Intracellular Retention and Degradation of the Epidermal Growth Factor Receptor, Two Distinct Processes Mediated by Benzoquinone Ansamycins*

Epidermal growth factor (EGF) stimulates the growth of various types of cells via its cell surface tyrosine kinase receptor. The EGF receptor (EGF-R) has an oncogenic potential when overexpressed in a wide range of tumor cells. Geldanamycin (GA) and herbimycin (HA), specific inhibitors of the cytosolic chaperone HSP 90 and its endoplasmic reticulum homologue GRP 94, were shown to accelerate degradation of the EGF-R and of its homologue p185c- erbB-2 . Here we compared the effects of GA and HA on intracellular degradation and maturation of EGF-R. By using an inhibitor of proteasomal degradation, we learned that GA, but not HA, blocks processing of newly synthesized EGF-R. The effects of GA and HA on receptor degradation are mediated by the cytosolic portion of EGF-R and could be conferred to the erythropoietin receptor (EPO-R), by employing the respective chimera. Neither HA nor GA affected stability of newly synthesized EGF-R lacking the cytosolic domain (Ex EGF-R), but GA caused intracellular retention of this mutant. Taken together, our results imply that GA has two distinct targets of action on the EGF-R, one for promoting its degradation and another for mediating its intracellular retention. Apparently, degradation of the EGF-R mediated by GA or HA requires the presence of the EGF-R cytosolic domain, whereas intracellular retention in the presence of GA is coupled to the extracellular domain of the EGF-R.

Herbimycin A (HA) 1 and geldanamycin (GA) are benzoquinone ansamycins that specifically inhibit the cytosolic chaperone HSP 90 and its endoplasmic reticulum (ER) homologue GRP 94. These compounds have antiproliferative and antitumor effects as they bind to HSP 90, inhibit the HSP 90-mediated conformational maturation/refolding reaction, and thus promote degradation of HSP 90 substrates (reviewed in Ref. 1). The molecular basis underlying this inhibition is still under intensive investigation (2). GA was shown to accelerate the degradation of cell surface proteins including receptor Tyr kinases (3) and the cystic fibrosis transmembrane conductance regulator (CFTR) protein (4). Enhanced degradation in the presence of GA was also observed for non-membrane proteins including Ser/Thr kinases (5), Tyr kinases (6), and mutated p53 (7).
The epidermal growth factor receptor (EGF-R) and its homologue p185 c-erbB-2 are well studied receptor Tyr kinases (8), which are degraded in the presence of GA and HA (9 -11), presumably via the proteasome (3,12). The cytosolic domain of the EGF-R contains Tyr kinase activity and may also contain determinants that are required for GA-and HA-mediated degradation. This is suggested by evidence that a soluble EGF-R, an EGF-R derivative containing only the extracellular domain of the receptor (11,13), and p185 c-erbB-2 lacking either the entire cytosolic domain or the kinase domain (10) were not degraded in the presence of HA (11,13) or GA (10,13). However, it is not clear whether Tyr kinase activity of the receptor or the presence of the entire kinase domain is required to confer GA-mediated degradation.
In the course of biosynthesis, the EGF-R is efficiently processed and is metabolically stable; thus, most of the receptor molecules are present on the cell surface at steady state (14 -16). An EGF-R mutant that lacks the cytosolic domain and contains the extracellular and the transmembrane domains (Ex EGF-R (17)) is also metabolically stable, although its trafficking to the plasma membrane is slower than that of wild type (wt) EGF-R (18,19). Ex EGF-R is therefore an appropriate experimental model system to address the net contribution of the EGF-R extracellular domain on receptor metabolism in the presence of GA and HA. The erythropoietin receptor (EPO-R) is a member of the cytokine receptor superfamily (20,21). Unlike receptor Tyr kinases, the EPO-R has no kinase activity in its cytosolic domain and is phosphorylated by Janus kinase 2 following ligand binding (22). It was previously demonstrated that the EPO-R localizes intracellularly, mainly in the ER (23)(24)(25)(26)(27), and it has been postulated that its retention in the ER may be the result of impaired folding of the extracellular domain of the receptor (24).
In the present study we compared the effects of GA and HA on maturation and degradation of the EGF-R. We show that newly synthesized Ex EGF-R (18,19) is retained intracellularly but is not degraded in the presence of GA. In the presence of HA, however, Ex EGF-R was efficiently transported to the cell surface, but, as with GA, its rate of degradation was not affected. No effect of GA was observed on degradation of newly synthesized EPO-R. Utilizing a chimera composed of the EPO-R extracellular domain and the EGF-R transmembrane and cytosolic domains (28), we demonstrate that the EGF-R cytosolic domain is sufficient to confer GA-mediated degradation. In addition, we show that GA-mediated degradation of the EGF-R does not require Tyr kinase activity of the receptor.
Antibodies-Monoclonal mouse antibodies directed against the extracellular part of the EGF-R were used at 1:2500 dilution for immunoprecipitation (monoclonal antibody 199.12, Neo Markers, Fremont, CA). Polyclonal antibodies directed against the cytosolic part of the EGF-R (29) were kindly provided by J. Schlessinger, New York University, and were used at 1:1500 dilution for immunoprecipitation. Polyclonal antibodies directed against the extracellular part of the EPO-R (26) were used at 1:250 dilution for immunoprecipitation.
Plasmids-Plasmids containing EPO-R/EGF-R chimera in pXM (28), wt EPO-R in pXM, and an EPO-R mutant containing only the extracellular domain in pXM (sEPO-R) were employed (30). The EGF-R mutant containing the extracellular and transmembrane domains (Ex EGF-R) in pCB6ϩ (17) was a generous gift from Dr. C. Carlin (Case Western Reserve University, Cleveland). pCDNA3 EGF-R and its derivative encoding a kinase-defective receptor (K721A) have been described (31).
Cell Culture and Transfection-COS 7 cells were maintained in Dulbeco's modified Eagle's medium supplemented with 10% fetal calf serum (v/v). Cells were cultured to 60% confluence and were transiently transfected using the DEAE-dextran/chloroquine method (32) with 5 g of plasmid containing the appropriate cDNA.
CHO cell lines stably expressing wt EGF-R or K721A EGF-R were maintained in Dulbeco's modified Eagle's medium/Ham's F-12 supplemented with 10% fetal calf serum.
Metabolic Labeling, Immunoprecipitation, and Endoglycosidase H (Endo H) Digestion-COS 7 cells transiently transfected with Ex EGF-R, EPO-R, sEPO-R, or EPO-R/EGF-R chimera cDNAs (2 ϫ 10 6 cells for each time point) or CHO cells expressing wt EGF-R or K721A EGF-R were labeled with [ 35 S]cysteine-methionine as described previously (25). Subsequently, cells were chased in growth medium. Solubilization of the cells, immunoprecipitation, and digestion with Endo H were performed as described previously (25,27). GA or HA was added to the cells during the period of methionine and cysteine starvation and were also present during the pulse labeling and chase incubations (final concentration 1 g/ml). Brefeldin A (BFA; Epicenter Technologies) was added to the cells at a final concentration of 5 g/ml during the chase periods. The proteasome inhibitor Z-L 3 VS (stock solution 5 mM dissolved in Me 2 SO) was added to a final concentration of 50 M to the cells during the period of methionine and cysteine starvation and was present during the pulse labeling and chase incubations as indicated.
Cell Surface Biotinylation-COS 7 cells transiently transfected with Ex EGF-R cDNA were labeled with [ 35 S]cysteine-methionine as described previously (25). After chase in growth medium, cells were rinsed twice with PBS supplemented with 0.1 mM CaCl 2 and 1 mM MgCl 2 (PBS 2ϩ ) and incubated with sulfosuccinimidobiotin (Pierce) at 0.5 mg/ml for 30 min on ice. The cells were rinsed three times with PBS 2ϩ and lysed in PBS containing 1% Triton X-100, 0.5% deoxycholate, and 5 mM EDTA supplemented with protease inhibitors. Isolation of biotinylated proteins was performed by immunoprecipitation with anti-EGF-R antibodies (monoclonal antibody 199.12) followed by protein A-agarose. Precipitates were washed three times with lysis buffer and twice with PBS and boiled in 50 l of 10% SDS. Subsequently, 450 l of lysis buffer was added, and the lysates were incubated with streptavidin agarose (Sigma) for 4 h at 4°C. Samples were boiled in Laemmli sample buffer, resolved by 7.5% SDS-PAGE, and subjected to autoradiography.

RESULTS
Metabolic Processing of EGF-R in the Presence of GA-It has previously been reported that GA affects both maturation and degradation of EGF-R and p185 c-erbB-2 (9, 10, 13) and that the cytosolic domain of p185 c-erbB-2 is required for receptor degradation. We addressed the question whether retarded maturation and enhanced degradation are two independent processes mediated by GA. To this end we compared the metabolic profile of an EGF-R mutant devoid of its cytosolic domain (Ex EGF-R (17)) to that of full-length EGF-R, in the presence of GA and HA, specific inhibitors of HSP 90 and GRP 94.
To enable comparison of EGF-R and Ex EGF-R metabolism in the same cell, we transiently expressed Ex EGF-R in COS 7 cells that also express an endogenous EGF-R. COS 7 cells were transiently transfected with Ex EGF-R, and after 48 h the cells were metabolically labeled in medium containing [ 35 S]cysteinemethionine and chased for periods up to 4 h (Fig. 1). In control non-treated cells newly synthesized EGF-R was stable throughout the chase periods and acquired resistance to Endo H (Fig. 1A), whereas in the presence of either GA or HA it was rapidly degraded (Fig. 1, B and C). In comparison, newly synthesized Ex EGF-R was metabolically stable, and typically 40 -50% of the receptor molecules acquired resistance to Endo H after a 4-h period of chase in the absence of inhibitors (Fig.  1A (19)). It should be noted that Endo H-resistant Ex-EGF-R runs as a more smeared band (probably due to a range of glycosylation products) as compared with Endo H-sensitive Ex EGF-R. Unlike EGF-R, in the presence of either GA or HA newly synthesized Ex EGF-R was metabolically stable throughout the chase periods (Fig. 1, B and C, respectively), suggesting a role for the EGF-R cytosolic domain for degradation in the presence of either GA or HA. After 4 h of chase in the presence of GA, the Ex EGF-R remained largely unprocessed and did not display Endo H-resistant forms (Fig. 1B), whereas processing of newly synthesized Ex EGF-R was practically identical in the presence of HA and in control cells (Fig. 1, C  and A, respectively). These results show that GA but not HA inhibited maturation of Ex EGF-R, suggesting that the extracellular domain of the receptor mediates this effect of GA.
The Effect of GA on Cell Surface Expression of Ex EGF-R-To determine whether GA treatment inhibited transport of Ex EGF-R or only affected its glycosylation pattern, cell surface expression of newly synthesized Ex EGF-R and EGF-R was examined in the presence of GA. COS 7 cells were transiently transfected with Ex EGF-R and were pulse-labeled in medium containing [ 35 S]cysteine-methionine in the presence of GA and compared with cells treated with HA or with no additives. BFA was employed as a control to inhibit overall protein transport to the cell surface (33). Following a 15-min pulse, the samples were chased for 4 h, and the cell surface proteins were labeled by biotinylation. Cell surface EGF-R and Ex EGF-R were isolated by immunoprecipitation of cell extracts, followed by incubation with streptavidin-agarose, and were then electrophoresed by 7.5% SDS-PAGE and subjected to autoradiography. As demonstrated in Fig. 2A, the expression level of Ex EGF-R at the cell surface was diminished in the presence of GA, whereas HA treatment reduced only slightly the cell surface levels of Ex EGF-R in comparison to control non-treated cells. The fact that the total levels of Ex EGF-R were not affected by either treatment is noteworthy (Fig. 2B). Newly synthesized EGF-R was not detected on the cell surface in the presence of either HA or GA ( Fig. 2A), since most of the EGF-R molecules were degraded under these conditions (Fig. 2B). Taken together, these results indicate that incomplete processing of Ex EGF-R in the presence of GA (Fig. 1) is due to retarded transport of Ex EGF-R to the cell surface.
The effect of HA and GA on the levels of total cell surface proteins was also addressed by isolating newly synthesized biotinylated cell surface proteins by streptavidin-agarose (Fig.  2C). Cell surface expression of newly synthesized proteins was reduced (Fig. 2C, asterisk), unchanged (Fig. 2C, triangle), or differentially affected by GA (Fig. 2C, circle). This observation will be extended in the future to examine the specific effects of GA and HA on selected cellular proteins.
Processing of EGF-R in the Presence of GA and Proteasomal Inhibition-It has previously been indicated that the proteasome is involved in degradation of p185 c-erbB-2 and of EGF-R in the presence of benzoquinone ansamycins (3,12). In order to focus on the effect of GA and HA on maturation of the EGF-R, and to minimize their effect on its degradation, we introduced the proteasomal inhibitor benzyloxycarbonyl-leucinyl-leucinylleucinal vinyl sulfone (Z-L 3 VS). To this end we examined the processing of EGF-R when Z-L 3 VS was applied together with HA or with GA. COS 7 cells were incubated in the absence or in the presence of GA or HA, with or without Z-L 3 VS. Metabolically labeled cells were chased for the indicated times in presence of the different inhibitors. The results displayed in Fig. 3 demonstrate that the unprocessed form of wt EGF-R was prominent in cells incubated with GA and Z-L 3 VS, indicating that the receptor was not modified by the Golgi-localized N-acetylglucosamine transferase I (34). However, in the presence of HA and Z-L 3 VS the metabolic profile of the wt EGF-R was indistinguishable from that of control non-treated cells (Fig. 3B,  lower panel), although the total levels of EGF-R were lower. These results further indicate that GA affects both maturation and degradation of EGF-R, whereas HA only accelerates the proteasomal degradation of this receptor. Since GA affects the maturation of both the full-length EGF-R and Ex EGF-R, but affects the degradation of only the full-length EGF-R, different domains of the receptor may be involved in mediating its action. Thus, the extracellular domain that is common to both receptor forms may be involved in its retention, whereas the intracellular domain, present only in the full-length receptor, probably plays a role in its degradation.
The Role of the Cytosolic Domain of EGF-R in GA-mediated Degradation-To investigate this latter possibility we used a chimeric protein composed of the extracellular domain of EPO-R linked to the transmembrane and cytosolic domains of the EGF-R (EPO-R/EGF-R). This chimera is largely retained in an early compartment of the secretory pathway (28), in contrast to the full-length EGF-R, which is efficiently transported to the cell surface. This system allows focusing on the net contribution of the EGF-R cytosolic domain to GA-mediated degradation, minimizing the effect of GA on receptor maturation. COS 7 cells were transiently transfected with cDNAs of the EPO-R, EPO-R/EGF-R, or a soluble EPO-R (sEPO-R), containing only the extracellular domain of the EPO-R (30). In control non-treated cells, the EPO-R/EGF-R chimera was more stable (t1 ⁄2 ϳ4 h) than the EPO-R (t1 ⁄2 ϳ2 h) (Fig. 4, A and B). Treatment with GA significantly enhanced degradation of the EPO-R/EGF-R chimera (t1 ⁄2 ϳ1.5 h; Fig. 4A), whereas it did not affect the degradation profile of EPO-R (Fig. 4B). Hence, the EGF-R cytosolic domain is sufficient to accelerate degradation mediated by GA. Furthermore, a specific action of GA on degradation of selected membrane proteins is implied. To exclude the possibility that degradation of EPO-R/EGF-R in the presence of GA was attributed to the absence of the EPO-R cytosolic  /ml) was added to the cells during starvation, and the inhibitors were present during the pulse and throughout the chase periods. BFA (5 g/ml) was added to the cells only during the chase period. After a 4-h chase period, the cells were subjected to cell surface biotinylation for 30 min. Samples were processed as follows prior to SDS-PAGE and autoradiography. A, cell surface biotinylated Ex EGF-R was immunoprecipitated with anti-EGF-R antibodies, followed by streptavidin-agarose. B, total EGF-R was immunoprecipitated using anti-EGF-R antibodies. C, biotinylated cell surface proteins were precipitated with streptavidin-agarose. Molecular weight markers are depicted on the right. The asterisk, circle, and triangle point at cell surface biotinylated proteins that were reduced by both HA and GA, reduced in size in the presence of GA, and remained unchanged by GA or HA treatment, respectively. domain, rather to the presence of the EGF-R cytosolic domain, the metabolic profiles of sEPO-R in the absence or the presence of GA were examined (Fig. 4C). The degradation kinetics of sEPO-R was essentially similar in the presence and absence of GA, supporting the notion that the EGF-R cytosolic domain is sufficient to confer GA-mediated degradation to the EPO-R/ EGF-R chimera.
To address whether kinase activity of the EGF-R is necessary for GA-mediated degradation of the receptor, we followed the metabolism of the EGF-R K721A mutant, which is devoid of Tyr kinase activity (Fig. 5). CHO cells stably expressing K721A EGF-R or wt EGF-R were pulse-labeled with [ 35 S]cysteinemethionine and chased in growth medium for 1 and 2 h in the absence or presence of GA. The kinetics of K721A EGF-R and wt EGF-R degradation in GA-treated cells was virtually identical, indicating that Tyr kinase activity of the EGF-R is not required for GA-mediated degradation. DISCUSSION Both HA and GA are inhibitors of HSP 90 and GRP 94, the cytosolic and ER-lumen chaperones, respectively. Here we demonstrate that the minute molecular differences between these compounds (35,36) are sufficient to elicit distinct biological effects on metabolism of the EGF-R. GA has two different effects on metabolism of the EGF-R. It enhanced degradation, and it inhibited maturation of the receptor. HA, however, only accelerated EGF-R degradation. Enhanced EGF-R degradation mediated by GA and HA is conferred by the cytosolic domain of the receptor, whereas GA-mediated intracellular retention requires its extracellular domain.
The EGF-R cytosolic domain is sufficient to confer degradation in the presence of GA, and this is supported by the rapid degradation of EPO-R/EGF-R chimera induced by this compound (Fig. 4). The specificity of GA was supported by the lack of its effect on degradation of full-length EPO-R and sEPO-R metabolism. Utilizing this EPO-R/EGF-R chimera provided further support for the notion that GA independently affects receptor maturation and degradation. Although present at low levels, cell surface EPO-R and EPO-R/EGF-R chimera are detected upon binding to radioactively labeled EPO (28). In the presence of GA, the expression levels of both cell surface EPO-R and EPO-R/EGF-R chimera were reduced by 50% (data not shown). This decrease in cell surface receptors may be due to accelerated degradation or inhibition of transport. The fact that the degradation of the chimeric receptor molecule was sensitive to GA, but that of the wild type EPO-R was not, indicates that degradation and retention are distinct processes mediated by GA. We speculate that retarded maturation of EGF-R in the presence of GA is mediated predominantly by inhibition of GRP 94 localized in the lumen of the ER and that enhanced degradation in the presence of HA and GA are mediated via inhibition of the cytosolic chaperone HSP 90. This possibility is currently under investigation.
It was previously reported that the cytosolic domains of EGF-R (13) and p185 c-erbB-2 (10) are required for degradation stimulated by GA and that the kinase domain of p185 c-erbB-2 is involved in this process (10). The data hereby presented demonstrate that a kinase-defective EGF-R is degraded similarly to wt EGF-R in GA-treated cells, ruling out the contribution of kinase activity per se to this process. This finding alludes to the existence of other, yet unidentified, sequence or structural motifs in the cytosolic domain of the EGF-R directing its degradation in the presence of GA.
Figs. 1 and 2 demonstrate the differential effects of GA and HA on turnover and processing of Ex EGF-R, an EGF-R mutant that lacks the cytosolic domain. Cell surface biotinylation experiments indicate that GA treatment significantly reduced expression levels of newly synthesized Ex EGF-R on the cell membrane, whereas the total levels of Ex EGF-R remained unchanged. Slightly lower levels of newly synthesized Ex EGF-R at the cell surface were observed in the presence of 1 g/ml HA, compared with control non-treated cells. It should be noted that no further reduction was observed at higher concentrations (up to 10 g/ml) of HA (data not shown). It was previously reported that a soluble EGF-R molecule was secreted in the presence of GA, although to a somewhat lower extent (13). It is conceivable that GA differentially affects membrane versus soluble secreted proteins. This might explain the differences in the level of inhibition of transport by GA observed for the soluble and membrane-bound forms of EGF-R. Furthermore, analysis of the general profile of newly synthesized biotinylated cell surface proteins indicates that GA affects only a subgroup of these proteins, whereas the others remain unaffected. Noteworthy is the fact that in the presence of BFA, newly synthesized cell surface proteins were barely detected. This confirms that indeed no cell surface labeling of EGF-R is detected when ER to Golgi transport is blocked. Hence, it seems that GA-mediated block of protein transport applies to proteins in addition to the EGF-R, yet it does not apply to all cell surface proteins.
The different activities of HA and GA were also demonstrated in experiments utilizing the combination of these inhibitors with the proteasomal inhibitor Z-L 3 VS. In the presence of Z-L 3 VS, HA treatment yielded fully processed EGF-R, whereas GA treatment under these conditions resulted in EGF-R forms impaired in their glycosylation. These data lend support to the conclusion that intracellular retention of the EGF-R mediated by GA is a separate effect from its effect on enhanced degradation of the receptor. A similar effect of GA in the presence of proteasomal inhibitors was also assessed for the CFTR (4), although the effects of HA in the presence of proteasomal inhibitors on CFTR degradation was not measured. Based on our results, a possibility exists that unlike GA, HA may not prevent maturation of the CFTR. The mechanism by which GA causes intracellular retention of the EGF-R and possibly other membrane proteins remains to be elucidated.