Cell, Vol. 61, 623-634,

May 18, 1990, Copyright

0 1990 by Cell Press

Kinase Activity Controls the Sorting of the Epidermal Growth Factor Receptor within the Multivesicular Body S. Felder: K. Miller,t G. Moehren,t A. Ullrich,* J. Schlessinger:§ and C. Ft. Hopkins* * Rorer Biotechnology, Inc. 660 Allendale Road King of Prussia, Pennsylvania 19406 t Imperial College London SW7 2AZ England *Max-Planck-lnstitut fur Biochemie Am Klopferspitz 16A 6033 Martinsried Federal Republic of Germany

Summary We compared the internalization and intracellular sorting of eptdermal growth factor receptor (EGF-R) and point mutant kinase-negative EGF-R separately expressed in NIH 3T3 cells lacking endogenous receptor. Both EGF-Rs internalized rapidly, but kinasenegative receptor was surface down-regulated only with monensin or at 20%. Furthermore, EGF internalized by mutant receptor alone was, in significant proportion, returned to the cell surface undegraded. Hence unlike wild-type receptor, kinase-negative EGFR recycles. By electron microscopy the early pathways of endocytosis for the two receptors were identical; however, after lo-20 min the pathways diverged at the multivesicular body (MVB). Wild-type EGF-R, destined for degradation, localized to internal vesicles, while kinase-negative EGF-R, destined for recycling, localized to surface membranes of the MVBs and moved to small tubulovesicles. We conclude that sorting of internalized receptor for degradation or recycling can occur through spatial segregation within the MVB, and sorting of EGF-R is controlled by tyrosine kinase activity. Introduction In a wide variety of animal cells the trafficking of the epidermal growth factor receptor (EGF-R) is as follows. EGF-R is initially monodisperse on the surface of target cells. When EGF binds to EGF-R, it rapidly becomes associated with and concentrated in clathrin-coated pits. After 1 to 2 min EGF-R and bound EGF are internalized, transit through the endosomal system, and are degraded within the lysosomal compartment with a half-time of degradation of 40-50 min (Schlessinger et al., 1976; Carpenter and Cohen, 1979; Haigler et al., 1979; Schlessinger, 1966). Degradation of EGF-R serves to reduce the surface-bound receptor pool and may play a regulatory role, modulating the stimulatory signal. 5 Present address: Department of Pharmacology, Medical Center, New York, New York 10016.

New York University

As soon as EGF binds, the kinase moiety of the cytoplasmic domain of EGF-R is activated, resulting in tyrosine phosphorylation of several residues near the carboxyterminal tail of EGF-R (Downward et al., 1964; Margolis et al., 1969) and the phosphorylation of tyrosine residues on several other proteins, some of which have been characterized (reviewed in Ullrich and Schlessinger, 1990). Within 2 min of EGF binding, phosphatidylinositol turnover is stimulated, Ca2+ is mobilized, and intracellular pH is increased (Moolenaar et al., 1967). With continued EGF stimulation for several hours, cells become committed to synthesize DNA and to divide. Intrinsic kinase activity appears necessary for stimulation of the rapid ionic and long-term mitogenic responses, as kinase-negative mutant EGF-Rs that bind EGF normally are inactive in signal transduction (Honegger et al., 1987a, 1967b; Chen et al., 1967; Moolenaar et al., 1966). Interestingly, intrinsic kinase activity also appears necessary for the normal trafficking of internalized receptor. Kinase-negative EGF-Rs are not surface down-regulated and are not degraded in response to EGF. This has been shown for a point mutant EGF-R in which lysine 721 was replaced with alanine (K721A; Honegger et al. [1987a] and used herein), for a double point mutant EGF-R with lysine 721 replaced with methionine and threonine 654 replaced with alanine (Glenney et al., 1968) and for a point mutant in which lysine 721 was replaced with methionine (Chen et al., 1989). The latter two studies concluded that kinasenegative EGF-R is neither down-regulated nor.degraded because it is not internalized. In contrast, we demonstrate rapid internalization for kinase-negative mutant receptor, both with radiolabeling studies and with ultrastructural studies. We have sought to determine the fate of internalized single point mutant K721A with binding studies using radiolabeled EGF and monoclonal anti-EGF-R antibodies, and with double-label morphological studies. We show that the kinase-negative receptor is internalized in response to EGF with kinetics similar to wild-type receptor; however, rather than being degraded, a large portion of the mutant receptor and a significant portion of EGF are recycled back to the cell surface. With electron microscopy, we show that for wild-type EGF-R and the K721A mutant the pathways of internalization are initially the same but diverge at the multivesicular body (MVB; defined as a circular vacuole of diameter 250-1000 nm, with interior vesicles of diameter 50-100 nm). We conclude that tyrosine phosphorylation within the MVB provides a sorting signal, selecting normal EGF-R for degradation. Results Monensin and Lowered Temperature Treatments Demonstrate Kinase-Negative EGF-R Is Recycled, as Is Transferrin Receptor Analysis of binding of 12sl-EGF (Figure 1A) shows that cells bearing the “wild-type” EGF-R (HER14) and cells expressing the kinase-negative EGF-R (K721A) internalize

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1 i

0

J

I 0

20

Time

10

40

Time

(min)

(min)

B With

M WT

WT I 40

20

Time Figure

1. Internalization

100

Time (mitt)

(min)

of EGF and Effect

of Monensin

HER14 (“m) and K721A (‘MN) cells were preincubated with 5 nM tssl-EGF at 4% for 90 min. The cells were rapidly warmed and incubated at 3pC for the times indicated. (A) Untreated cells. (B) Ceils pretreated and incubated with 50 ufvl monensin. Surface binding (dotted lines) was assessed by radioactive counts dissociated at pH 2. Initial counts bound on the surface of the two cells were 49,170 and 24,600 cpm for the HER14 and K721A cells, respectively, and did not change significantly for cells preincubated with monensin. Values are expressed as a percent of the total cell-associated counts at time 0 for comparison.

EGF with similar kinetics. The amount of internalized ligand (acid inaccessible) rose rapidly for both ceils, and reached near-plateau values by 15-20 min. In HER14 ceils surface binding was rapidly down-regulated in coordination with internalization, while for K721A cells little down-regulation occurred. Following treatment of cells with 50 uM monensin, accumulation of internal ligand increased approximately 15% in both cells, and most notably resulted in EGFinduced down-regulation of surface binding for the mutant receptor. Because monensin (in addition to other effects) has been shown to inhibit recycling of several receptors including receptors for low density lipoprotein (Basu et al., 1981), insulin (Whittaker et al., 1986) transferrin (Stein et al., 1984; and see below), asialoglycoprotein (Berg et al., 1983) and EGF in hepatocytes (Gladhaug and Christoffersen, 1988) without inhibition of internalization, we conclude that kinase-negative EGF-R is internalized normally and

Figure

2. Transferrin

Internalization

and Recycling

(A) HER14 (circles) and K721A (squares) cells were preincubated with 10 nM 1251-transferrin for 60 min at 4°C. Cells were washed and warmed and incubated in the absence of transferrin at 37% for the times indicated. Values represent the percent of the total cellassociated counts at time 0, dissociated at pH 2 from the surface (open symbols) or internal (filled symbols). (6) HER14 cells were preincubated with 10 nM 1251-transferrin for 60 min at 4%. Cells were warmed to 37°C (filled squares) or 20% (open squares) or were preincubated and assayed at 37% with 50 WM monensin (filled circles). Cells were incubated (in the continued presence of radioligand) for the times indicated. Values plotted represent the percent of cell-associated counts at each time point that were not dissociated at pH 2.

rapidly recycles in the absence of monensin. In addition, for cells expressing EGF-R with most of the cytoplasmic domain deleted (Na8-14 cells), EGF is very poorly internalized (Livneh et al., 1986) and monensin treatment does not potentiate internalization (data not shown). To explore the possibility that the K721A cell line was aberrant in some general way in its processing of recycling receptors, the ability of the two transfected cell lines to internalize and recycle surface-bound ‘%ransferrin was assessed. Transferrin was internalized and recycled with the same kinetics in both cell lines (Figure 2A). Similar results were obtained with untransfected cells (data not shown). We conclude that the internalization and recycling pathways for both cell types are similar and unaltered by transfection. As it did for EGF internalization of K721A

;CZ5F Receptor

Endosomal

Sorting

by Kinase

Activity

shown in Figure lA, monensin treatment increased the accumulation of internal transferrin (Figure 26). For untreated cells, after 20 min at 37% in the continued presence of transferrin, internalized label reached a steady state at roughly 60%. With monensin treatment, the initial rate of internalization was roughly the same, but at steady state the proportion internalized was increased to 95%. Hence monensin inhibited recycling. Similarly, incubation of cells at 20% increased the proportion of internalized transferrin at steady state, by decreasing the rate of recycling more than the rate of internalization. This effect was used (see below) to chase preinternalized EGF and receptor through subsequent processing steps. The influences of monensin and low temperature for internalization of transferrin receptor for the K721A cells were similar in all respects (data not shown).

K721A

I 5

10

Time

(min)

100

-0

Time c

(min)

1 /------

K721A

222 CO:” 0

1

HER

ii HER K721A

---

f 2

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-

----a

0

4 40

50

Time Figure

3. Uptake

and Processing

(min)

of rz51-MAb108

(A) Internalization. HER14 (filled circles) and K721A (open squares) cells were preincubated with 10 nM 1z51-MAb108 for 90 min at 4°C then warmed and incubated with 20 nM EGF in the continued presence of rz51-MAb108 at 37%. At the times indicated, cells were washed and internal counts (not dissociable at pH 2) were assessed. Total cell-associated counts after preincubation were 9,080 and 10,260 cpm for HER14 and Ki’21A cells, respectively. (6) Down-regulation. Cells treated with 20 nM EGF for the times noted at 37% were cooled to 4°C and washed twice, and surface binding sites were probed with 1251-MAb108. (C) Effects of monensin. Cells were preincubated at 37°C for 20 min with (solid lines, filled symbols) or without (dotted lines, open symbols) 50 uM monensin. Subsequently, 10 nM r2%MAb108 and 20 nM EGF were added for the times listed at 37%; surface counts were dissociated by a pH 2 wash, and counts remaining (internal) were determined.

Receptor Trafficking Followed with Monoclonal Antibody To follow EGF-R processing more directly than by using radiolabeled EGF, we used radiolabeled monoclonal antibody 106 (MAblOB). It is specific for the external domain of the human EGF-R, does not compete for binding with EGF, and does not induce internalization of EGF-R (Bellot et al., 1990). Additionally, MAb106 does not slow the internalization of occupied EGF-R, does not dissociate from EGF-R at pH above 4 (half-time of dissociation >l hr), and does not change the rate of EGF-R degradation for K721A or HER14 cells either in the presence or absence of EGF (Felder et al., unpublished data). These properties make it an excellent label to follow EGF-dependent internalization and subsequent trafficking of EGF-Ft. The data for internalization of receptor agree with those presented above for internalization of EGF. The initial kinetics of internalization of 1251-MAb106 were identical for both cell lines (Figure 3A) and were similar to those for EGF and transferrin. K721A cells showed much less surface down-regulation (Figure 38) than did HER14 cells. Furthermore, in the presence of monensin the accumulation of kinase-negative receptor to the internal compartment was greatly increased, while that for the wild-type receptor was increased much less (Figure 3C). These data are consistent with that shown above, supporting the conclusion that the kinase-negative receptor recycles following internalization, and suggest that MAb108 is a good probe for following trafficking of EGF-R. Internalized Kinase-Negative Receptor Is Not Degraded, but Recycled Since the data of Figure 28 showed that incubation of cells at 20% decreased recycling relative to internalization for transferrin receptor, this temperature was used for EGF internalization. Consistent with that data and with results for several cell types showing that incubation at 20% prevents transfer of ligand to the lysosome (Dunn et al., 1960; Miller et al., 1986) incubation of cells at 20°C resulted in intracellular accumulation of intact radioiodinated EGF (data not shown). We used this fact, along with the results presented in Figure 2 demonstrating increased accumulation of internal ligand at 20°C, to preload the in-

Cell 626

all time points from 5 to 30 min up to 50% of ligand was recycled. Consistent with this picture, in the presence of EGF, 1251-MAb108 was degraded in HER14 cells 4-fold more rapidly in the first 90 min (data not shown). It has been shown that the EGF-stimulated turnover of total cellular EGF-R is much more rapid for HER14 than for K721A cells (Honegger et al., 1987a). In morphological experiments presented below, we investigate the compartment loaded by the 20% preloading, and use the preloading procedure to follow the subsequent trafficking of the two EGF-Rs at 37%

20

Time

B

of

Chase

(min)

1

I

2 0

0

Figure

40

20

Time 4. Chase

of internalized

of

Chase

(min)

EGF

HER14 (A) and K721A (B) cells were preincubated for 60 min at 20% with 10 nM ?-EGF, washed, and then warmed and incubated without EGF at 37% for up to 30 min. At the times indicated, TCAprecipitable counts in the medium (“Released”), TCA-soluble counts in the medium (“Degraded”), counts released from cells at pH 2 (“Surface”), and counts remaining, solubilized with 0.1 M KOH (“Internal”). were assessed. By this analysis HER14 cells were more efficient at processing internalized EGF to TCA-soluble form than were K721A cells.

compartment with ligand and chase this label through subsequent processing steps upon warm-up to 37%. This allowed for a sensitive, time-synchronized test of the fate of internalized ligand (Figure 4). After 60 min at 20°C, 75% of the radioligand was internal for both cells. Following warm-up to 37oC, the amount of acid-inaccessible radioligand rapidly declined. For HER14 cells expressing wild-type EGF-R, this decline was due primarily to degradation, since 80% of the label appearing in the medium after 30 min was acid soluble. In K721A cells expressing mutant receptor, half of the radiolabel, initially acid inaccessible, was released intact (in TCA-precipitable form). Surface binding changed little during the warm-up, so the increased amount of label appearing in the medium could not have been due to dissociation. Hence, EGF accumulated in HER14 cells held at 20°C, and on transfer to 37oC it was rapidly degraded. In K721A cells, the ligand that was accumulated internally at 20°C was, on transfer to 37%, less efficiently degraded, and at ternal

Pathways of Internalization for Wild-Type and Kinase-Negative Receptors Diverge at a Late Stage of Endocytosis In preliminary studies, horseradish peroxidase-conjugated EGF (EGF-HRP) was used to outline the endocytic pathway in the two transfected cell lines using a 4°C pulse/ 37% chase protocol. These studies showed HRP reaction product initially bound at the cell surface, present within coated pits at 2 min of chase, within peripheral endosomes (vesicles, tubulovesicles, and vacuoles) at 10 min, and in MVBs at 10 to 20 min. From 15 min onward, MVBs were concentrated around the centrioles in the juxtanuclear area. These observations confirmed those of earlier studies on epithelioid cells (Miller et al., 1986; Haigler et al., 1979). In accord with the results shown above that trafficking of transferrin was the same in both cell types, there were no obvious differences between the two cell lines in the endocytic pathways outlined by transferrin conjugated to HRP (data not shown). To determine the distribution of EGF receptors following fixation and freezing, cryosections were labeled with antiEGF-R antibody either conjugated directly to colloidal gold or in two-step procedures with gold-conjugated protein A. In cells that were not treated with EGF, receptors were distributed predominantly on the plasma membrane. Within 2 min of EGF treatment, receptors were concentrated in coated pits and at 10 to 20 min were in elements of the endosome system (Figure 5), similar to the distribution of EGF-HRP Although labeling of cryosections was high enough to be qualitatively convincing and demonstrated the presence of EGF-R ih all of the compartments labeled by EGF-HRP in both HER14 and K721A cells, it was not high enough to allow comparative quantitative analysis. In addition, it did not allow the experimental manipulation of a pulse/chase protocol. To obtain a more strongly labeled EGF-R population, living cells were incubated with gold-MAblO8 conjugates. Cells were incubated with these conjugates at 4% for 30 min, rinsed, and then incubated with 37% medium containing 20 nM EGF. Without added EGF the gold-MAblOB particles remained for up to 30 min at 37% (the longest interval studied) randomly dispersed on the cell surface. On addition of EGF they became internalized via coated pits (Figure 6A) and followed the same time course through the endosome system as EGF-HRP and unlabeled EGF-R described above. The gold-MAb108 conjugate hence appears to be a reliable marker for following the EGF-induced redistribution of EGF-R in living cells. After 10 to 30 min of internalization at 37oC, EGF-HRP

;G&F Receptor

Figure

Endosomal

5. Localization

Sorting

of EGF-R

by Kinase

in MVBs

Activity

by Labeling

of Cryosections

HER14 (a) and K721A (b) cells were incubated with 20 nM EGF for 30 min at 4% rinsed, using an antibody to the EGF receptor followed by protein A-gold. Bars = 100 nm.

and gold-MAbl08 particles were localized to larger juxtanuclear endosomes, a population that included MVBs. Within these elements clear ‘differences in EGF-R localization were observed. Wild-type EGF-R was localized predominantly on the small internal vesicles of the MVBs (Figures 6C and 6D). At late stages mutant EGF-R was predominantly localized to the cell surface. Internal mutant EGF-R was localized in small tubulovesicles and in MVBs. Importantly, mutant EGF-R in MVBs remained largely on the limiting membrane rather than on internal vesicles (Figure 6B). EGF was sometimes distributed throughout the lumen of the MVB, but usually it was more closely associated with the limiting membrane within the immediate vicinity of the gold-labeled EGF-R. At late stages, larger MVBs containing EGF but no EGF-R were seen. In addition, some small vesicles, usually tubular in shape, appeared with EGF-R but no EGF. Chase of Internalized Receptor Reveals That Sorting Occurs at the MVB In a final series of experiments, we exploited the observations reported above, that 20% treatment prevented transfer to a degradative compartment and retarded recycling, to preload the endosome system with EGF-HRP and gold-MAb108. On transfer to 37% chase medium, it should then be possible to identify new labeled elements in HER14 cells as the degradative compartment. In K721A cells we would expect to see an amplification of the recycling arm of the pathway. In HER14 and K721A cells incubated with both EGFHRP and gold-MAb108 at 20% for 60 min. the distribution of the two labels was essentially the same (Figure 7). The majority of both labels was found in large (300-1000 nm) spherical, endosomal vacuoles distributed throughout the cell. Few tubular or tubulovesicular elements contained either tracer, and little label was localized to the plasma membrane. Fewer than 10% of the vacuoles contained internal vesicles. In most of the labeled vacuoles, gold parti-

and then warmed

to 3pC

for 20 min. Sections

were labeled

cles and HRP reaction product were arranged along the limiting membrane. Gold particles were usually evenly spaced and in single file. At lo-30 min of chase at 37%, the distribution and form of the labeled vacuoles changed dramatically. In HER14 cells (Figures 8A and 8B) the vacuoles concentrated around the centrioles in the juxtanuclear area and the majority now contained internal vesicles. The internal vesicles were labeled with EGF-HRP and gold-MAbl08. At later time points (30 min at 37%), vacuoles of lysosomal appearance in which HRP reaction product and gold particles were distributed throughout the lumena were seen (data not shown). In K721A cells (Figures 8C and 8D) the number of MVBs also increased at 37% but they remained in the minority. The most common labeled element after 10 min at 37% was highly pleomorphic and consisted of a vacuole with elaborate tubular and lamellar extensions (Figure 8C). In these structures the gold particles and the HRP reaction product were concentrated on the limiting membrane and in its tubular extensions. There were few internal vesicles within these vacuoles, but those present were labeled. In K721A cells after 20 min at 37% (Figure 8D), pleomorphic structures remained and there were also many small (70-200 nm diameter) vesicles and tubules free within the cytoplasm that contained both EGF-HRP and gold-MAblO6. Table 1 presents these data in quantitative form. The numbers of gold particles in three different compartments were counted: along the limiting membrane of the MVBs, along the surface membrane of the small internal vesicles, and inside small tubulovesicular elements (shortest diameter less than 200 nm). For both cell types before warm-up, most of the gold particles were localized to the limiting membrane of the MVBs. Following 20 min at 37%, most of the gold particles in HER14 cells were chased to the interior vesicles of the MVB. For the mutant cells, the percentage of particles localized to the interior vesicles of the MVB was not significantly different after warm-up; in-

Cell 628

Figure

6. Epon Sections

Demonstrating

Internalization

of the EGF-R and Its Ligand

at 37%

(A) K721A cells were prebound with 20 nM EGF-HRP and gold-MAblO8 for 30 min at 4OC, then warmed to 3PC for 2 min. inset: K721A cell similarly prebound and warmed with gold-MAblOB, but with unconjugated EGF at 20 nM. Both EGF and its receptor were localized to coated pits on the cell surface. Bars = 50 nm. (B) K721A cells were prebound with 20 nM EGF-HRP and gold-MAblO8 for 30 mm at 4% then warmed to 3PC for 30 min. EGF and its receptor were localized to the limiting membrane of large endosomes as shown. Surface membrane and small endosomes were labeled at this time as well (data not shown). Bar = 200 nm. (C and D) HER14 cells treated as in (B). EGF and its receptor were largely localized to the Intenor vesicles of large mumvesicular endosomes. Bars = 200 nm.

stead, particles that had been on the limiting membrane were chased to tubulovesicular elements. These data suggest a tendency for wild-type receptor to move centripetally from the limiting membrane of the late endosome to small vesicles within this compartment, and a tendency for the kinase-negative receptor to move centrifugally from the limiting membrane of the late endosome to separate, small vesicular elements. Discussion In agreement with the suggestions put forth earlier (Honegger et al., 1987a), we have shown that kinase-negative EGF-R is internalized in response to EGF binding, and in large proportion is recycled back to the cell surface. We have shown this by probing with radiolabeled EGF and with radiolabeled MAb108. The difference does not appear to result from a general trafficking defect in one of these cells lines, since both internalize and recycle transferrin with the same kinetics. In addition, the perturba-

tions of lowered temperature and monensin treatment were observed to inhibit recycling of transferrin in both cells and to enhance down-regulation of surface receptor effectively for the kinase mutant. However, while in the earlier work EGF was found to be degraded as rapidly by K721A cells as by wild-type cells, we have shown that approximately half of the EGF internalized by K721A cells at 20% for 60 min is returned to the medium undegraded. This difference may be due to differences in protocols. In this report for this particular experiment, we used internalization at 20% and chase (washout of label), while earlier we used incubation at 37% in the continued presence of ligand. It is important to note that the difference in trafficking between kinase-positive and kinase-negative receptors may not be absolute. A portion of kinase-positive receptors may escape selection for degradation, as suggested by experiments demonstrating recycling of that fraction of “wild-type” receptor not quickly degraded upon EGF presentation in the perfused liver (Dunn and Hubbard, 1984),

EGF Receptor 629

Figure

Endosomal

7. Preloading

Sorting

by Kinase

of Cells with EGF-HRP

Activity

and Gold-MAblOB

at 20%

(Left) Epon section of K721A cell incubated with both EGF-HRP and gold-MAblOB at 20% for 60 min. Both EGF and its receptor were localized to spherical vacuoles within the cytoplasm. Few particles and little peroxidase reaction product were seen in small tubulovesicles or at the cell sup face. Bar = 200 nm. (Right) Endosomal vacutoles from HER14 cell incubated as for the K721A cells. demonstrating the close colocalization of EGF and its receptor. Bar = 100 nm. Note the absence of vesicles within MVB lumena at this temoerature.

and one report of recycling of “wild-type” receptor in 3T3 cells (Murthy et al., 1988). Conversely, some kinasenegative receptor may follow the degradatory pathway, as evidenced by the fact that the rate of degradation of kinase-negative receptor is slightly elevated by EGF (Honegger et al., 1987a). Our data contrast on one specific point with the data of Glenney et al. (1988) and Chen et al. (1989). In agreement with our work, those investigators reported that kinasenegative receptor is inactive in signal transduction and is not down-regulated in response to EGF; however, they did not conclude that kinase-negative receptor is recycled after internalization, but rather that it is not internalized. The basis for this conclusion wast the short-term internalization assay without prebinding of ligand. We have shown that the rates of internalization of kinase-negative and wildtype receptors following prebinding of ligand to equilibrium are very similar. The difference in protocol and the specific assumptions of the kinetics analysis may account for the difference in conclusions from these data alone. We go on to show, however, that with two different methods that inhibit recycling (treatment with monensin and incubation at 20°C), kinase-negative receptor is surface down-regulated with the same time course as is wild-type receptor. Furthermore, the morphological pathways of endocytosis are the same to the point of the large juxtanuclear endosome for the two receptors. By the use of these additional tests, perturbation of recycling and morphology, we have clearly demonstrated that kinase activity is not essential for efficient, coated pit-mediated endocytosis of the EGF-R. This conclusion, in fact, is also supported by the work of Chen et al. (1989) who showed that

a C-terminal truncation of kinase-negative receptor produces a receptor that internalizes (and down-regulates). These further tests should be performed before conclusions can be drawn regarding inactivation of the process of endocytosis for a membrane receptor. Recently, the same group reported that EGF causes the transformation of cells transfected with a kinase-positive C-terminal deletion mutant EGF-R, reported not to be internalized (Wells et al., 1990). The conclusion reported, that internalization is not required for signal transduction, remains questionable, as the tests mentioned above were not reported for this receptor. Moreover, our preliminary data for an EGF-R with a similar C-terminal deletion suggests that it internalizes and recycles. Morphological studies using colloidal gold conjugated to anti-EGF-R antibodies to label the receptor and HRP conjugated to EGF to follow internalized ligand were consistent with conclusions drawn from the radiolabeling studies. Following internalization a clear difference in intracellular localization occurred, which is schematized in Figure 9. Wild-type and mutant receptors internalized along a similar pathway to the stage of the late juxtanuclear endosome, identified here as the MVB, and subsequently moved to different compartments. Wild-type receptor became localized to the surface of small vesicles within the MVB, while kinase-mutant receptor did not but instead was localized to the perimeter membrane of large endosomes and to small tubulovesicular elements that were both juxtanuclear and peripheral. These conclusions were reached from the experiments performed at physiological temperature (Figure 6). We next exploited the results of the radioligand experi-

Cell 630

Figure

8. Warm-Up

of Preloaded

Cells

(A and 6) Epon sections of HER14 cells incubated with EGF-HRP and gold-MAblOB for 60 min at 20% followed by 30 min at 37%. Receptors and ligand were found predominantly in vacuoles containing internal vesicles as shown and lysosome-like elements (not shown). Bars = 200 nm. (C) Epon section of K721A cell incubated with EGF-HRP and gold-MAblO8 for 60 min at 20% followed by 10 min at 37% demonstrating the appearance of vacuoles with tubular extensions. Gold particles were mainly on the limiting membrane of the vacuoles (large arrow) and within the tubular extensions (small arrow). Bar = 100 nm. (D) Epon section of K721A cell preloaded at 20% as in (C) and then warmed up to 37T for 20 min. Large vacuoles with tubular extensions were still apparent at this stage, and EGF and its receptor were often found in tubular extensions from the large vacuoles (small arrow), in small tubulovesicular elements, within the cytoplasm, and at the cell surface (large arrow). Bar = 100 nm

EGF Receptor 631

Endosomal

Table 1, Distribution and the Tubulovesicles

Sorting

by Kinase

of Gold-MAbl06 That Surround

within Them

Perimeter membrane Internal vesicles Tubulovesicles

-

37%.

20’

cles of this compartment. This suggests that lumenal vesicles of the MVBs arise directly from the perimeter membrane, rather than as a result of a complex fusion event. With the same time course we could chase kinase-negative receptor from the limiting membrane of the MVB into protrusions from this compartment and into small, tubulovesicular elements. This suggests that the small tubulovesicles seen containing kinase-negative receptor in these experiments and at late times in the 37°C experiments belong to the recycling pathway. Consistent with our data, EGF-Fl in A431 epidermoid carcinoma cells becomes localized to the interior vesicles of the MVB (Haigler et al., 1978; Miller et al., 1986), while recycling transferrin receptor is predominantly seen in small tubulovesicular elements, and when seen in MVBs following long incubations is only seen on the limiting membrane (Hopkins et al., 1983). We conclude that the MVB is an important organelle for intracellular membrane sorting, as it is equipped with a mechanism for specifically sequestering membrane proteins destined for lysosomes into its interior and with a mechanism for removal of limiting membrane for reuse. Specificity for the first, centripetal movement is concluded from the fact that wild-type but not mutant receptor was sequestered. Specificity for the second, centrifugal movement, i.e., selectivity for the removal and recycling of specific domains within the limiting membrane, may be suggested by some micrographs in which protrusions from the MVB contain a higher concentration of mutant receptor than the rest of the limiting membrane (Figures 8C and 8D, small arrows). Alternatively, the centrifugal movement may represent recovery of surface membrane with passive flow of protein components. Our description of EGF-R sorting is consistent with the proposed description of the sorting of recycling receptor from internalized ligand (Geuze et al., 1983). We would modify this proposal to encompass membrane proteins as follows. Endocytosed components that are destined for lysosomes enter the lumen of large vesicular endosomes; those to be shuttled to other compartments including Golgi or plasma membrane remain on the limiting membrane. Receptors may enter the lumen by invagination

MVSs Kinase

Wild Type 20%

Activity

20%

Mutant +

37%

67%

29%

62%

33%

26%

65%

18%

25%

7%

6%

20%

42%

20

Gold-MAblO6 and EGF were internalized for 60 min at 20% and cells were shifted to 37% for 20 min. Before or after the temperature shift cells were fixed, embedded, and thin-sectioned, and particles overlaying the components were counted for 500 HER14 and 750 K721A cell profiles.

ments, which demonstrated that receptor and ligand accumulate intracellularly at 20% and, furthermore, that following warm-up ligand was trafficked differently for the two cell types. This protocol allowed two advantages as compared with the 37% experiments. Since receptor and ligand were accumulated in the MVB compartment (presumably because steps downstream toward recycling or toward degradation were inhibited), the sensitivity with which the subsequent trafficking paths could be followed was improved, especially for the kinase-negative cells. In addition, the position of internalized receptor was now well synchronized, allowing for an effective intracellular chase experiment. Little label was seen on the cell surface or in small, peripheral endosomes after the 26% incubation for either cell, hence a different distribution following warm-up represented movement from the limiting membrane of the late endosome. Results of morphological studies using this temperature-shift protocol were qualitatively the same as those at 37°C. The same differences in localization between wild-type and mutant receptors were seen in both experiments. Hence the temperature-shift protocol did not artificially cause this difference in trafficking, but magnified it and allowed for the assignment of temporal relations. With this protocol we could chase wild-type receptor from the limiting membrane of the MVB to the interior vesi-

0 EGF o

0

IlUTANT EGF-R

o

0

Figure 9. Schematic Drawing of the Separate Pathways Followed by the Wild-Type and Kinase-Mutant Receptors It is proposed that following internalization, wild-type EGF-R (left) becomes sequestered into the lumenal space of the MVE by a pinching-off mechanism, and that the lumenal contents of this compartment are degraded following fusion with or possibly maturation to form a lysosomal vacuole. Either the mechanism of budding or the mechanism for localizing the EGF-R to the site of budding may be tyrosine kinase dependent. In contrast, the kinase-negative mutant receptor (right) does not enter the lumen of the MVS but is recycled to the plasma membrane by small tubulovesicular elements through an intrinsic process for recovery of surface membrane.

Cell 632

and pinching off of small vesicles. Ligand (or transported iron in the case of transferrin) may enter the lumen by dissociation from receptor at mildly acidic pH, or by remaining bound to a receptor that invaginates. During transit through the endosomal system, surface membrane is removed for shuttling to other cellular sites, including the cell surface. Removal of surface membrane may involve specific sequestration of membrane protein-rich domains. Our data suggest that unoccupied, and hence kinase-inactive, EGF-Rs inadvertently internalized may be among the membrane proteins recycled by this recovery process. Ligands and membrane proteins that have entered the lumena remain within the large, late endosomes, and these elements either mature to form of else fuse with lysosomes. On what basis the kinase-active and kinase-negative EGF-Rs are sorted is not clear. K721A and HER14 cells bind EGF identically (Honegger et al., 1987a), even at reduced pH (our unpublished data). As shown by crosslinking agents, both receptors aggregate in response to EGF (Honegger et al., 1990; Margolis et al., 1990). Hence, neither ligand binding nor receptor aggregation appears to provide the signal. Importantly, when both receptors are expressed in the same cell line, both receptors are phosphorylated (by the wild-type receptor), yet EGF stimulates the down-regulation and degradation of the wild-type receptor alone (Honegger et al., 1990). In addition, kinasepositive EGF-R mutants lacking any of the four C-terminal autophosphorylation sites are degraded as normal receptor (Honegger et al., 1988). Hence, autophosphorylation of EGF-R does not appear to be critical. We propose, then, that the intrinsic kinase activity, perhaps by effecting the phosphorylation of a localized substrate, provides the sorting signal for this receptor. The substrate may be a protein involved in pinching off of lumenal vesicles (which may explain why fewer intralumenal vesicles were seen for the K721A cells) or may bind to and bring EGF-R to sites from which vesicles pinch. This would require that EGF-Rs within endosomes remain catalytically active. This appears to be the case, as evidenced by the phosphorylation of a protein specific to the endosome, designated ~35 (Cohen and Fava, 1985). Furthermore, EGF-R in highly purified endosome fractions can autophosphorylate and phosphorylate endogenous and exogenous substrates (our unpublished data). It remains to be demonstrated whether this mechanism for sorting by invagination to vesicles interior to the late endosome is a general mechanism for sorting of peptide hormone receptors that are down-regulated in response to hormone. Comparison of the trafficking of PDGF receptor, a receptor kinase that is down-regulated, and insulin receptor, one that is recycled (for reviews see Williams, 1989; Ullrich and Schlessinger, 1990) will be important in this regard. Experimental

Procedures

Cell Lines NIH 3T3 cells, strain 2.2, transfected with a plasmid bearing either the full-length cDNA for the human EGF-R (HER14 cells) or the cDNA encoding the human EGF-R with lysine 721 replaced with alanine (K721A

cells), and each expressing roughly 400,000 receptors per cell, were used (Honegger et al., 1987a). The wild-type EGFR of HER14 cells is fully functional in signal transduction, while the point mutant receptor of K721A cells shows no tyrosine kinase activity as measured in immunoprecipitates or in living cells, and IS unable to elicit any cellular response to EGF (Honegger et al.. 1987a, 1987b; Moolenaar et al., 1988). Radiolabeling Studies For binding and internalization studies cells were plated on human fibronectin-coated 35 mm dishes or 24-well plates (Costar), and grown in Dulbecco’s minimal essential medium (DMEM) with 10% calf serum. Either just below or at confluence, cells were incubated with radioligands (mouse EGF, MAblOB, and bovine transferrin were iodinated with the lodogen method) in DMEM supplemented with 20 mM HEPES and 0.2% BSA (buffer A). Where indicated, cells were allowed to bind radioligands at 4°C. At the end of incubations, cells were washed three times with phosphate-buffered saline (PBS; with Gas+ and Mg*+). Surface-bound ligand was assessed by collecting two washes of cells with PBS adjusted to pH 2 with HCI for a total of 3 min at 4OC. Internal ligand was assessed by dissolving cells and ligand remaining after a pH 2 wash with 0.2 M KOH. Counts were quantitated with a gamma counter. Where indicated, cells were incubated at 37% for 30 min with 50 pM monensin (diluted from a 20 mM stock methanol) rn buffer A. For assessing degradation of EGF and MAblOB, incubation buffer A was removed and diluted with ice-cold TCA to make a 10% solution. BSA (1 mglml) was added as carrier, and precipitates were allowed to form for 1 hr at 4% and were then washed once wtth the TCA solution to improve separation. Precipitate (by this criterion, intact ligand) and supernatant (degraded ligand) were separated by centrifugation and counted. Preciprtatrons of intact ‘251-EGF and 1251-MAb108 were greater than 90%. Colloidal Gold Complexes Colloidal gold (5 nm and 8 nm diameter) sols were made as described by Slot and Geuze (1985). MAb108-8 nm colloidal gold (gold-MAblOB) and protein A-5 nm colloidal gold complexes were made according to standard methods (DeMey, 1986). For cell incubations the colloidal gold complexes were washed by centrifugation in a Beckman Airfuge at 150,000 x g for 5 min and resuspended in DMEM, 25 mM HEPES (pH 7.4) (for gold-MAblOB) or PBS, 0.1% BSA (pH 7.4) (for protein A). By electron microscopy the gold complexes were monodisperse. EGF-HRP Conjugate Human EGF and HRP, type II (Sigma, Poole, England), were dialyzed and then reacted with 3-(2.pyridyldithio)propionic acid N-hydroxysuccinimide ester (SPDP) heterobifunctional reagent as recommended by the manufacturer (Pharmacia, Milton Keynes, England). They were dialyzed and reacted together overnight at 20°C. The conjugate was then separated from unreacted EGF on a Sephadex G-100 column. Incubation of Cells with Tracers and Processing for Electron Microscopy For experiments at physiological temperature, cells grown on 3 cm ttssue culture dishes were rinsed twice with DMEM, 25 mM HEPES (pH 7.4) at 4OC and then incubated in the same buffer containing 100 pglml EGF-HRP conjugate and the gold-MAblO8 complex for 1 hr at 4%. The cells were then warmed to 37%’ by immersion of the plates in a water bath and incubated for various time intervals in the continued presence of ligands. For the temperature shift experiments, cells on the same dishes were rinsed with buffered medium at 20% and incubated in the same buffer and concentrations of conjugated ligands as above for 1 hr at 20%. The cells were rinsed twice and then warmed by incubating in buffered medium at 37°C for various time intervals in the absence of ligands. After the appropriate time of incubation at 3PC (for both kinds of experiments), cells were fixed in 2.5% glutaraldehyde. 2% formaldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 15 min. The cells were rinsed twice in 50 mM Tris-HCI buffer (pH 7.6) and incubated with 0.75 mglml diaminobenzidine (Sigma, Poole, England) to demonstrate peroxidase cytochemically. The cells were rinsed in 0.1 M sodium cacodylate buffer, postfixed in 1% 0~0~ rn the same buffer for 60 min at 4°C rinsed, and then scraped off the dish. The cells were pelleted in an Eppendorf microfuge. dehydrated, and

Ef3F Receptor

Endosomal

Sorting

by Kinase

Activity

embedded in Epon 812 (Polysciences. Northampton, England). Sections were cut on a Reichert Ultracut microtome, stained, and examined at 80 keV in a Philips CM 12 electron microscope. lmmunocytochemistry on Thin Frozen Sections Cells grown on tissue culture dishes were incubated in serum-free medium for 4 hr, and then in the presence of 10 nM EGF for 45 min at 4°C. The cells were rinsed twice at 4OC and warmed to 37°C for various times before fixing in 2% formaldehyde, 0.05% glutaraldehyde in phosphate buffer (pH 7.4) for 15 min. The cells were scraped off the dish, pelleted in an Eppendorf microfuge, infused with 2.3 M sucrose, and then frozen in liquid nitrogen. Thin frozen sections were cut on a Reichert FC4 cryoultramicrotome and immunolabeled essentially as described by Tokuyasu (1978). Sections were labeled with antibody to the EGF-R (antibody RI as reported in Miller et al., 1986) at 25OC for 60 min followed by protein A-gold for 20 min. Quantitation Quantitation of gold particles on Epon sections was carried out to determine changes in the distribution of EGF receptors on transfer from 20°C to 3PC. Sections (60 nm) were taken from blocks of Eponembedded cells that had been incubated for 60 min at 20°C in the presence of EGF-HRP and gold-MAblOB, or from cells incubated at 20°C for 60 min followed by 37oC for 20 min. Gold particles counted on random cell profiles were allocated to one of three internal compartments: the perimeter membrane of the vacuolarlmultivesicular endosome. the internal vesicles of MVB?.. or tubulovesicles. As the tubulovesicle population varied enormously in size, any gold-containing compartment whose smallest dimension was 250 nm or greater was classified as a vacuolar multivesicular endosome; all other labeled elements were put in the tubulovesicle category. Gold particles on 500 HER14 and 750 K721A cell profiles were counted, the amount of gold in each of the three compartments being expressed as a percentage of the total number of gold particles counted. Acknowledgments The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ‘advertisemenf” in accordance with 18 U. S. C. Section 1734 solely to indicate this fact. Received

January

29, 1990; revised

March

9, 1990.

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