became stained with trypan blue.
Time course of association of ~2SI-labelled EGF with 3T3 fibroblast We have studied the e~fect of various agents on the time c o u r ~ of the ~ s o -
196 60(]
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3 Time(hrs)
4
5
Fig. 4. T r y p s i n i z a t i o n o f cell b o u n d 1 2 S I - l a b e l l e d E G F . C o n f l u e n t m o n o l a y e r s o f 3 T 3 cells w e r e w a s h e d w i t h p h o p s h a t e b u f f e r e d saline c o n t a i n i n g 0 . 1 % b o v i n e s e r u m a l b u m i n a n d t h e n p r e i n c u b a t e d f o r 3 0 r a i n at 3 7 ° C w i t h m e d i u m c o n t a i n i n g 0 . 1 % b o v i n e s e r u m a l b u m i n a n d v a r i o u s d r u g s : e t h y l a m i n e (1); c b l o r q u i n e (o); 2 d e o x y g l u c o s e a n d s o d i u m a z i d e (A); a n d c o n t r o l e x p e r i m e n t (e). T h e n t h e cells w e r e e x p o s e d t o 1 0 n g / m l o f 1 2 5 i . l a b e l l e d E G F f o r t h e i n d i c a t e d p e r i o d s o f t i m e , w a s h e d w i t h i c e - c o l d Ca 2+a n d Mg 2+ free p h o s p h a t e - b u f f e r e d saline a n d t r e a t e d w i t h 0 . 4 m l Ca 2+ a n d Mg 2+ free p h o s p h a t e - b u f f e r e d saline c o n t a i n i n g 0 . 2 5 % t r y p s i n a n d 0 . 1 % E D T A . T h e cells w e r e s h a k e n g e n t l y a t 4 ° C f o r 1 5 r a i n a n d t r e a t e d w i t h f o e t a l c a l f s e r u m ( 1 2 % ) t o s t o p t r y p s i n i z a t i o n . T h e cells w e r e c e n t r i f u g e d f o r 7 m i n at 4 ° C in p h o s p h a t e - b u f f e r e d saline c o n t a i n i n g 2 5 % g l y c e r o l . F i n a l l y , t h e r a d i o a c t i v e c o n t e n t o f t h e p e l l e t a n d t h e s u p e r n a t a n t w e r e d e t e r m i n e d . N o n s p e c i f l c b i n d i n g w a s m e a s u r e d in t h e p r e s e n c e o f e x c e s s ( 5 0 0 n g / m l ) E G F a n d s u b t r a c t e d f r o m t h e c o u n t s o f t h e p e l l e t s a n d t h e s u p e r n a t a n t s . R e s u l t s are e x p r e s s e d as t o t a l c e l l - a s s o c i a t e d r a d i o a c t i v i t y ( A ) a n d as t h e f r a c t i o n o f cell a s s o c i a t e d r a d i o a c t i v i t y a c c e s s i b l e t o t r y p sin (B). T h e r e s u l t s are a v e r a g e o f f o u r m e a s u r e m e n t s .
ciation of 12SI-labelled EGF with 3T3 cells. Some of these results are shown in Fig. 4A. 0.1 mM chloroquine and 10 mM ethylamine prevent the loss of cell associated radioactivity. Similar results were obtained when the cells were pretreated with 0.1 mM dansylcadaverine, 10 mM ammonium chloride, 60 ~g/ml leupeptin and 1 mM procaine (data n o t shown). 10 mM putrescine, which has a low rate of uptake, and 0.1 mM di-densylcadaverine did not effect either the loss of cell associated radioactivity or the aggregation of EGF-receptors. The metabolic inhibitors 2-deoxyglucose and sodium azide (in glucose-deficient medium) did n o t inhibit the loss of cell associated radioactivity.
Release of cell associated ~2SI-labelled EGF and its degradative products 3T3 cells were preincubated with various agents for 30 min at 37°C 0.1 mM chloroquine, 60 ~g/ml leupeptin, 10 mM ammonium chloride, 10 mM ethylamine, 1 mM bacitracin and a mixture of 2
197 TABLE II HALF-TIME OF R E L E A S E (r 1/2) OF CELL-ASSOCIATED R A D I O A C T I V I T Y 3T3 cells were l i b e l e d with 5 n g / m l 12$1-libelled EGF, after 4 h at 37°C the cells were washed and the m e d i u m was replaced with 2 ml of fresh binding m e d i u m containlng the appropriate drug. The released and the cell associated specific radioactivities were separately collected and counted. Each point is an average of duplicate measurements. Tr eatment
Half-time for release TI/2 (h)
125 I-labelled EGF b o u n d pg/2 • 105 cells
Control E t h y l a m i n e ( I 0 mM) A m m o n i u m chloride ( I 0 mM) Leupep tin (60/~g/ml) Chloroquine (10 mM) Colehieine (10/~M) Bacitracin (1 mM) 2-Deoxyglucose (10 mM) and sodium azide (10 mM)
1.45 12.9 11.8 6.5 14.2 1.84 2.5 1.0
96 422 523 347 514 160 131 57
half time of release (T1/2) Was calculated from the initial part of the release curve. The cell associated radioactivity was also counted. Approx. 70% of the released radioactivity from both the amine-treated cells and the non-treated cells was soluble in cold 5% trichloroacetic acid. Table II summarizes the effect of the various agents. Ethytamine, chloroquine and ammonium chloride increase the cell-associated radioactivity and the half-time of release (rl/2) of radioactivity. Leupeptin and bacitracin have a partial effect on both rl/2 and the total cell-associated radioactivity. Colchicine enhances the cell-associated radioactivity and has an insignificant effect on rl/2. A mixture of sodium azide and 2~ieoxyglucose decreases both rl/2 and the cell-associated radioactivity.
The effect of amines on degradation of 12SI-labelled EGF We have examined the possibility that in the presence of amines the cell associated radioactivity is accumulated due to inhibition of hormone degradation. We have collected the binding media of cells which were exposed for 6 h to 12SI-labelled EGF and then compared the amount of trichloroacetic acid soluble and trichloroacetic acid-precipitable fractions. The data presented in Table III show that ethylamine, ammonium chloride and dansylcadaverine inhibit the degradation of EGF, while putrescine does n o t inhibit hormone degradation. Furthermore, the trichloroacetic acid soluble radioactivity is approx. 4-fold larger then the cell-bound radioactivity, suggesting that during the 6 h incubation with EGF, new receptors are inserted into the plasma membrane which also become rapidly internalized. This is the most likely interpretation, since there is no indication for recycling of EGF receptors or for receptor-independent internalization of EGF molecules. However, amines decrease the ratio of trichloroacetic acid~oluble radioactivity/cell-bound radioactivity by a factor of 6--8, suggesting that these drugs block the insertion of new EGF receptors into the plasma membrane.
198 T A B L E III T R I C H L O R O A C E T I C ACID P R E C I P I T A T E D R A D I O A C T I V I T Y IN B I N D I N G MEDIA 3 T 3 cells w e r e p r e i n c u b a t e d f o r 30 rain a t 3 7 ° C w i t h t h e a p p r o p r i a t e a m i n e . 5 n g / m l 1 2 S i . l a b e l l e d E G F w a s a d d e d a n d i n c u b a t e d f o r a d d i t i o n a l 6 h a t 3 7 ° C . Finally t h e cells w e r e w a s h e d a n d t h e w a s h i n g m e d i a w e r e m i x e d t o g e t h e r w i t h t h e b i n d i n g m e d i a . 5% T r i c h l o r o a c e t i c acid w a s a d d e d t o this s o l u t i o n a n d a f t e r 1 h at 4 ° C t h e s a m p l e s w e r e c e n t r i f u g e d a n d t h e specific r a d i o a c t i v e c o n t e n t of t h e s u p e r n a t e n t s was d e t e r m i n e d . T h e cell-associated r a d i o a c t i v i t y w a s c o u n t e d a f t e r r e m o v i n g t h e cells w i t h 0.1 N N a O H . As a c o n t r o l w e . h a v e i n c u b a t e d 5 n g / m l 1 2 5 i . l a b e l l e d E G F for 6 h in t h e a b s e n c e of cells a n d t h e n t r e a t e d t h e s o l u t i o n w i t h t r i e h l o r o a c e t i c acid a n d d e t e r m i n e d t h e r a d i o a c t i v e c o n t e n t of t h e s u p e r n a t a n t . E a c h p o i n t is an a v e r a g e o f f o u r m e a s u r e m e n t s . Treatment
Control E t h y l a m i n e (5 r a M ) C h l o r o q u i n e (0.1 m M ) A m m o n i u m c h l o r i d e ( 1 0 raM) D a n s y l c a d a v e r i n e (0.1 r a M ) P u t r e s c i n e (5 r a M )
Cell-associated 125i.labelled EGF ( p g / 2 • l 0 s cells)
113 213 275 232 124 101
T r i c h l o r o a c e t i c acid soluble r a d i o a c t i v i t y % of t o t a l radioactivity
% of cell-bound radioactivity
8.9 1.9 2.5 3 4.2 9
393 45 46 65 169 446
Proteolysis o f cell-associated radioactivity The location of the cell-associated hormone was followed by treating the cell with proteolytic enzymes at 4°C. We assume that the fraction of radioactivity accessible for proteolytic enzymes is proportional to membrane associated hormone, while the inaccessible portion is proportional to the internalized hormone. 3T3 cells were first exposed to 12SI-labelled EGF for various periods of time. After washing the cells were gently shaken with a solution of proteolytic enzymes containing 0.1% EDTA for 15 min at 4°C. The cells were separated from the medium by centrifugation through 25% glycerol in phosphate-buffered saline and the radioactivities of cells as well as media were separately determined. Cell viability, as determined by exclusion of trypan blue was not affected by this treatment. It is evident from Fig. 4B that EGF rapidly becomes trypsin inaccessible after binding to cells. The metabolic inhibitors sodium azide and 2-deoxyglucose slow this process whereas amines 10 mM ethylamine, and 0.2 mM dansylcadaverine (not shown) and 0.1 mM chloroquine somewhat decrease the trypsin accessible fraction. We were not able to increase significantly the fraction of radioactivity accessible for proteolysis; pronase (2 mg/ml), trypsin (2 mg/ml), ~-chymotrypsin (2.5 mg/ml) and various combinations of these proteolytic enzymes gave similar results (not shown). When the binding of EGF was performed at 4°C no difference could be detected between amine-treated and non-treated cells. In both experiments approx. 90% of the radioactivity was accessible for proteolysis with trypsin. Discrimination between cell-surface.bound and internalized hormone by treating the cells with acetic acid We have used an alternative method to follow the time course of the internalization of EGF in the presence of various drugs. According to this method
199
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i
i
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~ 50 ~
o
I
I
I
I B
Ioo "N
~
5C
\ I
I
180 Time(min)
I
240
Fig. 5. Time-course of association of 125 I-labelled EGF at 37°C w i t h 3T3 cells and the a m o u n t of radioactivity accessible to acetic acid at this temperature. Cells were pre i nc uba t e d for 30 mi n at 37°C with the following drugs: chloroquinc (o); e t h y l a m i n e ( s ) ; dansylcadaverine (4); 2 deoxyglucose and d o d i u m azide (A); and control e x p e r i m e n t (e). After hormone t r e a t m e n t the cells were washed and treated w i t h 0.5 N acetic acid (pH 2.7) for 6 rain at 4°C. The specific acid released and the t ot a l specific radioactivity were determined. Results are averages of four measurements.
cells were treated, after hormone binding, with acetic acid (pH 2.7) for 6 min at 4°C. The radioactivity released into the medium by this treatment is considered to be a reliable estimate of the cell~surface-bound hormone [12]. The time~ourse of association of 12SI-labelled EGF with cells and the amount of radioactivity which could be removed by acetic acid are shown in Fig. 5. It is clear that pretreating the cells with the metabolic inhibitors 10 mM sodium azide and 2
200 A
B
C
-~ 2OO
60
120
180
0
60 120 180 T,me(minutes)
120 180
Fig. 6. D o w n r e g u l a t i o n o f E G F r e c e p t o r s in t h e p r e s e n c e of a m i n e s . 3 T 3 cells w e r e w a s h e d w i t h 1 m l prew a r m e d p h o s p h a t e b u f f e r e d saline c o n t a i n i n g 0.1% b o v i n e s e r u m a l b u m i n a n d i n c u b a t e d f o r a n a d d i t i o n a l h o u r at 3 7 ° C in m e d i u m c o n t a i n i n g 0.1% b o v i n e s e r u m a l b u m i n . T h e cells w e r e f u r t h e r i n c u b a t e d f o r SO rain a t 3 7 ° C w i t h ( A ) E G F a l o n e (B) in t h e p r e s e n c e of 1 0 m M e t h y l a m i n e o r in t h e p r e s e n c e of 0.1 m M d a n s y l c a d a v e r i n e (C). T h e cells w e r e e x p o s e d t o 2 n g ] m l E G F (o); 1 0 n g ] m l E G F (m) o r b u f f e r a l o n e (o). A f t e r t h e i n d i c a t e d p e r i o d s of t i m e t h e cells w e r e w a s h e d w i t h p h o s p h a t e b u f f e r e d saline c o n t a i n i n g 0.1% b o v i n e s e r u m a l b u m i n a n d e x p o s e d t o 10 n g / m l o f 1 2 5 I - l a b e l l e d E G F f o r 4 5 rain at S7°C. T h e specific cell-associated r a d i o a c t i v i t y w a s d e t e r m i n e d . E a c h p o i n t is the a v e r a g e o f d u p l i c a t e s .
removed by treatment with acetic acid (not shown). The acetic acid-accessible radioactivity at 4°C was not affected by either time of incubation or by the various drugs employed in this study. Down-regulation of EGF receptors in presence of amines The phenomenon of decreased binding capacity of cell surface receptors after exposure to hormone is generally attributed to the endocytosis of hormone-receptor complexes following hormone binding [2,3]. Therefore, inhibitors of internalization of EGF receptor could in principle effect the so called 'down-regulation' of EGF receptors. The results in Fig. 6 demonstrate that 10 mM ethylamine and 0.1 mM dansylcadaverine, do not block the downregulation of EGF receptors induced by either 2 or 10 ng/ml EGF. Amines also did not block the process of hormone-independent receptor loss usually observed after replacement with fresh medium and designated as 'receptor degradation' [27]. However, the time dependence of EGF induced 'down regulation' is somewhat altered when the cells are treated with amines (Fig. 6). Discussion
Our goal in this study was to elucidate the effect of alkylamines on the binding, surface aggregation, internalization and degradation of EGF on cultured cells. In order to give quantitative answers to these controversial questions we have employed fluorescent and radiolabelled conjugates of EGF and various methods aimed at quantitating the cellular location of EGF. The conclusions drawn from this study are as follows: (1) Amines slow the formation of visible immobile patches of fluorescent EGF and ~2-macroglobulin on the cell surface, thus increasing the mobile fraction and the diffusion coefficients of EGF-receptor complexes on the cell surface.
201
(2) Amines do not block the clustering and endocutosis of EGF and a2macroglobulin. On most cells fluorescent EGF and a2-macroglobulin are clustered and endocytosed within 30--45 rain at 37°C. (3) Amines do n o t block the internalization [2,5,6] of 12sI-labelled EGF and the down regulation of EGF receptors. (4) Amines block the degradation [2;6] of the endocytosed EGF. Therefore we conclude that it is currently impossible to employ amines as a diagnostic tool for studying the site and mode of action of EGF [13] and other peptide hormones. All information concerning the effect of amines on the internalization of EGF into cells reported in this paper or from other laboratories [2,5,6,14,15] gives a clear and consistent picture. Amines do not effect the rate and the extent of internalization of EGF into 3T3 cells. However, our studies with the fluorescent conjugates of EGF and a2-macroglobulin show that amines slow, but do n o t prevent the formation of visible patches of EGF and a2-macroglobulin. FPR and video intensification microscopy cannot detect the formation of microclusters of EGF or a2-macrogiobulin on the cell surface. It was reported that ferritin-EGF forms microclusters composed of 2--6 molecules within 2 min of 37°C. Amines did not block this process [6,9]. Such microclusters are presumably mobile on the cell surface and capable of coalescing into patches visible by image intensification microscopy. It is also possible that some of the microclusters of EGF receptor complexes are endocytosed before forming visible patches and that the microclusters are less accessible to trypsin or acetic acid than dispersed receptors. It is frequently assumed that formation of visible patches of EGF-receptor complexes on the plasma membrane is a prerequisite for endocytosis. Our results suggest that EGF molecules become endocytosed prior to the formation of visible patches of EGF-receptor complexes and the visible patches appear as a consequence of fusion between vesicles containing microclusters of receptors invisible by fluorescence microscopy [6]. Inhibition of this fusion by amines could explain the effect of amines on the formation of visible patches. It is clear that the major effect of amines is inhibition of degradation of internalized EGF. Alkylamines are weak bases which bind lysosomal protons. Therefore they become trapped in lysosomes, thereby raising the intralysosomal pH from pH 4.7 -+ 0.2 to pH 6.2 ± 0.2 [28]. We have noticed that dansylcadaverine (Fig. 2E) is primarily localised within phase dense vesicles (lysosomes?). A rhodamine conjugate of bacitracin was also localised within similar vesicles (data n o t shown). Treatment with amines caused an extensive vacuolization in 3T3 cells; some of these vacuoles contained R-EGF or rhodamine~2macroglobulin. Mckanna et al. have shown that in the presence of amines ferri. tin-EGF remains localized within multivesicular bodies which do n o t fuse with lysosomes [6]. It is possible that the amine mediated increase in lysosomal pH inhibits the fusion between lysosomes and multivesicular bodies. It was recently shown that the fusion of Semliki forest virus with membranes exhibits a dependence on the pH of the environment [29] and therefore amines block the penetration of the virus through the lysosomal membrane [30]. Obviously more experiments are required in order to understand the effect of amines on
202
the insertion of EGF receptors and on the recycling o f receptors for a2-macroglobulin and mannose-glycoconjugates [31,32]. However, it is tempting to speculate that this effect is due to inhibition of fusion between the plasma membrane and vesicles containing nascent EGF or recycled a2-macroglobulin receptors. In qualitative terms all the effects of amines reported in this paper could be due to inhibition of fusion between cellular membranes containing EGF-receptors rather than inhibition of a transglutaminase which cross-links between occupied EGF-receptors [33]. Maxfield et al. [13] have shown that amines potentiate EGF-stimulated DNA synthesis by 3T3 cells. Since amines do not block EGF-clustering and internalization it is more reasonable to assume that the enhanced mitogenic response is due to inhibition of EGF degradation rather than effects on EGF internalization [13]. Several reports have pointed out that inhibition of degradation of EGF leads t o enhanced mitogenic response [34] and to the binding of EGF molecules to the nucleus [ 3 4 ] . It is clear from this and other studies that until the cellular site and mode of action of amines are fully understood it is impossible to employ these drugs as a diagnostic tool for studying the mechanism of EGF clustering, endocytosis [33] and action [13].
Acknowledgments We acknowledge the support by grants from NIH (CA-25820) and from the US-Israel Binational Science Foundation. References 1 Carpenter, G. and Cohen, S. (1979) Annu. Rev. Biochem. 48, 193--216 2 Carpenter, G. and Cohen, S. (1976) J. Cell Biol. 71,159--171 3 Sch!essinger, J., Shechter, Y., Wlllingham, M.C. and Pastan, I. (1976) Proc. Natl. Acad. Sci. U.S.A. 75, 2659--2663 4 Sehlessinger, J., Shechter, Y., Cuatrecasas, P., Willingham, M.C, and Pastan, I. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 5353---5357 5 Gorden, P., Carpentier, L.J., Cohen, S. and Orci, L. (1978) Proc. Natl. Acad. Sci. U.S.A. 76, 5025-5029 6 Mckanna, J.A., Haigler, H.T. and Cohen, S. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 5689--5693 7 Maxfield, F.R., w~Iningham, M.C., Davies, P.J.A. and Pastan, I. (1979) Nature 277,661--663 8 Willingham, M.C., Maxfield, F.R. and Pastan, I. (1979) J. Cell Biol. 82,614---625 9 Haigler, H.T., McKanna, J.A. and Cohen, S. ('1979) J, Cell BioL 81, 382--395 10 Aharonov, A., Pruss, R.M. and Herschman, H.R. (1976) J. Biol. Chem. 253, 3970--3977 11 Chang, S.Y., Maxfield, F.R., Robb, ins, J., Willingham, M.C. and Pastan, I. (1960) Proc. Natl. Acad. Sci. U.S.A. 77, 3425--3429 12 Haigler, H.T., Maxfleld, F.R., WUlingham~ M.C. and Pastan, I. (1979) J. Biol. Chem. 255, 1239--1241 13 Maxfield, F.R., Davies, P.J.A., Klempner, L., Willingham, M.C. and Pastan, I. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 5731---5735 14 Haigler, H.T., Willingham~ M.C. and Pastan, I. (1980) Biochem. Biophys. Res. C o m m u n . 94, 630--637 15 King, A.C., Hernaez-Davis, L. and Cuatrecasas, P. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 3283--3287 16 Savage, C.R., Jr. and Cohen, S. (1972) J. Biol. Chem. 247, 7609--7611 17 Hunter, M.W. and G r e e n w o o d , F.C. (1962) Nature ( L o n d o n ) 194, 495---496 18 Shechter, Y., Schlessinger, J., Jacobs, S., Chang, K.J. and Cuatreeasas, P. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 2135--2139 19 Shechter, Y., Hernaez, L., Schlessinger, J. and Cuatrecasas, P. (1979) Nature 278, 835--838 20 Levi, A., Neufeld, E.J., Sheehter, Y. and Sehlessinger, J. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 3465--3473
203 21 Schless~nger, J., Koppel, D.E., A xelrod, D., Jacobson, K., Webb, W.W. and Elson, E.J. (1976) Proc. Natl. Acad. Sci. U.S.A. 78, 2409--2413 22 Axelrod, D., Koppel, D.E., Schlesslnger, J., Elson, E.L. and Webb0 W.W. (1976) Biochys. J. 16, 1055--1069 28 Koppel, D.E., Axelrod, D., Schless~nger, J., Elson, E.L. and Webb, W.W. (1976) Biophys. J. 16, 1 8 1 5 - 1829
24 Edidin, M., ZqyJ~Isky, Y. and Lardner, T.J. (1976) Science 1 9 1 , 4 6 6 - - 4 6 8 25 Jacobson, K., Wu, E. and Poste, G. (1976) Biochim. Biophys. Acta 433, 215--222 26 Schlessinger, J., Axelrod0 D., Koppel, D.E., Webb, W.W. and Elson, E.L. (1977) Science 195, 307-8O9 27 Kahn, C.R., Freychet, O., E o t h , J. and Neville, D.M., Jr. (1974) J. Biol. Chem. 249, 2249--2257 28 Ohkuma0 S. and Poole, B. (1978) Proe. Natl. Acad. Sci. U.S.A. 75° 3327--3331 29 White, J. and Helenius0 A. (1960) Proc. Natl. Acad. Sci. U.S.A. 77, 3273--3277 (1980) 80 Helenius, A.0 Kartenbeck, J., Simons0 K. and Fries, E. (1980) J. Cell Biol. 64, 404--420 31 Tietze° C.° Schlesshlger° P. and Stahl0 P. (1980) Bioehem. Biophys. Res. C o m m u n . 98, 1--8 32 Van Leuven° F., Cassiman° J.-J. and van den Berghe, H. (1980) Cell 20, 37--42 83 Davies, P.J.A.0 Davies, D.R.° Levitzki° A.° Maxfleld° F.R., Milhaud, P., Win4n~ham, M.C. and Pastan, I. (1960) Nature 283, 162--167 84 Friedkin, M.° Legg, A. and Rozengurt° E. (1979) Proc. Natl. Acad. Sei. U.S.A. 76, 3909--3912 85 Savion, N.° Vlodavsky, I. and Gospodarowicz° D. (1980) Proc. Natl. Acad. Sci. U.S.A. 77° 1466--1470
Biochimica et Biophysica Acta, 6 7 4 ( 1 9 8 1 ) 1 8 8 - - 2 0 3 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press
BBA 29585
PRIMARY AMINES DO NOT PREVENT THE ENDOCYTOSIS OF EPIDERMAL GROWTH FACTOR INTO 3T3 FIBROBLASTS
Y. Y A R D E N , M. G A B B A Y a n d J. S C H L E S S I N G E R
Department of Chemical Immunology, The Weizmann Institute of Science, P.O. 26. Rehovot (Israel) (Received October 21th, 1980)
Key words: Primary amine; Endocytosis; Epidermal growth factor; (3T3 fibroblast)
Summary Various amines block the degradation of endocytosed epidermal growth factor (EGF) without affecting the binding of the hormone to its surface receptors. However, studies based on fluorescence microscopy demonstrate that amines block the internalization of a~-macroglobulin and EGF by preventing it from clustering in clathrin coated pits. In order to resolve this controversy we have studied in detail the effect of various amines on the localization and processing of fluorescent and radiolabelled EGF. We have explored the effect of amines on EGF binding and localization, receptor mobility, membrane fluidity, receptor down regulation, hormone degradation and release of degradative products as a function of time and temperature. Our conclusions are as follows. 1. Primary amines prevent the formation of visible patches of fluorescent EGF and a2-macroglobulin on the cell surface at least for 15 min, thus increasing the diffusion coefficients and the mobile fraction of EGF-receptor complexes on the cell surface. 2. Amines do not block the endocytosis of EGF and a2-macroglobulin. On most cells fluorescent EGF and a2-macroglobulin are clustered and endocytosed within 30--45 min at 37°C. 3. Amines do not effect the internalization of 12SI-labelled-EGF and the down regulation of EGF receptors. 4. Amines block the degradation of the endocytosed EGF.
Abbreviations: EGF, epidermal growth factor; R-EGF, EGF labelled at the free carboxyl groups with six rhodamine molecules; R-Iact-EGF, EGF conjugated to a-lactalbumin that is labelled with 7--8 rhodamine molecules; R2-CNBr-EGF, rhodamine labelled c y a n o g e n b r o m i d e cleaved E G F ; diI, 3,3'-dioctadecylindocarbocyanine iodide; FPR, f l u o r e s c e n c e p h o t o b l e a c h i n g recovery.
189 Furthermore, our results raise the possibility that EGF molecules become endocytosed prior to the formation of visible patches. We conclude that until the cellular site and mode of action of amines are fully understood it is impossible to use these drugs as a diagnostic tool for studying the mechanism of EGF clustering, endocytosis and mode of action.
Introduction
Epidermal growth factor (EGF) is a potent mitogen of epithelial and epidermal cells and many cells in culture [1]. Cultured fibroblasts, responsive to EGF, have specific, saturable plasma membrane receptors for this molecule [2]. Like EGF, insulin and a2-macroglobulin bind to their respective diffusely distributed, mobile receptors on the membrane of mouse 3T3 fibroblasts [3]. Upon warming the cells to 37°C the hormone receptor complexes cluster into visible, immobile patches above coated regions of the plasma membrane [3--9]. The coated pits are endocytosed and the internalised hormones are degraded by lysosomal hydrolases and their fragments are released into the medium. The internalization of hormone receptor complexes leads to receptor loss i.e., down regulation of receptors. This process is dependent on EGF concentration, time and temperature [2,9]. Various amines block the degradation of the endocytosed EGF without affecting the binding of the hormone to its surface receptors [2]. This leads to a prolonged retention of intracellular intact 12SI-labelled EGF. Several reports presented data indicating that amines inhibit the formation of visible patches of EGF, a2-macroglobulin and 3,3',5-triiodo-L-thyronine and their subsequent internalization [7,11,12]. Amines at doses which inhibit receptor clustering potentiate EGF stimulated DNA synthesis by 3T3 cells [13]. This suggests that receptor clustering and internalization are related to the removal of the hormone from the cell surface rather than to their mode of action [13]. Recently Haigler et al. [14] reported that amines do block the endocytosis of a:-macroglobulin but do n o t block the endocytosis of EGF into 3T3 cells. Previous studies have shown that amines do not inhibit the endocytosis of EGF into human epithelial carcinoma cells (A-431) and fibroblasts [2,5,6] and that the main action of amines is inhibition of hormone degradation [2,5,6,15]. In order to resolve these conflicting reports we have studied in detail the effect of various amines on the localization and processing of fluorescent and radiolabeUed EGF on 3T3 cells. We have studied the effect of various amines on EGF localization, mobility, receptor down regulation, hormone degradation and release as a function of time. We conclude that primary amines block hormone degradation, prevent receptor patching fQr at least 15 rain, but have virtually no effect on EGF binding, internalization and down regulation of EGF receptors. Materials and Methods Materials. EGF was purified from male mouse submaxillary glands by the procedure of Savage and Cohen [16]. l:SI-labelled EGF (100000--120000
190 cpm/ng) was prepared by the chloroamine-T m e t h o d of Hunter and Greenwood [17]. Rhodamine ~-lactalbumin-EGF conjugate was prepared as previously described [18]. Rhodamine conjugate of cyanogen bromide cleaved EGF (R2CNB~-EGF} was prepared as previously reported [19]. Biologically active fluorescent conjugate of EGF labelled with six rhodamine molecules at the free carboxyl groups of EGF was prepared according to a modified procedure (Y. Shechter, unpublished data) developed for labelling nerve growth factor [20]. All sera, growth media, antibiotics and trypsin solutions were from BioLab Laboratories. Na12SI was purchased from Amersham. Bovine serum albumin, protease (type VI, 5 U/mg), becitracin, dansylcadaverine and di-dansylcadaverine were from Sigma. a-Chymotrypsin (49 U/mg) and trypsin were from Worthington. 3,3'-Dioctadecylindocarbocyanine iodide was a generous gift from Dr. A. Waggoner. Cell and cell cultures. The cells studied were mouse Balb 3T3 clone c/3 fibroblasts. All experiments were performed on stationary confluent cells. The cells were plated at (3--5) • 104 cells in 1 ml of Dulbecco's modified Eagle's medium with 10% foetal calf serum i n 1.5 cm diameter disposable multi-dish trays (Costar) and grown under a humidified atmosphere of 95% air/5% CO2 at 37 °C. Binding studies. Binding studies were performed in multi-dish trays. Each 1.5 cm well contained approx. 3 . l 0 s cells. Before experiments the cells were washed with 1.0 ml prewarmed phosphate buffered saline containing 0.1% bovine serum albumin. The binding was performed in 0.4 ml medium with 0.1% bovine serum albumin. Monolayers of 3T3 cells were preincubated with the indicated concentration of drug for 30 min at 37°C prior to the addition of the labelled hormone. Nonspecific binding was measured in the presence of excess unlabelled hormone (usually 500 ng/ml). U n b o u n d radioactivity was removed by washing with 3 ml ice-cold phosphate buffered saline. The cells were solubilized with 1.0 ml of 0.1 N NaOH and the radioactive content was determined. The binding results are the averages of 2--4 measurements for each data point. The variability in all the binding studies was less than 15% of the corresponding values. Visualization o f receptor distribution and measurement o f receptor mobility. Receptor mobility was measured by the fluorescence photobleaching recovery m e t h o d (FPR) [21--25]. We have added to the fluorescence photobleaching recovery apparatus an intensified silicon intensified target or silicon intensified target camera. This camera can detect very low levels of light and is employed to localize the fluorescent reagent on the cells. We also use this camera in order to align and focus the laser beam (Argon, 514 nm, approx. 5 pW) on the cell surface. After the laser beam is focused, a brief (10 ms) intense pulse irreversibly bleaches the fluorescence in a small region (approx. 3/~m 2) on the cell surface. The time course for recovery of fluorescence in the bleached region due to the replenishment of fresh fluorophores from adjacent regions of t h e cell membrane was recorded as previously described [23]. Diffusion coefficients (D) were calculated as previously reported [21--23]. Incomplete fluorescence recovery was interpreted as an indication that a fraction of the fluorophores are immobile on the time scale of the experiments. D < 3 • 10 -~2 cm2/s is considered to indicate immobility [21]. Our fluorescence microscope
191 (Zeiss, Universal) is equipped with three filter sets for selective observation of fluorescein, rhodamine or dansyl fluorescence. Results
In order to understand the effect of amines on EGF clustering, endocytosis and degradation we have performed a detailed study to determine their effect on the localization and processing of fluorescent and radioactively-labelled EGF on 3T3 fibroblasts and other cells. We have compared the effect of various amines, agents which increase intracellular pH, to the effect of either chloroquine, an inhibitor of hormone degradation, or sodium azide and 2-deoxyglucose, metabolic inhibitors, which were claimed to be inhibitors of cellular endocytosis.
The effect of amines on receptor clustering and mobility 3T3 cells were incubated for 30 rain at 37°C with 25 ng/ml R-EGF. As previously reported, the membrane-bound fluorescent hormone appears as visible immobile patches which subsequently become endocytosed [3] (Fig. 1, A,B). When the cells were incubated with 0.1 mM dansylcadaverine (or with either 10 mM ethylamine or methylamine) for 30 rain at 37°C and then with the fluorescent hormone, the hormone was homogeneously distributed over the cell surface (Fig. 1, C,D) [7]. When the buffer was replaced with buffer that did not contain amine, visible patches were seen after 10 min at 23°C. When the cells were pretreated with dansylcadaverine, labelled with R-EGF, washed and then incubated with 0.1 ~M of anti-EGF antibodies, a different result was obtained. Anti-EGF antibodies induced the formation of visible patches of R-EGF after 5--10 rnin at 37°C (Fig. 1, E,F). This result indicates that after 10 rain in the presence of dansylcadaverine, a substantial fraction of the fluorescent EGF molecules is still on the cell surface since the antibodies cannot penetrate into living cells. In order to assess the possibility that the free amine of dansylcadaverine is the molecular moiety responsible for inhibition of receptor clustering, we have compared the effects of monodansylcadaverine and di-dansylcadaverine (two fluorescent amines) on receptor clustering. Dansylcadaverine, which blocks receptor clustering (Fig. 1, C,D), was localised by fluorescence microscopy in phase dense vesicles (presumably lysosomes) and was also diffusely distributed in the cytoplasm (Fig. 2, D,F). However, di-dansylcadaverine, which did not inhibit patch formation (Fig. 2, A,C), was localised in the cytoplasm but did not appear in the phase dense vesicles (Fig. 2A). Clearly a free amino group is required for inhibition of receptor clustering. We measured the diffusion coefficients of R-EGF and the lipid probe 3,3'dioctadecylindocarbocyanine iodide on 3T3 fibroblasts using the fluorescence photobleaching recovery (FPR) method combined with an image intensified video camera. This system allows the correlation between mobility and localization of specific fluorescent markers on a single viable .cell [21]. The results are summarized in Table I. 3T3 cells were incubated for 30 rain at 37°C with 25 ng/ml of R-EGF. As previously reported, most of the hormone-receptor complexes appear as visible,
192
Fig. 1. T h e d i s t r i b u t i o n of R - E G F o n 3 T 3 cells in t h e p r e s e n c e or a b s e n c e o f a m i n e a n d a n t i - E G F antib o d i e s . (a) 3 T 3 cells w e r e i n c u b a t e d f o r 30 rnin at 3 7 ° C w i t h 2 5 n g / m l of R - E G F . T h e cells w e r e w a s h e d a n d v i e w e d as viable cells in D v o r a k c h a m b e r (A p h a s e a n d B f l u o r e s c e n c e m i c r o g r a p h s o f t h e s a m e field). (b) 3 T 3 cells w e r e p r e i n c u b a t e d f o r 30 rain a t 3 7 ° C w i t h 0,1 m M d a n s y l c a d a v e r i n e a n d for an a d d i t i o n a l 30 rain w i t h 2 5 n g / m l R - E G F . T h e cells w e r e w a s h e d a n d v i e w e d as viable cells (C p h a s e a n d D fluoresc e n c e m i c r o g r a p h s o f t h e s a ~ e field). (c) 3 T 3 cells w e r e p r e t r e a t e d w i t h d a n s y l c a d a v e r i n e a n d labelled w i t h R - E G F as d e s c r i b e d in (b), t h e n w a s h e d a n d i n c u b a t e d w i t h 0.1 /~M a n t i - E G F a n t i b o d i e s . P a t c h e s w e r e seen a p p r o x , a f t e r 5 rain at 3 7 ° C (E p h a s e a n d F f l u o r e s c e n c e m i c r o g r a p h s of t h e s a m e field). (Magnification, X1700).
immobile patches [3]. Only 15 -+ 7% of the occupied receptors were mobile with D = (3.2 + 0.7) • 10 -1° cm2/s. When the cells were preincubated with 10 mM m e t h y l a m i n e f or 30 min at 37°C and t hen with the fluorescent h o r m o n e , t h e h o r m o n e was m o r e mobile and in m o s t of the cells h o m o g e n e o u s l y distributed over the cell surface. Approx. 75% of the occupied receptors are mobile with a diffusion coefficient D = (5.1 -+ 0.6) • 10 -1° cm2/s. Clearly, m et hyl am i ne slows th e f o r m a t i o n o f visible patches and the immobilization of E G F - r e c e p t o r complexes. Similar results were obtained when the cells were labeled with
193
Fig. 2. T h e c e l l u l a r l o c a l i z a t i o n o f d a n s y l e a d a v e r i n e a n d d i - d a n s y l c a d a v e r l n e a n d t h e i r e f f e c t o n r e c e p t o r c l u s t e r i n g . 3 T 3 cells w e r e p r e i n c u b a t e d f o r 3 0 r a i n a t 3 7 ° C w i t h 0 . I m M o f e i t h e r d a n s y l e a d a v e r i n e o r d i - d a n s y l c a d a v e r l n e a n d t h e n f o r a n a d d i t i o n a l 3 0 r a i n w i t h 2 5 n g / m l R - E G F . T h e cells w e r e w a s h e d in p h o s p h a t e b u f f e r e d sal/ne a n d v i e w e d viable. P a n e l A s h o w s p h a s e p a t t e r n , p a n e l B s h o w s t h e d i s t r i b u t i o n o f d i - d a n s y l c a d a v e r i n e a n d p a n e l C s h o w s t h e d i s t r / b u t i o n o f R - E G F o f t h e s a m e field, S i m i l a r l y , p a n e l D is a p h a s e m i c r o g r a p h , p a n e l E s h o w s t h e d i s t r i b u t i o n o f d a n s y l c a d a v e r l n e a n d p a n e l F s h o w s t h e d i s t r i b u t i o n o f R - E G F o f t h e s a m e field. ( M a g n i f i c a t i o n , × 1 9 0 0 . )
R2-CNBr-EGF. This analogue binds to EGF receptors b u t does not induce patch formation and it is also devoid of biological activity [19]. After incubation for 1 h at 37°C in the presence of 200 ng/ml R2-CNBr-EGF the hormone. derivative was homogeneously distributed over the cell surface. The diffusion coefficient of this derivative (D = (6.9 ± 1.2) • 10 -1° cm2/s) is larger than the diffusion coefficient o f R-EGF in the presence of amine. The diffusion coefficient of EGF receptors labelled with R2-CNBr-EGF is presumably the closest value to the diffusion coefficient of the unoccupied receptors.
194 TABLE EFFECT PROBE
I
OF METHYLAMINE
ON THE LATERAL
DIFFUSION
OF EGF RECEPTORS
AND
A LIPID
V a l u e s o f l a t e r a l d i f f u s i o n c o e f f i c i e n t m e a s u r e d b y F P R w e r e o b t a i n e d f r o m t h e average o f m e a s u r e m e n t s t a k e n f r o m t e n d i f f e r e n t c e l l s , a t least t e n m e a s u r e m e n t s p e r cell. A l l the i n c u b a t i o n s for labelling o f f l u o r e s c e n t h o r m o n e s w e r e c a r r i e d o u t at 3 7 ° C and all F P R m e a s u r e m e n t s w e r e c a r r i e d o u t at r o o m t e m perature. Marker and binding conditions
D (cm 2/s)
R-EGF R-EGF. methylamine R2-CNBr-EGF diI diI, methylamine
(3.2 (5.1 (6.9 (8.II (9.28
-+ 0 . 7 ) -+ 0 . 8 ) + 1.2) -+ 1 . 5 4 ) -+ 1 . 5 )
• • • • •
10 -10 10 -10 10 -10 10 -9 10 -9
Mobile fraction (% r e c o v e r y )
Cellular distribution
1 5 -+ 7 75 + 11 8 0 -+ 1 5 ~-I00 ~100
patches d i f f u s e o n m o s t cells diffuse diffuse diffuse
In order to examine the possible effect of amines on the viscosity of the lipid matrix we measured the diffusion coefficient of the lipid probe 3,3'-dioctadecylindocarbocyanine iodide in the presence of absence of amine. The cells were pretreated with 10 mM methylamine and then labelled with 3,3'-dioctadecylindocarbocyanine iodide as previously described [26]. The lipid probe was homogeneously distributed on both the amine treated and non-treated cells. Table I summarizes the results. In absence of amine D = (8.11 -+ 1 . 5 4 ) . 10 -9 cm2/s; in presence of amine the diffusion coefficient of 3,3'-dioctadecylindocarbocyanine iodide was D = (9.28 + 1.5) • 10 -9 cm:/s. Under both conditions the fractional recovery was 100%, indicating the continuity of the lipid matrix for the diffusion of this probe. This result indicates that the viscosity of the lipid matrix is n o t significantly altered b y amines and that the change in mobility o f R-EGF in the presence of amine is n o t the consequence of alterations in lipid fluidity.
The effect o f amines on the patching and internalization o f fluorescent conjugates o f EGF We have explored the effect of amines on the patching and internalization of R-EGF, R-lact-EGF and rhodamine~2-macroglobulin on 3T3 and human epithelial carcinoma (A-431) cells. 3T3 cells were incubated for 30 min at 37°C with 10 mM ethylamine (or 0.1 mM dansylcadaverine) and then for another 20 min at 37°C with R-EGF. Then the buffer containing R-EGF was replaced with buffer containing amine and the cellular location of R-EGF was observed as a function of time. Initially the fluorescent conjugate appeared diffusely distributed on most of the cells (Fig. 1 C and D). However, after 5--10 min at 37°C patches of R-EGF were seen on many cells. After an additional 10 min at 37°C patches of R-EGF were seen all over the cells. Fig. 3 (A and B) shows the distribution of R-EGF after 1 h at 37°C in the presence of 0.1 mM of dansylcadaverine. Similar results were obtained when human epithelial carcinoma cells (A-431) were incubated with R-EGF in the presence of 10 mM ethylamine or when 3T3 cells were exposed to either R-EGF, R-lact-EGF or rhodamine-a2-macroglo-
195
Fig. 3. Dansylcadaverine d o e s n o t prevent the e n d o e y t o s i s o f R-EGF. 3T3 cells were pretreated w i t h 0.1 mM dansylcadaverine for 30 min at 37°C and then for an additional 30 ml n with 25 ng/ ml R-EGF. T h e cells were washed and maintained in buffer containing dansylcadaverine, (A) Phase and (B) fluorescence m i c r o g r a p h s o f cells observed after o n e h o u r at 37 ° C. (Magnification, × 1615.)
bulin in the presence of 10 mM ethylamine or other amines such as dansylcadaverine or methylamine. In all our experiments amines slow rather than block the formation of visible patches of fluorescent conjugates of EGF and a2-macroglobulin. Furthermore, amines did n o t inhibit the subsequent endocytosis of EGF and a:-macroglobulin.
Binding of ~2sI-iabelled EGF to 3T3 cells at 4°C We have tested the effect o f various amines on the binding of EGF to 3T3 cells. The cells were first incubated for 30 min at 37°C with buffer containing the amines. The cells were then cooled to 4°C and incubated for 2 h with 5 ng/ml ~2SI-labelled EGF at this temperature, then the specific cell-associated radioactivity was determined. Similar amounts of ~2SI-labelled EGF bind to the cells (53 ± 4 pg EGF/3 • l 0 s cells) in the presence of 5 mM ethylamine, 0.1 mM chloroquine, 5 mM methylamine, 10 mM ammonium chloride or in the absence of amines. We have also tested the effect of these drugs on cell viability and adhesion to the substrate. The same concentrations of amines did n o t alter cell morphology, adhesion to the substrate or the exclusion of trypan blue from the cells for more than 4 h. However, higher concentrations of amines: 30 mM ethylamine, 1 mM chloroquine, and 0.5 mM dansylcadaverine were toxic to the cells. At these concentrations the cells started to detach from the substrate and became stained with trypan blue.
Time course of association of ~2SI-labelled EGF with 3T3 fibroblast We have studied the e~fect of various agents on the time c o u r ~ of the ~ s o -
196 60(]
~
.......
2oo
-
0
i
I
I
I
I
B 75
•! ~ ~c ~_
25
0
I
~
3 Time(hrs)
4
5
Fig. 4. T r y p s i n i z a t i o n o f cell b o u n d 1 2 S I - l a b e l l e d E G F . C o n f l u e n t m o n o l a y e r s o f 3 T 3 cells w e r e w a s h e d w i t h p h o p s h a t e b u f f e r e d saline c o n t a i n i n g 0 . 1 % b o v i n e s e r u m a l b u m i n a n d t h e n p r e i n c u b a t e d f o r 3 0 r a i n at 3 7 ° C w i t h m e d i u m c o n t a i n i n g 0 . 1 % b o v i n e s e r u m a l b u m i n a n d v a r i o u s d r u g s : e t h y l a m i n e (1); c b l o r q u i n e (o); 2 d e o x y g l u c o s e a n d s o d i u m a z i d e (A); a n d c o n t r o l e x p e r i m e n t (e). T h e n t h e cells w e r e e x p o s e d t o 1 0 n g / m l o f 1 2 5 i . l a b e l l e d E G F f o r t h e i n d i c a t e d p e r i o d s o f t i m e , w a s h e d w i t h i c e - c o l d Ca 2+a n d Mg 2+ free p h o s p h a t e - b u f f e r e d saline a n d t r e a t e d w i t h 0 . 4 m l Ca 2+ a n d Mg 2+ free p h o s p h a t e - b u f f e r e d saline c o n t a i n i n g 0 . 2 5 % t r y p s i n a n d 0 . 1 % E D T A . T h e cells w e r e s h a k e n g e n t l y a t 4 ° C f o r 1 5 r a i n a n d t r e a t e d w i t h f o e t a l c a l f s e r u m ( 1 2 % ) t o s t o p t r y p s i n i z a t i o n . T h e cells w e r e c e n t r i f u g e d f o r 7 m i n at 4 ° C in p h o s p h a t e - b u f f e r e d saline c o n t a i n i n g 2 5 % g l y c e r o l . F i n a l l y , t h e r a d i o a c t i v e c o n t e n t o f t h e p e l l e t a n d t h e s u p e r n a t a n t w e r e d e t e r m i n e d . N o n s p e c i f l c b i n d i n g w a s m e a s u r e d in t h e p r e s e n c e o f e x c e s s ( 5 0 0 n g / m l ) E G F a n d s u b t r a c t e d f r o m t h e c o u n t s o f t h e p e l l e t s a n d t h e s u p e r n a t a n t s . R e s u l t s are e x p r e s s e d as t o t a l c e l l - a s s o c i a t e d r a d i o a c t i v i t y ( A ) a n d as t h e f r a c t i o n o f cell a s s o c i a t e d r a d i o a c t i v i t y a c c e s s i b l e t o t r y p sin (B). T h e r e s u l t s are a v e r a g e o f f o u r m e a s u r e m e n t s .
ciation of 12SI-labelled EGF with 3T3 cells. Some of these results are shown in Fig. 4A. 0.1 mM chloroquine and 10 mM ethylamine prevent the loss of cell associated radioactivity. Similar results were obtained when the cells were pretreated with 0.1 mM dansylcadaverine, 10 mM ammonium chloride, 60 ~g/ml leupeptin and 1 mM procaine (data n o t shown). 10 mM putrescine, which has a low rate of uptake, and 0.1 mM di-densylcadaverine did not effect either the loss of cell associated radioactivity or the aggregation of EGF-receptors. The metabolic inhibitors 2-deoxyglucose and sodium azide (in glucose-deficient medium) did n o t inhibit the loss of cell associated radioactivity.
Release of cell associated ~2SI-labelled EGF and its degradative products 3T3 cells were preincubated with various agents for 30 min at 37°C 0.1 mM chloroquine, 60 ~g/ml leupeptin, 10 mM ammonium chloride, 10 mM ethylamine, 1 mM bacitracin and a mixture of 2
197 TABLE II HALF-TIME OF R E L E A S E (r 1/2) OF CELL-ASSOCIATED R A D I O A C T I V I T Y 3T3 cells were l i b e l e d with 5 n g / m l 12$1-libelled EGF, after 4 h at 37°C the cells were washed and the m e d i u m was replaced with 2 ml of fresh binding m e d i u m containlng the appropriate drug. The released and the cell associated specific radioactivities were separately collected and counted. Each point is an average of duplicate measurements. Tr eatment
Half-time for release TI/2 (h)
125 I-labelled EGF b o u n d pg/2 • 105 cells
Control E t h y l a m i n e ( I 0 mM) A m m o n i u m chloride ( I 0 mM) Leupep tin (60/~g/ml) Chloroquine (10 mM) Colehieine (10/~M) Bacitracin (1 mM) 2-Deoxyglucose (10 mM) and sodium azide (10 mM)
1.45 12.9 11.8 6.5 14.2 1.84 2.5 1.0
96 422 523 347 514 160 131 57
half time of release (T1/2) Was calculated from the initial part of the release curve. The cell associated radioactivity was also counted. Approx. 70% of the released radioactivity from both the amine-treated cells and the non-treated cells was soluble in cold 5% trichloroacetic acid. Table II summarizes the effect of the various agents. Ethytamine, chloroquine and ammonium chloride increase the cell-associated radioactivity and the half-time of release (rl/2) of radioactivity. Leupeptin and bacitracin have a partial effect on both rl/2 and the total cell-associated radioactivity. Colchicine enhances the cell-associated radioactivity and has an insignificant effect on rl/2. A mixture of sodium azide and 2~ieoxyglucose decreases both rl/2 and the cell-associated radioactivity.
The effect of amines on degradation of 12SI-labelled EGF We have examined the possibility that in the presence of amines the cell associated radioactivity is accumulated due to inhibition of hormone degradation. We have collected the binding media of cells which were exposed for 6 h to 12SI-labelled EGF and then compared the amount of trichloroacetic acid soluble and trichloroacetic acid-precipitable fractions. The data presented in Table III show that ethylamine, ammonium chloride and dansylcadaverine inhibit the degradation of EGF, while putrescine does n o t inhibit hormone degradation. Furthermore, the trichloroacetic acid soluble radioactivity is approx. 4-fold larger then the cell-bound radioactivity, suggesting that during the 6 h incubation with EGF, new receptors are inserted into the plasma membrane which also become rapidly internalized. This is the most likely interpretation, since there is no indication for recycling of EGF receptors or for receptor-independent internalization of EGF molecules. However, amines decrease the ratio of trichloroacetic acid~oluble radioactivity/cell-bound radioactivity by a factor of 6--8, suggesting that these drugs block the insertion of new EGF receptors into the plasma membrane.
198 T A B L E III T R I C H L O R O A C E T I C ACID P R E C I P I T A T E D R A D I O A C T I V I T Y IN B I N D I N G MEDIA 3 T 3 cells w e r e p r e i n c u b a t e d f o r 30 rain a t 3 7 ° C w i t h t h e a p p r o p r i a t e a m i n e . 5 n g / m l 1 2 S i . l a b e l l e d E G F w a s a d d e d a n d i n c u b a t e d f o r a d d i t i o n a l 6 h a t 3 7 ° C . Finally t h e cells w e r e w a s h e d a n d t h e w a s h i n g m e d i a w e r e m i x e d t o g e t h e r w i t h t h e b i n d i n g m e d i a . 5% T r i c h l o r o a c e t i c acid w a s a d d e d t o this s o l u t i o n a n d a f t e r 1 h at 4 ° C t h e s a m p l e s w e r e c e n t r i f u g e d a n d t h e specific r a d i o a c t i v e c o n t e n t of t h e s u p e r n a t e n t s was d e t e r m i n e d . T h e cell-associated r a d i o a c t i v i t y w a s c o u n t e d a f t e r r e m o v i n g t h e cells w i t h 0.1 N N a O H . As a c o n t r o l w e . h a v e i n c u b a t e d 5 n g / m l 1 2 5 i . l a b e l l e d E G F for 6 h in t h e a b s e n c e of cells a n d t h e n t r e a t e d t h e s o l u t i o n w i t h t r i e h l o r o a c e t i c acid a n d d e t e r m i n e d t h e r a d i o a c t i v e c o n t e n t of t h e s u p e r n a t a n t . E a c h p o i n t is an a v e r a g e o f f o u r m e a s u r e m e n t s . Treatment
Control E t h y l a m i n e (5 r a M ) C h l o r o q u i n e (0.1 m M ) A m m o n i u m c h l o r i d e ( 1 0 raM) D a n s y l c a d a v e r i n e (0.1 r a M ) P u t r e s c i n e (5 r a M )
Cell-associated 125i.labelled EGF ( p g / 2 • l 0 s cells)
113 213 275 232 124 101
T r i c h l o r o a c e t i c acid soluble r a d i o a c t i v i t y % of t o t a l radioactivity
% of cell-bound radioactivity
8.9 1.9 2.5 3 4.2 9
393 45 46 65 169 446
Proteolysis o f cell-associated radioactivity The location of the cell-associated hormone was followed by treating the cell with proteolytic enzymes at 4°C. We assume that the fraction of radioactivity accessible for proteolytic enzymes is proportional to membrane associated hormone, while the inaccessible portion is proportional to the internalized hormone. 3T3 cells were first exposed to 12SI-labelled EGF for various periods of time. After washing the cells were gently shaken with a solution of proteolytic enzymes containing 0.1% EDTA for 15 min at 4°C. The cells were separated from the medium by centrifugation through 25% glycerol in phosphate-buffered saline and the radioactivities of cells as well as media were separately determined. Cell viability, as determined by exclusion of trypan blue was not affected by this treatment. It is evident from Fig. 4B that EGF rapidly becomes trypsin inaccessible after binding to cells. The metabolic inhibitors sodium azide and 2-deoxyglucose slow this process whereas amines 10 mM ethylamine, and 0.2 mM dansylcadaverine (not shown) and 0.1 mM chloroquine somewhat decrease the trypsin accessible fraction. We were not able to increase significantly the fraction of radioactivity accessible for proteolysis; pronase (2 mg/ml), trypsin (2 mg/ml), ~-chymotrypsin (2.5 mg/ml) and various combinations of these proteolytic enzymes gave similar results (not shown). When the binding of EGF was performed at 4°C no difference could be detected between amine-treated and non-treated cells. In both experiments approx. 90% of the radioactivity was accessible for proteolysis with trypsin. Discrimination between cell-surface.bound and internalized hormone by treating the cells with acetic acid We have used an alternative method to follow the time course of the internalization of EGF in the presence of various drugs. According to this method
199
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i
A 2OO LLI
,2..' N ~ "GI50 "O--~__OI 0 0
~ 50 ~
o
I
I
I
I B
Ioo "N
~
5C
\ I
I
180 Time(min)
I
240
Fig. 5. Time-course of association of 125 I-labelled EGF at 37°C w i t h 3T3 cells and the a m o u n t of radioactivity accessible to acetic acid at this temperature. Cells were pre i nc uba t e d for 30 mi n at 37°C with the following drugs: chloroquinc (o); e t h y l a m i n e ( s ) ; dansylcadaverine (4); 2 deoxyglucose and d o d i u m azide (A); and control e x p e r i m e n t (e). After hormone t r e a t m e n t the cells were washed and treated w i t h 0.5 N acetic acid (pH 2.7) for 6 rain at 4°C. The specific acid released and the t ot a l specific radioactivity were determined. Results are averages of four measurements.
cells were treated, after hormone binding, with acetic acid (pH 2.7) for 6 min at 4°C. The radioactivity released into the medium by this treatment is considered to be a reliable estimate of the cell~surface-bound hormone [12]. The time~ourse of association of 12SI-labelled EGF with cells and the amount of radioactivity which could be removed by acetic acid are shown in Fig. 5. It is clear that pretreating the cells with the metabolic inhibitors 10 mM sodium azide and 2
200 A
B
C
-~ 2OO
60
120
180
0
60 120 180 T,me(minutes)
120 180
Fig. 6. D o w n r e g u l a t i o n o f E G F r e c e p t o r s in t h e p r e s e n c e of a m i n e s . 3 T 3 cells w e r e w a s h e d w i t h 1 m l prew a r m e d p h o s p h a t e b u f f e r e d saline c o n t a i n i n g 0.1% b o v i n e s e r u m a l b u m i n a n d i n c u b a t e d f o r a n a d d i t i o n a l h o u r at 3 7 ° C in m e d i u m c o n t a i n i n g 0.1% b o v i n e s e r u m a l b u m i n . T h e cells w e r e f u r t h e r i n c u b a t e d f o r SO rain a t 3 7 ° C w i t h ( A ) E G F a l o n e (B) in t h e p r e s e n c e of 1 0 m M e t h y l a m i n e o r in t h e p r e s e n c e of 0.1 m M d a n s y l c a d a v e r i n e (C). T h e cells w e r e e x p o s e d t o 2 n g ] m l E G F (o); 1 0 n g ] m l E G F (m) o r b u f f e r a l o n e (o). A f t e r t h e i n d i c a t e d p e r i o d s of t i m e t h e cells w e r e w a s h e d w i t h p h o s p h a t e b u f f e r e d saline c o n t a i n i n g 0.1% b o v i n e s e r u m a l b u m i n a n d e x p o s e d t o 10 n g / m l o f 1 2 5 I - l a b e l l e d E G F f o r 4 5 rain at S7°C. T h e specific cell-associated r a d i o a c t i v i t y w a s d e t e r m i n e d . E a c h p o i n t is the a v e r a g e o f d u p l i c a t e s .
removed by treatment with acetic acid (not shown). The acetic acid-accessible radioactivity at 4°C was not affected by either time of incubation or by the various drugs employed in this study. Down-regulation of EGF receptors in presence of amines The phenomenon of decreased binding capacity of cell surface receptors after exposure to hormone is generally attributed to the endocytosis of hormone-receptor complexes following hormone binding [2,3]. Therefore, inhibitors of internalization of EGF receptor could in principle effect the so called 'down-regulation' of EGF receptors. The results in Fig. 6 demonstrate that 10 mM ethylamine and 0.1 mM dansylcadaverine, do not block the downregulation of EGF receptors induced by either 2 or 10 ng/ml EGF. Amines also did not block the process of hormone-independent receptor loss usually observed after replacement with fresh medium and designated as 'receptor degradation' [27]. However, the time dependence of EGF induced 'down regulation' is somewhat altered when the cells are treated with amines (Fig. 6). Discussion
Our goal in this study was to elucidate the effect of alkylamines on the binding, surface aggregation, internalization and degradation of EGF on cultured cells. In order to give quantitative answers to these controversial questions we have employed fluorescent and radiolabelled conjugates of EGF and various methods aimed at quantitating the cellular location of EGF. The conclusions drawn from this study are as follows: (1) Amines slow the formation of visible immobile patches of fluorescent EGF and ~2-macroglobulin on the cell surface, thus increasing the mobile fraction and the diffusion coefficients of EGF-receptor complexes on the cell surface.
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(2) Amines do not block the clustering and endocutosis of EGF and a2macroglobulin. On most cells fluorescent EGF and a2-macroglobulin are clustered and endocytosed within 30--45 rain at 37°C. (3) Amines do n o t block the internalization [2,5,6] of 12sI-labelled EGF and the down regulation of EGF receptors. (4) Amines block the degradation [2;6] of the endocytosed EGF. Therefore we conclude that it is currently impossible to employ amines as a diagnostic tool for studying the site and mode of action of EGF [13] and other peptide hormones. All information concerning the effect of amines on the internalization of EGF into cells reported in this paper or from other laboratories [2,5,6,14,15] gives a clear and consistent picture. Amines do not effect the rate and the extent of internalization of EGF into 3T3 cells. However, our studies with the fluorescent conjugates of EGF and a2-macroglobulin show that amines slow, but do n o t prevent the formation of visible patches of EGF and a2-macroglobulin. FPR and video intensification microscopy cannot detect the formation of microclusters of EGF or a2-macrogiobulin on the cell surface. It was reported that ferritin-EGF forms microclusters composed of 2--6 molecules within 2 min of 37°C. Amines did not block this process [6,9]. Such microclusters are presumably mobile on the cell surface and capable of coalescing into patches visible by image intensification microscopy. It is also possible that some of the microclusters of EGF receptor complexes are endocytosed before forming visible patches and that the microclusters are less accessible to trypsin or acetic acid than dispersed receptors. It is frequently assumed that formation of visible patches of EGF-receptor complexes on the plasma membrane is a prerequisite for endocytosis. Our results suggest that EGF molecules become endocytosed prior to the formation of visible patches of EGF-receptor complexes and the visible patches appear as a consequence of fusion between vesicles containing microclusters of receptors invisible by fluorescence microscopy [6]. Inhibition of this fusion by amines could explain the effect of amines on the formation of visible patches. It is clear that the major effect of amines is inhibition of degradation of internalized EGF. Alkylamines are weak bases which bind lysosomal protons. Therefore they become trapped in lysosomes, thereby raising the intralysosomal pH from pH 4.7 -+ 0.2 to pH 6.2 ± 0.2 [28]. We have noticed that dansylcadaverine (Fig. 2E) is primarily localised within phase dense vesicles (lysosomes?). A rhodamine conjugate of bacitracin was also localised within similar vesicles (data n o t shown). Treatment with amines caused an extensive vacuolization in 3T3 cells; some of these vacuoles contained R-EGF or rhodamine~2macroglobulin. Mckanna et al. have shown that in the presence of amines ferri. tin-EGF remains localized within multivesicular bodies which do n o t fuse with lysosomes [6]. It is possible that the amine mediated increase in lysosomal pH inhibits the fusion between lysosomes and multivesicular bodies. It was recently shown that the fusion of Semliki forest virus with membranes exhibits a dependence on the pH of the environment [29] and therefore amines block the penetration of the virus through the lysosomal membrane [30]. Obviously more experiments are required in order to understand the effect of amines on
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the insertion of EGF receptors and on the recycling o f receptors for a2-macroglobulin and mannose-glycoconjugates [31,32]. However, it is tempting to speculate that this effect is due to inhibition of fusion between the plasma membrane and vesicles containing nascent EGF or recycled a2-macroglobulin receptors. In qualitative terms all the effects of amines reported in this paper could be due to inhibition of fusion between cellular membranes containing EGF-receptors rather than inhibition of a transglutaminase which cross-links between occupied EGF-receptors [33]. Maxfield et al. [13] have shown that amines potentiate EGF-stimulated DNA synthesis by 3T3 cells. Since amines do not block EGF-clustering and internalization it is more reasonable to assume that the enhanced mitogenic response is due to inhibition of EGF degradation rather than effects on EGF internalization [13]. Several reports have pointed out that inhibition of degradation of EGF leads t o enhanced mitogenic response [34] and to the binding of EGF molecules to the nucleus [ 3 4 ] . It is clear from this and other studies that until the cellular site and mode of action of amines are fully understood it is impossible to employ these drugs as a diagnostic tool for studying the mechanism of EGF clustering, endocytosis [33] and action [13].
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