Farnesyltransferase inhibitors disrupt EGF receptor traffic through modulation of the RhoB GTPase

Matthew Wherlock1, Alexandra Gampel1, Clare Futter2 and Harry Mellor1,*

1 Mammalian Cell Biology Laboratory, Department of Biochemistry, School of Medical Sciences, University of Bristol, University Walk, Bristol, BS8 1TD, UK
2 Institute of Ophthalmology, University College London, 11-43 Bath Street, London, EC1V 9EL, UK

* Author for correspondence (e-mail: h.mellor{at}bristol.ac.uk)

Accepted 1 March 2004


    Summary
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The Rho family of small GTPases play a pivotal role in the dynamic regulation of the actin cytoskeleton. Recent studies have suggested that these signalling proteins also have wide-ranging functions in membrane trafficking pathways. The Rho family member RhoB was shown to localise to vesicles of the endocytic compartment, suggesting a potential function in regulation of endocytic traffic. In keeping with this, we have previously shown that expression of active RhoB causes a delay in the intracellular trafficking of the epidermal growth factor (EGF) receptor; however, the site of action of RhoB within the endocytic pathway is still unknown. RhoB exists as two prenylated forms in cells: geranylgeranylated RhoB (RhoB-GG) and farnesylated RhoB (RhoB-F). Here we use farnesyltransferase inhibitors (FTIs) to show that prenylation specifies the cellular localisation of RhoB. RhoB-GG localises to multivesicular late endosomes and farnesylated RhoB (RhoB-F) localises to the plasma membrane. The gain of endosomal RhoB-GG elicited by FTI treatment reduces sorting of EGF receptor to the lysosome and increases recycling to the plasma membrane. Ultrastructural analysis shows that activation of RhoB through drug treatment or mutation has no effect the sorting of receptor into late endosomes, but instead inhibits the subsequent transfer of late endosomal receptor to the lysosome.

Key words: Rho GTPase, Endocytosis, FTI, Multivesicular body, Trafficking, EGF


    Introduction
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Farnesyltransferase inhibitors (FTIs) are a novel class of cancer therapeutics designed to target the Ras small GTPase. Ras regulates cell proliferation, differentiation and survival and its activity is frequently deregulated in tumour cells; either indirectly or through oncogenic mutations that lock the protein in a constitutively active state (Shields et al., 2000Go). Ras undergoes post-translational modification by farnesyltransferase I, which covalently attaches a farnesyl group to the C-terminus of the protein (Casey et al., 1989Go). This 15-carbon isoprenoid tail acts as a membrane anchor for Ras, directing it to the plasma membrane. The demonstration that this farnesylation is crucial for Ras oncogenicity (Kato et al., 1992Go) has led to the design of a wide range of specific inhibitors of farnesyltransferase I, with the aim of reversing the contributions of active Ras to tumour growth (Cox and Der, 2002Go; Ohkanda et al., 2002Go; Tamanoi et al., 2001Go). FTIs have been shown to potently inhibit farnesylation of the H-Ras isoform in vitro and in mouse tumour models (Sebti and Hamilton, 2000Go). True to the original hypothesis, this is accompanied by phenotypic reversion of H-Ras-transformed cell lines, inhibition of tumour formation in mouse xenograft models and regression of tumours in mice harbouring an oncogenic H-Ras mutation (Haluska et al., 2002Go; Sebti and Hamilton, 2000Go).

Despite the seemingly straightforward effectiveness of these drugs against H-Ras transformed cells, the identities of the relevant farnesylated proteins are not fully resolved. FTIs clearly have the potential to reverse oncogenic H-Ras signalling; however, these compounds also act on tumour cell lines with wild-type Ras status (End et al., 2001Go; Sepp-Lorenzino et al., 1995Go). These and other data suggest that the clinical actions of FTIs extend outside of the Ras family (Tamanoi et al., 2001Go). Several other farnesylated proteins have been suggested as potential relevant FTI targets (Cox and Der, 2002Go; Prendergast, 2001Go; Sebti and Hamilton, 2000Go), the best characterised being RhoB, a small GTPase of the Rho family. RhoB is unique in being the only protein known to exist as both farnesylated and geranylgeranylated forms within the cell (Adamson et al., 1992aGo). This distinguishes it from the highly homologous RhoA and RhoC isoforms, which are solely geranylgeranylated (Adamson et al., 1992aGo). Treatment of cells with FTIs causes a loss of farnesylated RhoB (RhoB-F) and a consequent increase in geranylgeranylated RhoB (RhoB-GG) as newly synthesized protein is efficiently prenylated by geranylgeranyltransferase I (Lebowitz et al., 1997aGo). Prendergast and co-workers have proposed the `FTI-Rho hypothesis' of FTI action, which states that at least some actions of these drugs are mediated by increasing the cellular pool of RhoB-GG (Prendergast, 2001Go). Work from this group suggests that the cellular functions of the two prenylated forms of RhoB are distinct; RhoB-F has a pro-growth activity, whereas RhoB-GG has a pro-apoptotic role that is triggered on FTI treatment (Du et al., 1999Go; Du and Prendergast, 1999Go; Liu et al., 2000Go). While aspects of this hypothesis have been challenged (Chen et al., 2000Go; Sebti and Hamilton, 2000Go), studies from the RhoB knockout mouse provide compelling evidence for a role for RhoB in at least some of the spectrum of FTI actions (Liu et al., 2000Go; Liu et al., 2001Go).

As FTIs approach wider clinical use, it is clearly important to understand the molecular basis of their actions, particularly with respect to non-Ras targets. Work from Prendergast and colleagues supports the gain of RhoB-GG elicited by FTI as an important mediator of drug action; however, it has been unclear why RhoB-F and RhoB-GG should function differently in cells. Our work on RhoB has focussed on its role in the regulation of endocytic traffic. RhoB localises to endocytic vesicles (Adamson et al., 1992bGo) and is activated as internalised EGF receptor passes through this compartment through the actions of the Vav2 exchange factor (Gampel and Mellor, 2002Go). In our previous studies we have shown that activated RhoB appears to slow the intracellular trafficking of internalised EGF receptor to the lysosome (Gampel et al., 1999Go); however it has been unclear what specific step in receptor sorting is affected. Here we examine the localisation of endogenous RhoB and show that FTI treatment defines two cellular pools: the gain of RhoB-GG elicited by FTI corresponds to a gain in the endocytic pool of this signalling protein, with a corresponding loss of plasma membrane RhoB. Further, we show that this FTI-induced redistribution of RhoB leads to retention of internalised receptor within multivesicular late endosomes. These studies define the site of action of RhoB within the endocytic pathway and suggest a basis for the differential cellular functions of the two prenylated forms of this signalling protein.


    Materials and Methods
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Materials
Human recombinant epidermal growth factor (EGF) and L-744,832 were purchased from Calbiochem. Na-[125I] (3.7 GBq/ml) was from Amersham. Iodobeads and D-Salt Dextran desalting columns were from Pierce. Monoclonal antibodies to RhoB (C-5), the myc-epitope (9E10) and epidermal growth factor receptor (EGFR1), and a goat polyclonal antibody to the EGF receptor were from Santa Cruz Biotechnology. The EEA1 monoclonal antibody (clone 14) was from Becton-Dickinson. The CD63 monoclonal antibody (RFAC4) was from Biogenesis. Phospho-specific antibodies to JNK, p38, Erk1/2 and PKB were from Cell Signalling Technology. A LAMP-1/lgp120 polyclonal antibody was raised in rabbits to a peptide with the sequence KRSHAGYQTI. Cy2- and Cy3-conjugated donkey anti-IgG antibodies were from Jackson Laboratories. ToPro3 and biotinylated EGF complexed with Alexa 488-streptavidin were from Molecular Probes. Monoclonal antibody to the extracellular domain of the EGF receptor (clone 108) was a generous gift from J. Schlessinger (Yale University School of Medicine, Newhaven) and was coupled to colloidal gold (British BioCell) as described by Slot and Geuze (Slot and Geuze, 1985Go). Mammalian expression vectors encoding myc-epitope tagged RhoB (N-terminus), the constitutively-active RhoB-G14V mutant and the HR1 domain of PKN/PRK1 were previously described (Adamson et al., 1992bGo; Mellor et al., 1998Go).

Cell culture and transient transfection
Heb7a HeLa cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% foetal bovine serum (FBS), 100 units/ml penicillin, 100 units/ml streptomycin and 2 mM glutamine. For transfection, HeLa cells were plated to give a cell confluency of 30% and were transiently transfected the following morning, using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Where required, cells were then serum starved overnight. Treatment with 10 µM L-744,832 or vehicle (DMSO) was for the same overnight period (16 hours).

Immunofluorescence microscopy
HeLa cells were plated onto acid-washed glass coverslips and allowed to adhere overnight. Following any treatments described in the figure legends, cells were fixed for 15 minutes in 4% fresh paraformaldehyde in PBS, washed in PBS and then permeabilised in 0.2% Triton X-100 in PBS for 5 minutes. The cells were then washed again in PBS and incubated with 0.1% sodium borohydride for 10 minutes. For the LAMP-1 antibody, cells were permeabilised with 0.5% saponin in PBS; the solutions containing sodium borohydride or antibodies contained 0.1% saponin. The cells were washed three times in PBS then incubated with primary antibody in 1% BSA for 1 hour. The cells were washed three times in PBS then incubated for 45 minutes with secondary antibody in PBS. The cells were washed three times in PBS and mounted over MOWIOL 4-88 (Calbiochem) containing 0.6% 1,4-diazabicyclo-(2.2.2) octane (Sigma) as an anti-photobleaching agent. Confocal microscopy was performed using a Leica TCS-NT confocal laser-scanning microscope with an attached Leica DMRBE upright epifluorescence microscope under a Plan Apox63/1.32 oil-immersion objective. Fluorophores were excited using the 488 nm (Alexa 488, Cy2), 568 nm (Cy3) and 647 nm (ToPro3) lines of a krypton-argon laser. A series of images were taken at 0.5 µm intervals through the Z-plane of the cell and, unless stated otherwise, were processed to form a projected image.

Cell lysis and western blotting
Cells grown in 6-well tissue culture plates were washed three times in PBS and then scraped into 800 µl SDS-PAGE sample buffer (250 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 20 mM DTT and 0.01% bromophenol blue). Samples were heated to 90°C for 5 minutes for complete lysis. Proteins were resolved by SDS-PAGE and transferred onto Polyvinylidene Fluoride (PVDF) membrane for immunoblotting.

Radio-iodination of EGF
Purified recombinant human EGF was covalently radiolabelled with Na[125I] using Iodobeads according to the manufacturer's instructions. The iodinated EGF was separated from unincorporated 125iodide by gel filtration using D-Salt Dextran desalting columns and stored in PBS supplemented with 0.1% fatty acid-free BSA. The [125I]EGF was iodinated to a stoichiometry of less than one and typically had a specific activity of approximately 330 Bq/ng EGF.

EGF Receptor trafficking assay
HeLa cells grown in 6-well tissue culture plates were serum-starved overnight in DMEM supplemented with 0.1% fatty-acid-free BSA. These incubations contained either 10 µM L-744,832 or vehicle (DMSO). Cells were incubated with 100 ng/ml [125I]-EGF in working buffer (DMEM with 0.1% fatty-acid-free BSA and 20 mM HEPES, pH 7.3) for 10 minutes at 37°C to allow EGF receptor internalization. Cells were transferred to ice, washed three times in ice-cold working buffer, and then incubated with acidic buffer (0.1 M glycine pH 3, 150 mM NaCl) twice for 3 minutes on ice to remove residual surface bound ligand. Cells were then washed three times in working buffer at 37°C and incubated with working buffer containing 100 ng/ml unlabeled EGF at 37°C. At each time point medium was collected from the cells and intact EGF precipitated with 20% (w/v) trichloroacetic acid for 30 minutes at 4°C. Fresh working buffer containing unlabeled EGF was added back to the cells. Precipitates were cleared by centrifugation at 23,000 g for 10 minutes at 4°C, and dissolved in 1 M NaOH. Solubilised precipitates and supernatants were analyzed on a {gamma}-counter. Supernatants were used to calculate the amount of degraded [125I]-EGF while precipitates were used to calculate recycled ligand. At the end of the time course, cells were solubilised in 1 M NaOH and analysed to determine the remaining amount of intact internalised radio-ligand.

Electron microscopy
HEp2 cells were transfected with RhoB constructs by electroporation as described previously (Stinchcombe et al., 1995Go). Cells grown on thermanox coverslips were incubated with gold probes under different conditions as described in the text and were then fixed, processed and treated with tannic acid as described previously (Stinchcombe et al., 1995Go). Where pre-embedding immuno-labelling was performed, cells were permeabilised with digitonin, fixed and immuno-labelled as described in Futter et al., (Futter et al., 1998Go). Coverslips were embedded on Epon stubs (Taab Laboratories) and then peeled from the stubs after heating. Cells were sectioned facing forwards, stained with lead citrate and viewed in a Jeol 1010 electron microscope. For quantification, the number of EGF receptor-conjugated gold particles in multivesicular late endosomes (defined as vacuole of greater than 200 nm and containing monodisperse gold) and lysosomes (defined as containing aggregated gold) was counted. Very few gold particles were found in any other cellular compartment.

Cell proliferation assay
HeLa cells were seeded into 6-well tissue culture plates at a density of 1.5x105 cells per well. Following adherence, cells were transferred to DMEM containing 10% FBS (fed) or 0.1% fatty acid free BSA (starved) with 10 µM L-744,832 and/or 25 ng/ml EGF as appropriate. Medium, L-744,832 and EGF were refreshed every 24 hours. Adherent cells were harvested by trypsinization at each time point and the number of viable cells (that excluded Trypan Blue) was determined using a Neubauer counting chamber.


    Results
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
FTI treatment defines two cellular pools of RhoB
Previous studies of cells overexpressing epitope-tagged RhoB have assigned the protein largely to endocytic vesicles, with a small amount of the protein at the plasma membrane (Adamson et al., 1992bGo; Robertson et al., 1995Go). Overexpression can lead to redistribution and/or mislocalization of proteins and we therefore used a highly specific RhoB antibody (Gampel and Mellor, 2002Go) to determine the cellular localization of endogenous RhoB (Fig. 1). In keeping with studies using expression of epitope-tagged RhoB, the endogenous protein showed a punctate staining pattern; however, there was also significant staining of the plasma membrane in most cells (Fig. 1B,c). A similar distribution was observed in other RhoB expressing cells (MDCK, MCF-7, HUVEC and Hep2, data not shown). Quantification of cell populations showed that the relative distribution of these compartments is approximately 60:40 (plasma membrane:vesicle), although this varied somewhat between individual cells (Fig. 1C). The endocytic pathway comprises a number of sub-compartments that mediate the sorting and processing of internalised cargo (Sorkin, 2000Go). We used markers of the various endocytic sub-compartments to obtain a more precise definition of the localization of intracellular RhoB. Cells were incubated in the presence of fluorescent-labelled transferrin to saturate the early and recycling endocytic compartments, and cells were then fixed and co-stained for endogenous RhoB. No colocalization between transferrin+ and RhoB+ vesicles was observed (Fig. 1A,a), similarly, RhoB+ vesicles did not colocalise with the early endosomal marker EEA1 (data not shown). The intracellular RhoB compartment showed extensive colocalization with LAMP-1/lgp120 (Fig. 1A,b), a marker of the late endosomal/lysosomal compartment (Fukuda, 1991Go). Taken together, these data demonstrate compartment specific localization of endogenous RhoB within the endocytic pathway, with restriction to the degradative late endosomal branch. This is consistent with previous detection of epitope-tagged RhoB on the bounding membranes of multivesicular late endosomes by immuno-electron microscopy (immuno-EM, (Robertson et al., 1995Go) and also with the observed arrival of EGF receptor in the RhoB+ compartment 30 minutes after receptor internalization (Gampel et al., 1999Go; Waterman and Yarden, 2001Go).



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Fig. 1. FTI treatment defines two cellular pools of RhoB. (A) (a) HeLa cells were allowed to accumulate fluorescent-labelled transferrin (red) for 1 hour and then fixed and stained for endogenous RhoB (green). (b) Cells were co-stained for endogenous RhoB and LAMP-1 (red). Endogenous RhoB did not colocalise with early or recycling endosomes as marked by transferrin (a) but showed clear colocalization with the late endosome/lysosome marker LAMP-1 (b). Scale bar, 10 µm. (B) Single confocal sections taken through the broadest part of the cells showed that endogenous RhoB (green) has a heterogenous distribution between endocytic and plasma membrane pools (c). FTI treatment caused a complete loss of the plasma membrane pool, with a corresponding gain in endocytic staining (d). Myc-epitope tagged RhoB (green) localised almost entirely to endosomal structures (e), a fraction of which became clustered near the nucleus with FTI treatment (f). Scale bars, 10 µm. (C) The relative distribution of RhoB between plasma membrane and endosomal pools was quantified from confocal sections of FTI-treated and untreated cells using Scion Image. The data represents the mean±s.e.m. of three independent experiments, where 10 c ells were quantified for each condition.

 

Previous studies showed that the amounts of farnesylated and geranylgeranylated RhoB within cells are approximately equal (Lebowitz et al., 1997bGo), i.e. broadly similar to the relative distribution between plasma membrane and endosomal pools. We were therefore interested in the potential relationship between the cellular localisation of RhoB and its prenyl status. Treatment of cells with the potent and specific peptidomimetic FTI L-744,832 (Kohl et al., 1995Go) caused RhoB distribution to become homogenous across the cell population with a loss of plasma membrane staining and a corresponding increase in localization to the endocytic compartment (Fig. 1B,c,d). This change in distribution of RhoB in response to FTI treatment occurred in the absence of any net change in total RhoB levels within the cell (data not shown). From these data we infer that the two cellular pools of RhoB correspond to the two different prenylated forms of the protein, with farnesylated RhoB localised to the plasma membrane and geranylgeranylated RhoB to the endocytic compartment. Consistent with this, RhoB mutants engineered to be either solely farnesylated or solely geranylgeranylated (Baron et al., 2000Go) target to the plasma membrane and endocytic compartments respectively (Gilles Favre, personal communication). Prendergast and co-workers have previously examined the effects of FTI treatment on RhoB localization in cells overexpressing epitope-tagged RhoB and seen relatively minor changes to the morphology of the intracellular compartment with some increase to the number of RhoB+ vesicles (Lebowitz et al., 1995Go). This would seem to differ from the behaviour of endogenous RhoB protein. We repeated these experiments and also saw only small changes in localization of epitope-tagged RhoB in response to FTI treatment, corresponding to minor clustering of a proportion of RhoB+ vesicles around the nucleus on FTI treatment (Fig. 1B,e,f). However, epitope-tagged RhoB showed little localisation to the plasma membrane in untreated cells (Fig. 1B,e) and so any net translocation on FTI treatment would be hard to detect. Untagged RhoB localised to the plasma membrane and to endocytic vesicles, although this latter compartment was significantly disrupted on overexpression of untagged RhoB (data not shown). It seems that the epitope-tag in some way perturbs the cellular distribution of RhoB, underscoring the importance of examining the behaviour of the endogenous protein where possible.

FTI treatment blocks EGF receptor traffic
The EGF receptor is internalised on activation through a process of clathrin-mediated endocytosis and enters early endosomes. The bulk of the receptor is then sorted to the late endocytic compartment, before eventual degradation in the lysosome (Waterman and Yarden, 2001Go). In our previous studies we have shown that the internalised EGF receptor activates endogenous RhoB on arriving at the RhoB+ endosomal compartment (Gampel and Mellor, 2002Go), and that overexpression of active RhoB retards the intracellular trafficking of the receptor (Gampel et al., 1999Go). Given that FTI treatment increases the pool of endosomal RhoB, we were interested in determining the effect of this drug on receptor traffic. In control cells, the majority of the EGF receptor is degraded within 1hour of EGF stimulation (Fig. 2a). Overnight treatment with FTI for 16 hours led to a marked inhibition of EGF receptor degradation, with a significant pool of receptor left intact 4 hours after internalization (Fig. 2b). This treatment time was previously shown to be sufficient to allow complete turnover of farnesylated RhoB with no significant effect on the farnesylation of Ras (Lebowitz et al., 1995Go). The FTI-induced trafficking defect was examined in greater detail using a quantitative biochemical assay of receptor sorting. Cells were pulse-labelled with [125I]EGF and the fate of internalised ligand was followed over time. In control cells approximately 10% of the internalised receptor was recycled to the cell surface within 30 minutes of stimulation, whereas the remaining 90% was degraded in the lysosomal compartment over a longer period (Fig. 2c). Treatment with FTI had no effect on the amount of EGF internalised (data not shown) but significantly decreased receptor degradation (Fig. 2d). This was accompanied by a corresponding increase in receptor recycling, but also by the appearance of an intracellular pool of ligand that was not resolved to either pathway within the time course of the experiment (Fig. 2d). This latter pool resolved to the degradative pathway on FTI washout over a time course (4 hours) that paralleled the recovery of the normal cellular distribution of RhoB (data not shown).



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Fig. 2. FTI blocks the degradation of the EGF receptor in EGF stimulated HeLa cells. Serum-starved HeLa cells incubated without (a), or with (b) FTI were stimulated with 100 ng/ml EGF for the indicated times. Total EGF receptor content at each time point was analyzed by western blot. The positions of the intact EGF receptor and an intermediate degradation product are indicated to the left of the figure, as is the position of a non-specific band (*). Serum starved cells incubated without (c) or with (d) FTI were pulse-labelled with [125I]EGF. Following the removal of surface bound ligand, cells were chased with unlabeled EGF and medium was collected at the indicated times. Medium was analyzed for intact and degraded EGF and after 4 hours the cells were lysed and assayed for retained radio-ligand. Error bars represent standard error of the mean (s.e.m.) based on four independent experiments, with three replicates per experiment. Differences between EGF receptor degradation in control and FTI treated cells were found to be significant by two-way ANOVA (P<0.001).

 

FTI treatment results in a specific block in late endosome/lysosome transfer
We examined the FTI-induced trafficking defect in greater detail by probing the precise location of the arrested receptor. Cells were stimulated with EGF and the intracellular distribution of internalised EGF receptor was followed over time by confocal immunofluorescence microscopy. Cells treated with FTI showed retention of EGF receptor+ vesicles at late time points compared with control cells (Fig. 3). These vesicles showed no colocalization with the early endosomal marker EEA1 (Fig. 3d), however the majority of these structures co-stained with CD63 (Fig. 3e), a protein localised at steady state to the internal vesicles of late endosomes (Escola et al., 1998Go). These structures also showed significant colocalisation with the late endosomal marker Rab7 (data not shown). A smaller proportion of EGF receptor+ vesicles co-stained with LAMP-1/lgp120 (Fig. 3f), which is localised to the bounding membrane of late endosomes and lysosomes (Fukuda, 1991Go). These data are consistent with an FTI-induced block in traffic through the late endosomal/lysosomal branch of the endocytic pathway.



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Fig. 3. EGF receptor is predominantly retained in CD63+ endosomes in FTI treated cells. HeLa cells were serum starved overnight in the presence or absence of FTI and then incubated with 100 ng/ml EGF for 2 hours. Cells were fixed and stained for endogenous EGF receptor (green), nuclei (blue) and endosomal markers (red). EEA1 was used as a marker for early endosomes, CD63 for late endosomes and LAMP-1 for late endosomes/lysosomes. FTI-treatment led to retention of EGF receptor+ vesicles (compare top and bottom panels). In FTI-treated cells EGF receptor did not colocalise with EEA1 (d), almost completely colocalised with CD63 (e) and partially colocalised with LAMP-1 (f). The merged image in panel (e) is split in panels (g,h) to allow clearer visualisation of the EGF receptor (g) and CD63 (h) signals in FTI treated cells; colocalising vesicles (green arrowheads), non-overlapping signals (red arrowheads). Scale bar, 10 µm.

 

The resolution of light microscopy places limitations on the precise localization of proteins to intracellular compartments. Similarly, few markers map precisely to a unique cellular compartment. We therefore sought an unambiguous definition of the FTI-induced traffic-defect through ultrastructural analysis. Cells were pre-incubated with an anti-EGF receptor antibody conjugated to colloidal gold to surface label the EGF receptor. This pre-labelling is without affect on ligand binding or subsequent endocytic traffic of the receptor (Futter et al., 2001Go). Cells were then stimulated with EGF and the progression of the internalised receptor through the various endocytic compartments was followed by EM. The receptor reached multi-vesicular late endosomes within 30 minutes (data not shown). These are morphologically distinctive structures by EM and consist of a bounding membrane enclosing a number of smaller internal vesicles (Fig. 4a). EGF receptor arriving in late endosomes is sorted into these internal vesicles by a process of involution of the bounding membrane (Katzmann et al., 2002Go). This sorting step has a direct relevance to receptor signalling as it marks the sequestration of the cytoplasmic tail of the receptor from the cytoplasm. Fusion between the bounding membrane of a multivesicular late endosome and a lysosome then allows transfer of the internal vesicles containing the EGF receptor to this degradative compartment. By 45 minutes the receptor had begun to enter lysosomes and by 90 minutes delivery to this compartment was essentially complete (Table 1). Again, lysosomes are morphologically distinctive structures by EM, that present as electron dense bodies, often containing internal membranous structures (Fig. 4a). Examination of receptor traffic in FTI-treated cells revealed the defective trafficking step. FTI treatment had no effect on the sorting of the receptor to multivesicular late endosomes; however, there was a marked inhibition of subsequent transfer to the lysosomal compartment (Fig. 4, Table 1). EGF receptor accumulated in the internal vesicles of late endosomes, with no apparent residence in the bounding membrane (Fig. 4), ruling out a defect in the involution step. This precisely defines the site of action of FTI as inhibition of the final step of endocytic traffic in the degradative pathway: fusion between multivesicular late endosomes and lysosomes. To our knowledge, this is the first compound to be shown to inhibit this specific trafficking step.



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Fig. 4. HEp2 cells were incubated overnight in serum-free medium in the absence (a and c) or the presence (b and d) of FTI. Cells were then stimulated with EGF in the presence of anti-EGF receptor-gold (10nm) for 45 minutes (a and b) or 90 minutes (c and d) in the continued absence or presence of inhibitor. Multivesicular late endosomes (MVBs) and lysosomes (Lys) are indicated. Anti-EGF receptor-gold in MVBs is monodisperse while that in lysosomes is aggregated owing to proteolysis of the anti-EGF receptor antibody (arrows). In the absence of FTI, gold can be found in both MVBs and lysosomes 45 minutes after EGF treatment, whereas in the presence of FTI the majority of gold is in MVBs. Ninety minutes after EGF stimulation, the majority of gold is in lysosomes in the absence of FTI, whereas gold is still frequently found in MVBs in the presence of inhibitor. There is no evidence that FTI affects sorting of EGF receptor onto internal vesicles of MVB or internal vesicle formation. Scale bar, 100 nm.

 

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Table 1. Quantification of EM analysis of EGF receptor traffic in FTI-treated cells and cells expressing constitutively-active RhoB

 

FTI action on EGF receptor traffic is mediated by RhoB
Our previous studies showed that overexpression of active RhoB retards the intracellular trafficking of the internalised EGF receptor to the lysosome (Gampel et al., 1999Go). In this respect, the effects of FTI treatment on receptor traffic are consistent with a gain of RhoB function through increased recruitment to the endocytic pool. We therefore revisited our previous work in greater detail, using immuno-EM to analyse the effects of expressing a constitutively active RhoB mutant on EGF receptor traffic. Expression of RhoB-G14V had no effect on receptor internalization (data not shown), but like FTI treatment, caused a pronounced defect in traffic to the lysosome that was associated with an accumulation of receptor in the internal vesicles of multivesicular late endosomes (Fig. 5, Table 1). RhoB itself localised to the bounding membrane of late endosomes (Fig. 5), as seen with previous EM analysis (Robertson et al., 1995Go). The quantitative effects of the RhoB-G14V mutant were greater than those of FTI-treatment (Table 1), consistent with the constitutive activation of this mutant protein.



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Fig. 5. HEp2 cells were electroporated with RhoB-G14V cDNA and incubated overnight in medium containing 10% serum before incubation with serum-free medium for 1 hour. Cells were then stimulated with EGF in the presence of anti-EGF receptor-gold (10 nm) for 45 minutes (a) or 90 minutes (b and c). RhoB-G14V-expressing cells were identified by immuno-labelling with anti-myc antibody (5 nm gold). The majority of anti-EGF-receptor gold in RhoB-G14V-expressing cells is found in MVBs, even after 90 minutes of EGF stimulation. RhoB-G14V is found on EGF-receptor-containing MVBs as well as on small vesicles and/or tubules in the vicinity of MVBs. Scale bar, 100 nm.

 

Although the effects of FTI on EGF receptor traffic were entirely consistent with the effects of RhoB activation, clearly it was possible that other farnesylated proteins might be involved. We therefore examined whether blocking the activity of endogenous RhoB would rescue EGF receptor traffic in FTI treated cells. Substitution of a conserved threonine residue in small GTPases (equivalent to Thr17 in Ras) prevents GTP binding and additionally can confer dominant negative properties in many cases, presumably through sequestration of upstream exchange factors (Feig, 1999Go). The equivalent RhoB mutant (RhoB-T19N) appears inactive, rather than having any obvious inhibitory properties, and does not affect EGF receptor traffic (Gampel et al., 1999Go). We therefore attempted to sequester endogenous active RhoB through overexpression of a recombinant Rho-binding domain. A similar approach was proven effective with Cdc42, which can be specifically inhibited by expression of the CRIB (Cdc42/Rac interactive binding) domain of its effector protein WASP (Nobes and Hall, 1999Go). The HR1 domain of the PRK1/PKN kinase binds to active RhoA, RhoB and RhoC (Amano et al., 1996Go; Flynn et al., 1998Go) and is targeted to endosomes when co-expressed with active RhoB (Mellor et al., 1998Go). RhoA, RhoB and RhoC are highly homologous (approximately 87% identical); however, because RhoA and RhoC are solely geranylgeranylated and therefore unaffected by FTI (Adamson et al., 1992aGo), we reasoned that any effects on FTI action seen by expressing the HR1 domain should be specific to RhoB. Cells transiently transfected with the PRK1 HR1 domain were stimulated with EGF, then fixed at various time points and processed for confocal immunofluorescence microscopy. The total fluorescence signal of the endogenous EGF receptor was quantified on a cell-by-cell basis at each time point and HR1-transfected and control cells were compared. HR1 expression had no effect on EGF receptor internalization (data not shown); however, HR1 expression fully reversed the effects of FTI treatment on receptor degradation (Fig. 6). If any part of these effects were through inhibition of RhoA or RhoC, then a reduction in receptor signal should also be observed in untreated cells. This was not the case (Fig. 6), and we conclude that the effects of FTI treatment on EGF receptor traffic are mediated solely through RhoB.



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Fig. 6. Expression of the RhoB-binding HR1 domain from PRK1 overcomes the FTI-induced block on EGF-receptor degradation. Cells were transiently transfected with a HA-tagged HR1 domain construct from PRK1/PKN. Cells were starved overnight in the absence or presence of FTI and then stimulated with 100 ng/ml EGF for 2 hours. Cells were fixed and stained for endogenous EGF receptor and for expression of the HR1 construct. Cells were imaged by confocal microscopy, and the mean fluorescence signal for the EGF receptor within a fixed area was determined from 30 cells for each condition. Error bars represent standard error of the mean (s.e.m.) (n=3; 30 cells each experiment). The negation of the FTI induced block of EGF receptor by expression of the HR1 domain was found to be significant by the Student's t-test (P<0.001).

 

Effects of perturbation of EGF receptor traffic by FTI treatment on signalling outcomes
There is growing evidence for interplay between receptor trafficking pathways and receptor signalling output (Sorkin and Von Zastrow, 2002Go). We were therefore interested in examining whether the perturbation of EGF receptor traffic caused by FTI treatment was associated with any change in receptor signalling. The EGF receptor signals to a number of branching pathways (Carpenter, 2000Go). We used antibodies specific to the active (phosphorylated) form of various signalling proteins to assay the effects of FTI on the Erk1/2, JNK, p38 and phosphatidylinositol 3-kinase pathways. As FTI treatment delays EGF receptor degradation and significantly increases the pool of recycling receptor, it seemed possible that the drug would increase the magnitude and/or duration of signalling response. This was not the case, and indeed no apparent differences in receptor signalling were observed on FTI treatment (Fig. 7). Further, no differences in signalling were observed when cells were stimulated with a short 10 minutes pulse of EGF and then re-stimulated 1hour later (data not shown). This would seemingly discount any short-term consequence of the increase in receptor recycling observed in FTI-treated cells.



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Fig. 7. FTI treatment has no obvious short-term effect on signalling through the EGF receptor. Cells were serum starved overnight in the absence or presence of FTI and then stimulated with 100 ng/ml EGF for the indicated times. Activation of Erk1/2, PKB, JNK and p38 was analyzed by western blot using phospho-specific antibodies. The blots are representative of at least three separate experiments.

 

Single cycles of receptor activation are commonly used for experimental analysis of cell signalling; however, these generally give information about acute outputs, rather than longer-term responses. Indeed, stimulation of cell proliferation by EGF can require occupation of the EGF receptor for as long as 8 hours (Waterman and Yarden, 2001Go). We examined cell proliferation on the basis that this is a process with the potential to integrate over time small changes in cell signalling. FTI treatment reduced cell proliferation in serum-supplemented and serum-deprived cells (Fig. 8a). This was accompanied with an accumulation of cells in the G2 phase of the cell cycle (data not shown), as observed in a number of other FTI-sensitive cell lines (Tamanoi et al., 2001Go). We next examined the effects of FTI treatment on cell proliferation when accompanied by long-term EGF receptor activation. Addition of EGF alone to serum-deprived cells had a mild stimulatory effect on proliferation (Fig. 8b); however, when combined with FTI, EGF markedly potentiated the anti-proliferative effects of the drug, leading to a complete loss of cell proliferation (Fig. 8b). This, somewhat surprising, result suggests that continued activation of the EGF receptor in the presence of FTI generates a cytostatic and/or cytotoxic signal.



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Fig. 8. FTI treatment inhibits cell proliferation. Cells were maintained in DMEM containing 10% FBS (fed) or 0.1% fatty acid free BSA (starved) with 10 µM L-744,832 and/or 25 ng/ml EGF as indicated. The medium, L-744,832 and EGF were refreshed every 24 hours. Trypan blue-excluding cells were counted every 24 hours. Bars represent standard error of the mean (s.e.m.) based on five independent experiments. Differences in cell proliferation induced by FTI treatment in each cell culture condition were found to be significant by two-way ANOVA (P<0.001). FTI treatment had a significant effect on cell growth in serum-starved cells incubated with FTI, with or without EGF (P<0.005).

 


    Discussion
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The Rho family of small GTPases play a pivotal role in dynamic regulation of the actin cytoskeleton (Bishop and Hall, 2000Go; Ridley, 2001Go). Recent studies have suggested that these signalling proteins also have wide-ranging functions in membrane trafficking pathways (Qualmann and Mellor, 2003Go; Symons and Rusk, 2003Go). Our previous studies showed that expression of active RhoB causes an unspecified defect in the trafficking of internalised EGF receptor to the lysosomal compartment (Gampel et al., 1999Go). Here, we have identified the trafficking step controlled by RhoB as being transfer of the contents of multivesicular late endosomes to the lysosome. Further, we have shown that the two prenylated forms of RhoB are differently localised in cells, with geranylgeranylated RhoB comprising a late endosomal pool and farnesylated RhoB a plasma membrane localised one. Regulation of receptor traffic by RhoB then depends not only on the level of activated RhoB in the cell, but also on the relative abundance of the two prenylated forms.

Sorting mechanisms in the late endocytic compartment have recently received considerable attention. One important site of regulation of late endocytic sorting is the internalization process that transfers EGF receptors to internal vesicles. This process requires ubiquitylation of the cytoplasmic domain of the receptor by the ubiquitin ligase Cbl, which is recruited to the activated receptor through an SH2 domain interaction (Katzmann et al., 2002Go). The ubiquitinated EGF receptor interacts with multimeric protein assemblies on the surface of the late endosome termed ESCRT complexes (for endosomal sorting complex required for transport) that mediate sorting of the receptor into internal vesicles (Katzmann et al., 2002Go). Disruption of Cbl function leads to decreased receptor degradation and promotes recycling to the cell surface (Levkowitz et al., 1998Go). In this study, we found that the gain of endosomal RhoB elicited by FTI treatment also leads to an increase in EGF-recycling from late endosomes and an inhibition of lysosomal degradation. While these net effects are qualitively similar to the effects of inhibiting the involution process, ultrastructural analysis of cells treated with FTI or of those expressing active RhoB, revealed no apparent defect in the formation of internal vesicles, or the sorting of EGF receptors into these structures. Instead, these studies showed that the receptor accumulated in internal vesicles within the late endosome, thus defining the RhoB-induced trafficking switch as an inhibition of the subsequent lysosomal transfer.

The mechanisms controlling fusion between late endosomes and lysosomes are only partially understood, but analogies have been drawn to the process of homotypic vacuole fusion in budding yeast (Luzio et al., 2001Go). In yeast, this is an actin-dependent process that requires two members of the Rho GTPase family: Cdc42p and Rho1p (Eitzen et al., 2001Go; Muller et al., 2001Go). Cdc42p regulates Arp2/3-dependent actin remodelling on the surface of docked vacuoles prior to fusion (Eitzen et al., 2002Go). Rho1p is also required for docking of tethered vacuoles (Eitzen et al., 2001Go); however, its precise function is unknown. Because Rho1p is the yeast homologue of the mammalian RhoA, RhoB and RhoC isoforms it is tempting to speculate that RhoB may be performing a function in endocytic sorting that is analogous to that of Rho1p in yeast. Certainly, several studies point to a role of actin filaments in late endosome to lysosome transfer (Barois et al., 1998Go; Durrbach et al., 1996Go; Raposo et al., 1999Go; van Deurs et al., 1995Go).

Regulation of EGF receptor traffic at the level of lysosomal transfer has both unique properties and properties shared with regulation of the late endosomal involution step. Blocking either step clearly has a similar trafficking outcome – both lead to an inhibition of degradation and an increased recycling to the surface. However, there are important qualitative differences in terms of receptor signalling in the two processes: our data show that the increased recycling promoted by RhoB activation occurs in the absence of any detectable change in receptor signalling, whereas inhibition of the involution step generally potentiates mitogenic signalling. One example of this effect occurs with transforming growth factor-alpha (TGF-{alpha}). The EGF receptor can be activated by TGF-{alpha}; however, unlike EGF, this ligand dissociates from the receptor in the endosomal compartment (French et al., 1995Go). In the absence of continued ligand engagement, the receptor fails to recruit Cbl to the late endosome and does not enter internal vesicles (Longva et al., 2002Go). This then leads to the recycling of the receptor from the bounding membrane of the late endosome (Longva et al., 2002Go; Waterman and Yarden, 2001Go). TGF{alpha} causes a much more potent signalling response than EGF, which has been ascribed to its ability to promote receptor recycling over degradation (Waterman et al., 1998Go). Similar changes in receptor signalling occur when the sorting of EGF receptor into internal vesicles is blocked by mutations of the sorting machinery. Such mutations have been shown to increase mitogenic signals from receptor activation and to lead to tumour formation in animals (Katzmann et al., 2002Go; Waterman and Yarden, 2001Go; Wiley, 2003Go). Indeed, Cbl was first isolated as an oncogenic, dominant-negative truncation (v-Cbl) from a naturally occurring murine retrovirus (Blake et al., 1991Go). Similarly, one component required for recognition of the ubiquitylated EGF receptor is the human tumour susceptibility protein Tsg101 (Bishop et al., 2002Go); Tsg101-deleted fibroblasts give rise to metastatic tumours when introduced to nude mice (Li and Cohen, 1996Go). The most obvious difference between the effects of RhoB on EGF receptor traffic and those of TGF{alpha} or transforming mutations in Cbl and Tsg101 is that the latter leave the receptor with its kinase domain exposed to the cytoplasm. The EGF receptor is still capable of signalling from the late endosomal membrane, and indeed, kinase activity is required for sorting into internal vesicles (Felder et al., 1990Go). This would suggest that signalling from the surface of multivesicular late endosomes makes a significant contribution to the effects of oncogenic Cbl mutants.

A discriminating attribute of RhoB then, is that it allows recycling of late endosomal cargo without prolonged receptor signalling. As the EGF receptor recycles from the bounding membrane of multivesicular late endosomes (Longva et al., 2002Go), it is not immediately clear how RhoB-triggered accumulation of receptor in internal vesicles can lead to increased recycling. However, the internal vesicles of late endosomes are capable of re-fusion with the bounding membrane in some circumstances to allow retrieval of content to the plasma membrane (Murk et al., 2002Go; Piper and Luzio, 2001Go). This mechanism allows regulated surface presentation of processed antigen from a specialised late endocytic compartment (the MHC II compartment) that is present in B lymphocytes, macrophages and dendritic cells (Murk et al., 2002Go). The existence of this retrieval pathway, therefore presents a potential route for the RhoB-dependent increase in EGF receptor recycling seen in FTI-treated cells.

Regulation of receptor sorting through RhoB seems to have two potential governing factors: the activity of the endosomal RhoB pool and its concentration. The EGF receptor triggers RhoB activation on entering the late endosomal compartment through the Vav2 exchange factor (Gampel and Mellor, 2002Go). This is probably a fairly fixed signal, in that the receptor recruits a stoichiometric amount of Vav2 on activation. A more variable factor is the concentration of RhoB in the endosomal pool. The FTI studies show that, increasing the level of endosomal RhoB by even a small factor leads to a significant change in receptor sorting. RhoB is an acutely regulated protein, whose expression is rapidly induced in response genotoxic stress (Fritz et al., 1995Go) or to growth factors such as EGF (Jahner and Hunter, 1991Go). In this way, the effect of RhoB on late endocytic traffic could therefore be accessed either directly through receptors that couple to RhoB exchange factors, or indirectly through extracellular signals that increase RhoB expression.

Finally, given the lack of detectable effect of FTI treatment on EGF receptor signalling, we need to consider possible explanations for the synergistic growth-inhibitory effects of long-term EGF stimulation in the presence of FTI. One attractive possibility is that prolonged feeding of EGF receptor through a defective endocytic pathway leads to a chronic trafficking defect that triggers a cytostatic and/or apoptotic response. However, while we have been able to establish that the effects of FTI on EGF receptor traffic are mediated through RhoB, we do not know whether this is also true for the effects on growth. With this in mind, we are currently trying to establish epithelial cell lines from the transgenic RhoB null mouse (Liu et al., 2001Go). In either case, given the wild-type Ras status of HeLa cells, it is clear that the synergistic anti-proliferative effects of EGF on FTI-treatment are part of the spectrum of activities of this class of drug that fall outside of their original cellular target.


    Acknowledgments
 
We thank David Stephens and Pete Cullen for critical reading of the manuscript, and Gilles Favre for sharing unpublished data. This work was funded by a Wellcome Trust University Award to H.M. M.W. was supported by a BBSRC Committee Studentship.


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