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Endocrinology Vol. 148, No. 6 2944-2954
Copyright © 2007 by The Endocrine Society

Compartmentalization of Signaling-Competent Epidermal Growth Factor Receptors in Endosomes

Alejandro Balbis, Amanda Parmar, Ye Wang, Gerardo Baquiran and Barry I. Posner

Polypeptide Hormone Laboratory, Faculty of Medicine, McGill University, Montreal, Quebec, Canada H3A 2B2

Address all correspondence and requests for reprints to: Barry I. Posner, Polypeptide Hormone Laboratory, Faculty of Medicine, McGill University, 3640 University Street, Suite W315, Montreal, Quebec, Canada H3A 2B2. E-mail: barry.posner{at}mcgill.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In this study, the preparation of detergent-resistant membranes (DRMs) and the immunoisolation of intracellular vesicles enriched in raft markers were used to investigate the effect of physiological doses of epidermal growth factor (EGF) in vivo on the compartmentalization and activation of EGF receptor (EGFR) in rat liver endosomes. Both of these techniques show that after EGF administration, a distinctive population of intracellular EGFR, which was characterized by a high level of tyrosine phosphorylation, accumulated in endosomes. EGFR recruited to early endosomes were more tyrosine phosphorylated than those from late endosomes. However, the level of tyrosine phosphorylation of EGFR in DRMs isolated from early and late endosomes was comparable, suggesting that EGFR in endosomal DRMs are more resistant to tyrosine dephosphorylation. In accordance with the higher level of Tyr phosphorylation, EGF induced an augmented recruitment of Grb2 and Shc to endosomal DRMs compared with whole endosomes. Furthermore, a proteomic analysis identified a selective increase of many {alpha}-subunits of heterotrimeric G proteins in endosomal DRMs in response to EGF. These observations suggest that a distinctive pool of endocytic EGFR, potentially competent for signaling, is actively trafficking through intracellular compartments with the characteristic of lipid rafts.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
BINDING OF EPIDERMAL growth factor (EGF) to its cell surface receptor (EGFR) is followed by tyrosine phosphorylation, activation, and internalization of the EGFR into early endosomes from where they are recycled to the plasma membrane (PM) or sorted to late endosomes for degradation (1, 2, 3). Although it has been suggested that endocytosis attenuates EGFR signaling (4), other studies have demonstrated augmented tyrosine phosphorylation of and association of Shc and Grb2 with endosomal EGFRs (5, 6) indicating their participation in signaling. Furthermore, the selective activation of endosomal insulin receptor kinase (IRK) and EGFR has been shown to entrain their respective signaling pathways (7, 8). These findings support the view that endocytosis of activated receptors provides temporal and spatial regulation in the signaling cascade (9, 10).

Spatial control could be further modulated by activation of signaling complexes in subcompartments such as lipid domains within a given organelle (10). Membrane lipid microdomains (lipid rafts), rich in sphingolipid and cholesterol, were initially proposed to play a role in sorting Golgi proteins to the apical PM of polarized MDCK epithelial cells (11). In subsequent studies, the preparation of detergent-resistant membranes (DRMs) from cell lysates was used to characterize rafts biochemically (12, 13). Although it is unlikely that DRMs represent intact rafts in vivo, it seems clear that proteins copurifying with DRMs have a high affinity for these structures (14). A number of studies, based on the isolation of DRMs, have suggested that lipid rafts play a role in various cellular processes (12, 15). The use of techniques that do not involve extraction with detergent suggest that in resting cells rafts are very small (nanoscale structures) and short lived (16, 17, 18).

Caveolae are invaginated cell surface microdomains, enriched in sphingolipids, cholesterol, and caveolin oligomers, which have been considered to constitute a morphological identifiable subset of lipid rafts (19). Caveolae/rafts have been implicated in endocytosis and intracellular trafficking of viruses, toxins, and glycosyl-phosphatidylinositol-anchored proteins and may also function as signaling platforms (12, 15, 19, 20). Several studies have implicated caveolae/rafts in the modulation of EGFR signaling. In cells, cholesterol depletion increased EGFR tyrosine kinase activity and EGF-induced ERK activation and DNA synthesis (21, 22). In fibroblasts, EGFR appeared initially to be concentrated in caveolae/rafts and then exited this compartment after EGF stimulation (23). Thus, a model was proposed wherein EGFR signaling is negatively regulated by association with caveolae/rafts and positively regulated by internalization through a clathrin-dependent mechanism (24). Recent work has, however, showed that a fraction of EGFR in the PM can be internalized through a clathrin-independent, lipid raft-dependent pathway (25). Furthermore, upon EGF stimulation, EGFR is recruited to coated pits that also contain raft domains (26). These results suggest that a pool of EGFR at the PM might be sorted to specific intracellular compartments through a raft-mediated route. Thus, different endocytic pathways could contribute to intracellular EGFR trafficking. To investigate the possible compartmentalization of EGFR in rat liver endosomes, we analyzed the characteristics of the EGFR recruited to both DRMs and vesicles enriched in raft markers after EGF stimulation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Female Sprague Dawley rats, 10 wk of age and 160–180 g body weight (BW), were purchased from Charles River Canada Ltd. (St. Constant, Quebec, Canada), housed in an animal facility with 12-h light, 12-h dark cycles at 25 C and fed ad libitum on Purina chow. Animals were fasted overnight (16–18 h) before each study. All animal procedures were performed in accordance with the standards of the Canadian Animal Care Committee.

Materials
Porcine insulin was a gift from Eli Lilly and Co. (Indianapolis, IN). EGF and antibodies against caveolin (610059, used for immunoblotting and immunoisolation), flotillin-1 (610820, used for immunoblotting), early endosome antigen 1 (EEA1) (610456), GM130 (610822), and Rab11 (610656) were from BD Biosciences (Mississauga, Ontario, Canada). Antibodies against c-Src (05-184), Shc (06-203), Gi1&2{alpha} (06-236), and Na/K ATPase (05-369) were from Upstate (Lake Placid, NY). Antibodies against Rab5a (sc-309), Rab7 (sc-10767), Grb2 (sc-255), ubiquitin (sc-8017), EGFR (sc-03, used for immunoblotting), phosphotyrosine (PY) proteins (PY99, sc-7020), and flotillin-1 (sc-25506, used for immunoisolation) were from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against Gs{alpha} (371732) and lysosomal-associated membrane protein-1 (LAMP-1) (428017) were from Calbiochem (La Jolla, CA). An antibody against the transferrin receptor (13-6800) was from Zymed Laboratories (San Francisco, CA). Antimouse IgG developed in goat (M-8642), antirabbit IgG developed in goat (R-2004), an antibody against actin (A-4700), and most of the chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). Goat antirabbit and goat antimouse were labeled with Na125I as described before (27) and were used as secondary antibodies for immunoblotting. An antibody raised against a peptide corresponding to residues 942–969 of the juxtamembrane region of the IRK ß-subunit (anti-960) was prepared and purified as previously described (28) and used for immunoblotting. A monoclonal antibody directed against the extracellular domain of the EGFR (used for immunoprecipitation) was kindly provided by Dr. Bergeron (McGill University, Montreal, Canada). An antibody against Rab7 was kindly provided by Dr. Marino Zerial (Max Planck Institute, Dresden, Germany). Reagents for electrophoresis and for measuring the protein content of the liver fractions were from Bio-Rad (Richmond, CA). Polyvinylidene difluoride Immobilon-P transfer membranes were from Millipore Ltd. (Mississauga, Ontario, Canada). Magnetic beads (Dynabeads M-280 and Dynabeads Protein A) were from Dynal (Lake Success, NY).

Preparation of subcellular fractions
Rats were anesthetized and killed by decapitation after intrajugular injections at the indicated times as described in the appropriate figure legends. Livers were exsanguinated, rapidly excised, and minced at scissor point in ice-cold buffer (5 mM Tris-HCl buffer, pH 7.4, containing 0.25 M sucrose, 1 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, 1 mM MgCl2, 2 mM NaF, and 2 mM Na3VO4). Microsomes, PMs, and combined endosomes were prepared as previously described (29). In some experiments (see Fig. 7Go), combined endosomes were further fractionated into early and late endosomes by Percoll density gradient centrifugation as previously described (30).


Figure 7
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  		FIG. 7. EGFR Content in immunoisolated caveolin- and flotillin-1-containing structures of hepatic endosomes. After an overnight fast, rats received a single dose of EGF (1 µg/100 g BW) and were killed after 5 min. Combined endosomes were prepared and immediately incubated with magnetic beads (M. Beads) coated with an antibody against caveolin, flotillin-1, or IgG as described in Materials and Methods. A, Endosomes (END), immunoisolated caveolin-containing structure (CAV), and the negative control (IgG) were subjected to SDS-PAGE and analyzed by immunoblotting with antibodies against caveolin, EGFR, Rab5, and flotillin-1. The immunoisolations from three different preparations of endosomes (EXP. 1–3) are shown. B, Endosomes (END), immunoisolated flotillin-1-containing structure (Flot), and the negative control (IgG) were subjected to SDS-PAGE and analyzed by immunoblotting with antibodies against EGFR, PY proteins, flotillin-1, and the transferring receptor (TfR). A representative immunoblot from a duplicate experiment is shown.

 
Isolation of DRMs
DRMs were isolated by a modification of the method of Liu et al. (31). Briefly, combined endosomes from four rats (~8 mg protein) were pelleted by centrifugation at 200,000 x g for 1 h in 0.25 M sucrose and mixed with 2.2 ml ice-cold 1% Triton X-100 in buffer A [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, and 2 mM Na3VO4]. The samples were homogenized (10 strokes in a glass homogenizer), incubated on ice for 1 h, adjusted to the same amount of protein, and diluted 1:1 with 80% sucrose in buffer B [50 mM Tris-HCl (pH 7.5), 150 mM NaCl]. The extract (4 ml, ~7 mg protein) was loaded at the bottom of a 12-ml ultracentrifuge tube and overlaid with 4 ml each of 30 and 10% sucrose in buffer B. The gradient was centrifuged for 21 h at 29,000 x g in a SW40 Ti rotor (Beckman Instruments, Fullerton, CA), and 4 ml (corresponding to the middle of the 10–30% sucrose gradient) was collected and further centrifuged at 200,000 x g for 1 h. The resultant pellet, resuspended in 0.5 ml PBS, constitutes the DRM fraction (~300 µg protein). Soluble proteins in the residual 40% sucrose layer (endosomes post centrifugation) and the pellet (~75 µg protein), resuspended in 0.5 ml of ice cold PBS, were also collected. Samples (7 µg protein) were subsequently analyzed by SDS-PAGE and immunoblotting. In some experiments, DRMs were prepared from 1.2 mg early and late endosomes, which yielded between 30–40 µg protein in each DRM fraction. Each preparation (2.5 µg protein) was subsequently analyzed by SDS-PAGE and immunoblotting.

Immunoisolation of caveolin and flotillin-enriched membrane domains
Magnetic beads (Dynabeads M-280) were coated with an antibody against caveolin or IgG (negative control) as specified by the manufacturer. Coated beads were incubated with combined endosomes for 2 h at 4 C and subsequently washed four times with Tris-buffered saline (pH 7.5) before boiling for 5 min in Laemmli sample buffer and subjecting to SDS-PAGE. For the isolation of flotillin-1-containing vesicles, an antibody against this protein or IgG was bound to magnetic beads coated with protein A (Dynabeads Protein A) as specified by the manufacturer. Magnetic beads coated with these antibodies were incubated with combined endosomes as indicated above. It is pertinent to note that Dynabeads M-280 coated with the antiflotillin antibody were unable to bind flotillin-enriched vesicles. The attachment of the antibody to M-280 beads required a 48-h incubation at room temperature, a procedure that impaired the binding capacity of the antiflotillin antibody but not that of the anticaveolin antibody. For this reason, we used Dynabeads Protein A, which required only a 2-h incubation at 4 C for the suitable attachment of competent antiflotillin antibodies. However, with this procedure, a strong nonspecific band migrating at 20–30 kDa was observed in the immunoblots, rendering it impossible to detect caveolin.

Proteomic sample preparation
Each sample (75 µg protein) was separated on a 1-mm-thick 7–12% gradient one-dimensional SDS-PAGE and stained with Coomassie-G. The gel lane was horizontally cut into 86 slices. Each slice was further diced into 1-mm cubes that were transferred to a 96-well tray. Reduction and alkylation of disulfide bridges, trypsin digestion, and peptide extraction were done using a MassPrep Workstation (Micromass, Manchester, UK) as described before (33). A volume of 20 µl of the peptide solution was injected on a trapping guard column (Zorbax 300SB-C18, 5 x 0.3 mm, 5 µm, 300 Å) by the autosampler of an Agilent 1100 Series Nano Pump (Agilent Technologies, Palo Alto, CA). After 5 min of washing with 0.1% formic acid (aqueous) at 15 µl/min, the six-port valve was actuated so the acetonitrile gradient would elute the peptides at a flow rate of 200 nl/min to the PicoFrit column (75 µm inner diameter; New Objective, Woburn, MA) filled with BioBasic (10 µm, 5 µm, 300Å). Mass spectrometric data were acquired on a Q-Tof micro (Micromass, UK) by automatic function switching (34), employing the Data Directed Analysis feature available on MassLynx 4.0 (Micromass, UK). The MS-Survey scan was from 350-1600 mass-to-charge ratio (m/z) for 1 sec. One precursor was selected for tandem mass spectrometry (MS/MS) at a time based on a threshold of 25 counts. Each MS/MS scan of 100-1990 m/z was 1.35 sec long and would be allowed to continue until the total ion current would reach 2800 counts or a maximum of 4 sec. Doubly and triply charged ions were preferentially selected using the Data Directed Analysis option. MS/MS raw data were transferred to a 50-terabyte server and manipulated by Toolbox for Mass Spectrometry Data Analysis (TOMAS) 1 to generate peaklists (Mascot Distiller; Matrix Science, London, UK) and perform database searching (Mascot Cluster version 1.9.03; Matrix Science). TOMAS used the temperature information tagged in all scans to apply a correction factor of 160 ppm/°C on the peaklists generated by the distiller for the data shown in supplemental Table 2 (C4,5,6 and I1,2,3), published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org.

The peaklist parameters used were as follows: maximum interaction, 500; correlation threshold, 0.7; minimum peak, 50 m/z; maximum peak, 100,000 m/z; minimum S/N, 2; minimum peak width, 0.01; maximum peak width, 1; expected peak width, 0.1; maximum allowed precursor difference, 3 Da. Regridding was performed with a value of 20 data points/Da.

TOMAS (see Footnote 1) then submitted peaklisted data to Mascot Cluster and searched against a locally frozen copy of the NCBInr (National Center for Biotechnology Information nonredundant) database from March 18, 2004. Parameters for Mascot searching were as follows: monoisotopic, one missed trypsin cleavage allowed, fixed carbamidomethyl alkylation of cysteines, variable oxidation of methionine, and 0.5 mass unit tolerance on parent and fragment ions.

All peptide identifications of at least 95% confidence level were transferred (by TOMAS) to an in-house clustering software 2 that matches peptides to their corresponding proteins. Proteins were clustered and redundancy removed as previously described (35, 36). Peptide counts were performed as described before (37) and correspond to the spectral count method of Liu et al. (38). Peptide sequences that are shared between multiple identified proteins (shared peptides) have been taken into account according to Blondeau et al. (37), Liu et al. (38) (peptide counts = spectral counts), and Pang et al. (39) (peptide counts = relative amounts). Briefly, shared peptides are apportioned to their cognate proteins based on the total number of unique peptides identifying each cognate protein.

Protein identifications from each gel lane were then compared against each other using CD-HIT (40) at 90% or greater. This both collapsed protein identifications from a single lane that were at least 90% identical to each other and also matched protein identifications that were at least 90% identical to each other across all replicates and conditions. Peptide counts and apportioning of shared peptides were recalculated for this data set using the in-house software described above.

Statistics
Differences between detergent-resistant and detergent-soluble fractions were analyzed by Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
EGFR and IRK in DRMs
Recent evidence suggesting that receptor kinases could exploit different endocytic pathways (25, 41) prompted us to determine the possible compartmentalization of EGFR and IRK in rat liver endosomes. To accomplish this, we carried out a biochemical characterization of DRMs isolated from rat liver endosomes. Although isolated DRMs do not represent a preexisting membrane structure in intact cells, this technique allows us to identify proteins with a potential affinity for a distinctive membrane domain, like rafts (14). Therefore, hepatic combined endosomes, isolated from rats receiving either EGF (1.0 µg/100 g BW), insulin (1.5 µg/100 g BW), or buffer (basal), 5 min before killing, were homogenized in 1% Triton X-100 and fractionated by sucrose gradient centrifugation, as described in Materials and Methods. The DRMs (D), solubilized endosomal components in the load zone (pE), and the Triton-insoluble pellet (P), obtained after centrifugation (Fig. 1AGo), were analyzed in parallel with aliquots of the original combined endosomal fraction (E) and microsomes (M). As shown previously, the content of IRK and EGFR increased in combined endosomes after stimulation with their respective ligands (5) (Fig. 2AGo, left panel). Similarly, EGFR also increased in DRMs and in the pellet after 5 min of EGF stimulation (Fig. 2AGo, left panel). At this time of EGF stimulation, EGFRs are located at the PMs and endosomes. The combined endosome fraction used in this study has been extensively characterized in previous work and has been shown to be free of PMs (29, 42, 43). In further support for the distinctiveness of these fractions, Fig. 1BGo shows that actin and the Na/K ATPase (PM markers) are highly concentrated in PM, and the endosomal marker Rab5 is found concentrated in endosomes. Thus, we conclude that the EGFR recruited to DRMs in response to EGF derive from endosomes. In contrast, after insulin stimulation, the level of IRK in DRMs was low compared with that in whole endosomes (Fig. 2AGo, right panel). This is in accordance with previous studies showing little or no IRK in DRMs isolated from rat liver PMs (44) or adipocytes (45).


Figure 1
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  		FIG. 1. Preparation of subcellular components. A, After an overnight fast, rats received a single dose of EGF (1 µg/100 g BW) or insulin (1.5 µg/100 g BW) and were killed after 5 min. Livers were removed, and combined endosomes (E), isolated from microsomes (M), were homogenized in 1% Triton X-100 at 4 C for subsequent isolation of DRMs (D) as described in Materials and Methods. The residual proteins in the 40% sucrose layer (pE) and the pellet (P) were also collected. These fractions were subjected to SDS-PAGE (7 µg protein per lane) and analyzed by immunoblotting with specific antibodies against the proteins as specified in the subsequent figures. The bands were analyzed using a Bio-Rad densitometer (model GS-800), and the data are expressed as percentage of the maximal value in each individual experiment. B, Homogenate (H), microsomes (M), PM, and endosomes (EN) were purified from rat liver. Samples (40 µg protein) were subjected to SDS-PAGE followed by immunoblotting with antibodies specific for actin, Na/K ATPase, and rab5.

 

Figure 2
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  		FIG. 2. EGFR and IRK in DRMs. After an overnight fast, rats received a single dose of EGF (1 µg/100 g BW) or insulin (1.5 µg/100 g BW) and were killed after 5 min. Livers were removed, and subcellular fractions were prepared and subjected to SDS-PAGE as described in Materials and Methods (see Fig. 1Go for abbreviations). Fractions were analyzed by immunoblotting with antibodies against EGFR and IRK (A) and caveolin, flotillin-1, and c-Src (B). Each bar is the mean ± SEM of three independent experiments. The value for each protein is expressed as a percentage of the maximal value obtained for that protein in each of the three individual experiments performed. *, P < 0.05 vs. pE.

 
Characterization of DRMs isolated from rat liver endosomes
Biochemical characterization of the endosomal DRMs demonstrated that they were highly enriched in typical raft markers caveolin, flotillin-1, and c-Src compared with the other fractions generated (Fig. 2BGo). These proteins did not increase significantly after ligand stimulation with the exception that caveolin increased in DRMs by 100% after insulin administration (Fig. 2BGo). In previous work, we showed that insulin did not appear to induce internalization of caveolin from the liver PM (44). The increase of caveolin observed in endosomal DRMs in response to insulin might be a consequence of a redistribution of caveolin between intracellular membranes as described previously in skeletal muscle in response to insulin (46). The Triton-insoluble pellet (P) showed an almost complete absence of raft markers (Fig. 2BGo). This contrasts with our observations in hepatic PM where we demonstrated, with the same technique, that caveolin-1 is highly enriched in the Triton-insoluble, actin-enriched pellet (44).

The presence of raft markers in endosomal DRMs (Fig. 2BGo), together with previous findings that internalized caveolae/rafts fuse with the classical early endosomes (47, 48), prompted us to assess the presence of endosomal markers in our DRM preparations. As observed in Fig. 3AGo, Rab5, -7, and -11, but not EEA1, were enriched in endosomes relative to microsomes. Rab 5 was enriched in DRMs before and after ligand stimulation. EEA1 was also enriched in this fraction but only after EGF, not insulin, stimulation (Fig. 3AGo). These results suggest that at least a portion of the DRM fraction has originated from early endosomes. In contrast, there was no enrichment of either Rab7 or -11 in DRMs compared with endosomes. Indeed, DRMs were impoverished in these putative markers of late and recycling endosomes (Fig. 3AGo). This does not mean that DRMs do not originate from late or recycling endosomes. Triton X-100 treatment could exclude these endosomal markers from DRMs, but we cannot rule out the enrichment of other endosomal components in this fraction. In fact, as we will see later, DRMs do originate from late endosomes.


Figure 3
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  		FIG. 3. Endosomal and cytoskeletal markers in DRMs. After an overnight fast, rats received a single dose of EGF (1 µg/100 g BW) or insulin (1.5 µg /100 g BW) and were killed after 5 min. Livers were removed and subcellular fractions prepared and subjected to SDS-PAGE as described in Materials and Methods (see Fig. 1Go for abbreviations). A, Fractions were analyzed by immunoblotting with antibodies against Rab5, Rab7, Rab11, and EEA1. Each bar is the mean ± SEM of three independent experiments. The values for each protein are expressed as a percentage of the maximal value obtained in each individual experiment of the three experiments performed. *, P < 0.05 vs. pE. B, Subcellular fractions were prepared as indicated above and analyzed by immunoblotting with antibodies against actin and tubulin. A representative immunoblot is shown.

 
In view of the data indicating an association of liver PM DRMs with cytoskeletal proteins, in particular actin (44), we investigated whether cytoskeletal markers associated with endosome-derived DRMs. We found a low level of association of actin with DRMs and the pellet compared with that seen in microsomes (Fig. 3BGo). In contrast, higher levels of tubulin were observed in association with DRMs and with the Triton-insoluble pellet relative to that seen in microsomes (Fig. 3BGo). Our results, indicating the association of DRMs with the actin cytoskeleton at the PM (44) and with microtubules in endosomes, are consistent with the previous suggestion of dual control of caveolar membrane trafficking noted by Mundy et al. (49).

Ubiquitination of EGFR in DRMs
Previous studies have shown that EGF treatment is followed by rapid ubiquitination of the EGFR (50). Ubiquitination seems to play an important role in trafficking of the EGFR between early and late endosomal compartments (50). Recent work showed that ubiquitinated EGFR is internalized almost exclusively via a non-clathrin-, lipid-raft-dependent route (25). Thus, we evaluated the level of protein ubiquitination in general and EGFR ubiquitination in particular in DRMs after EGF stimulation. As seen in Fig. 4AGo, endosomes and DRMs showed comparable levels of overall ubiquitination. Proteins in microsomes and the Triton-insoluble pellet were only weakly ubiquitinated (Fig. 4AGo). These results suggest that DRMs constitute an important compartment involved in the intracellular trafficking of ubiquitinated proteins. We next immunoprecipitated EGFR from DRMs and the residual endosomal fraction, present in the load zone (pE, see Fig. 1Go) after sucrose gradient centrifugation. These immunoprecipitates were run on SDS-PAGE, transferred to polyvinylidene difluoride membranes, and immunoblotted with antibodies against the EGFR and ubiquitin. As observed in Fig. 4BGo, EGFRs are ubiquitinated in both of these fractions (see the broad band between 180–250 kDa). However, the ratio of ubiquitinated EGFR/EGFR was 3-fold higher in DRMs (Fig. 4CGo). Thus, DRMs contain a pool of EGFR with a higher proportion of ubiquitinated receptors and/or EGFR with a higher level of ubiquitin per receptor.


Figure 4
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  		FIG. 4. EGFRs localized in DRMs are highly ubiquitinated. Rats received a single dose of EGF (1 µg/100 g BW) and were killed after 5 min. Hepatic subcellular fractions were prepared and subjected to SDS-PAGE as described in Materials and Methods (see Fig. 1Go for abbreviations). A, Fractions were analyzed by immunoblotting with an antibody against ubiquitin; B, DRMs from 12 rats were pooled and solubilized with 1% Triton X-100 in the presence of protease inhibitors for 30 min at room temperature followed by centrifugation for 10 min at 15,000 x g. The supernant [solubilized DRMs (D)] and pE (see Fig. 1Go) were subjected to immunoprecipitation (IP; 200 µg protein) with an antibody (ab) against the extracellular domain of the EGFR attached to protein A-Sepharose beads. Immunoprecipitates were analyzed by immunoblotting (IB) with antibodies against the carboxy terminus of the EGFR and ubiquitin (Ub). Two negative controls were prepared: 1) uncoated protein A-Sepharose beads incubated with 200 µg pE and 2) protein A-Sepharose beads coated with the antibody against EGFR incubated with PBS buffer. These two controls were carried out to investigate the source of the band migrating at 170 kDa (and below) in the immunoblots with the anti-ubiquitin antibody. The immunoblot on the right shows that this is a nonspecific band that did not derive from the sample. C, Ratio of ubiquitinated EGFR (Ub-EGFR)/EGFR. Each bar is the mean ± half the range from two experiments.

 
Activation and translocation of signaling proteins to DRMs after EGF stimulation
Recent studies have suggested that kinase receptors, internalized through caveolae/rafts, might be directed to the degradative pathway rather than having a function in signaling (25, 41). Our results showing that EGFRs in DRMs are more ubiquitinated are consistent with this hypothesis. Thus, to gain more insight into the role of the pool of EGFR in endosomal DRMs, we examined the impact of EGF administration on the distribution of Tyr-phosphorylated EGFR and its associated signaling proteins in endosomes, DRMs, and the other fractions (see Fig. 1Go). In response to EGF, EGFRs of both DRMs and the pellet were more tyrosine phosphorylated than EGFR in whole endosomes (Fig. 5AGo). Thus, the ratio of PY-EGFR/EGFR was 2.5-fold increased in both DRMs and the pellet compared with endosomes (Fig. 5BGo). Furthermore, the concentrations of Grb2 and Shc in DRMs and the pellet were comparably augmented when compared with their concentrations in whole endosomes (Fig. 5CGo). This correlation between PY-EGFR and Grb2/Shc enrichment is consistent with their association in a multimeric complex as previously shown in liver endosomes (5).


Figure 5
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  		FIG. 5. DRMs are enriched in Tyr-phosphorylated EGFR and signaling proteins. Rats received a single dose of EGF (1 µg/100 g BW), and hepatic subcellular fractions were prepared and subjected to SDS-PAGE as described in Materials and Methods (see Fig. 1Go for abbreviations). A–C, Fractions were analyzed by immunoblotting with antibodies against PY and EGFR (A), and the ratio of PY-EGFR (PY)/EGFR was determined (B) as well as the distribution of Grb2 and Shc (C). D, The relative concentrations of GS{alpha}, and Gi1&2{alpha} were determined by immunoblotting of cell fractions from control and EGF-treated rats (d). In B and C, each bar is the mean ± SEM of three independent experiments. *, P < 0.01 vs. pE.

 
We next investigated the role of DRMs in EGF signaling by carrying out a proteomic analysis of this preparation after EGF treatment. DRMs from three independent preparations (control and 5 min post EGF) were run on one-dimensional SDS-PAGE. Each lane (three control and three EGF stimulated) was cut into 86 horizontal gel slices and processed as indicated in Materials and Methods. To identify EGF-associated signaling proteins, we considered only those signaling proteins whose peptide number increased significantly after stimulation with EGF (Table 1Go and supplemental Table 1, published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). There was a good correlation between the quantification of signaling proteins by peptide number and immunoblotting [compare Table 1Go (EGFR, Grb2, and Shc) with Fig. 5Go, A and C]. There was also a good correlation when the peptide numbers of caveolin, flotillin-1, and rab proteins were compared with their corresponding immunoblots (data not shown). Interestingly, we found that the peptide number of the {alpha}-subunit of several heterotrimeric G proteins increased significantly after EGF stimulation (Table 1Go) and confirmed this EGF-induced increase of Gs{alpha} and Gi1&2{alpha} in DRMs by immunoblotting (Fig. 5DGo). These G proteins might be involved specifically in EGFR signaling because a proteomic analysis of DRM/rafts after insulin stimulation showed no increase of these {alpha}-subunits (supplemental Table 2). Thus, our results demonstrate that in response to EGF, endosomal DRMs recruit highly Tyr-phosphorylated EGFR along with various signaling proteins, indicating a likely role for this compartment in signal transduction.


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  		TABLE 1. Identification by MS of selected signaling proteins in DRMs that increase after EGF stimulation

 
Isolation of DRMs from early and late endosomes
To define more precisely the distribution of DRMs in the intracellular compartments, endosomes were further fractionated in early and late endosomes by Percoll density gradient centrifugation (30). This technique allows us to separate early and late endosomes in a light and dense Percoll fraction, respectively (51, 52). As shown in Fig. 6AGo, early endosomal markers were enriched in fraction 2 and 3 of the Percoll gradient; however, LAMP-1 (a marker of late endosomes and lysosomes) was enriched in the denser fraction 4. EGFR rapidly appeared in fraction 2 by 2 min after EGF administration and remained in this fraction for about 30 min (Fig. 6BGo). However, EGFR in the late endosomal fraction (fraction 4 of the Percoll gradient) was detected transiently between 5 and 15 min after EGF (Fig. 6BGo). At 5 min after EGF stimulation, EGFR in early endosomes was more tyrosine phosphorylated than those in late endosomes (Fig. 6CGo), indicating that EGFR might be partially dephosphorylated before accessing the late compartment. However, EGFR in DRMs prepared from early and late endosomes showed a similar level of tyrosine phosphorylation (Fig. 6DGo), suggesting that EGFRs in endosomal DRMs are relatively resistant to dephosphorylation. Interestingly, DRMs from early and late endosomes showed a different distribution of caveolin and flotillin-1; the former was more abundant in early endosomes and the latter in late endsomes (Fig. 6DGo).


Figure 6
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  		FIG. 6. DRMs isolated from early and late endosomes. A, After an overnight fast, rats were killed, and combined endososmes were prepared and immediately subjected to centrifugation in a self-generating Percoll gradient as indicated in Materials and Methods. Four fractions were obtained, membranes in each fraction were concentrated by centrifugation, and proteins in the pellets were subjected to SDS-PAGE (same amount of protein in each lane) and analyzed by immunoblotting with antibodies against EEA1, transferring receptor (TfR), and LAMP-1. B, After an overnight fast, rats received a single dose of EGF (1 µg/100 g BW) and were killed at the indicated times. Percoll fractions were obtained and subjected to SDS-PAGE (same amount of protein in each lane) as indicated above. Proteins were analyzed by immunoblotting with antibodies against the EGFR and PY proteins. A representative immunoblot of three independent experiments is shown. C, The ratio of PY-EGFR (PY)/EGFR was determined in fraction 2 (enriched with early endosomes) and fraction 4 (enriched with late endosomes) 5 min after EGF. The bar is the mean ± SEM of five independent experiments. *, P < 0.01. D, Rats were killed at 5 min after receiving EGF. Early and late endosomes were prepared as indicated above, and 1.2 mg protein (pool of 12 rats) was used for the preparation of DRMs as indicated in Materials and Methods. DRMs (2.5 µg protein) were subjected to SDS-PAGE and immunoblotted with antibodies against EGFR, PY, caveolin, and flotillin-1. The graph on the right represents the ratio of PY-EGFR (PY)/EGFR. The bar is the mean ± half the range from two independent experiments.

 
Immunoisolation of endosomal vesicles enriched in raft markers
Previous work has shown the existence of different clathrin-independent endocytic pathways characterized for the presence of caveolin (53), flotillin-1 (54) and other raft markers also found in DRM (55, 56). Thus, we decided to test the possibility that the EGFR found in DRMs might be located in a distinctive endocytic compartment enriched with raft markers. To accomplish this, endosomes, prepared from rats 5 min after EGF, were incubated with magnetic beads coated with a specific antibody against caveolin or flotillin-1, permitting the immunoisolation of structures enriched, respectively, in these markers. As shown in Fig. 7AGo, caveolin-enriched vesicles specifically isolated from endosomes were depleted of EGFR and the early endosomal marker, rab5. Although flotillin-1 was found in this fraction, the content of this raft marker was low compared with endosomes (Fig. 7AGo). For the isolation of flotillin-1-containing vesicles, we use a specific antibody that recognizes the C terminus of this protein because previous work has shown that the C terminus of flotillin-1 is exposed to the cytoplasm (57). We found a low content of EGFR with a high level of tyrosine phosphorylation in flotillin-1-enriched vesicles in comparison with endosomes (Fig. 7BGo). Thus, this pool of EGFR shares similar characteristics with that found in endosomal DRMs (Fig. 5AGo).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we demonstrate EGF-induced compartmentalization of a distinctive population of EGFR in endosomes, as assessed using detergent extraction and immunoisolation of endosomal vesicles enriched in flotillin-1. The combined endosome fraction used in this study has been extensively characterized in previous work and shown to be composed of endosomes contaminated by Golgi and endoplasmic reticulum elements but free of contamination by PM (29, 42, 43). Furthermore, previous work demonstrated that in response to EGF, this endosomal fraction accumulated EGFR in endosomes and not in Golgi components (42). Hence, we conclude that the EGFR found in DRMs must come from endosomes. Also the concentration of rab5 in DRMs suggests that at least some of the DRMs originated from early endosomes. DRMs do not reflect a preexisting membrane organization, and different structures like endosomes and Golgi can contribute to DRM formation (14). However, it is accepted that the presence of a protein in DRMs is indicative of a potential affinity of that protein for lipid rafts (14). Thus, the association of highly Tyr-phosphorylated and ubiquitinated EGFR in DRMs suggests that this population of EGFR might be compartmentalized in a distinctive pool of endocytic vesicles or membrane domains enriched with raft markers. This hypothesis is strengthened by the presence of highly Tyr-phosphorylated EGFR in flotillin-1-enriched vesicles. In support of this observation, an endocytic pathway that depends on flotillin-1 but not on clathrin or caveolae was recently described (54).

Corresponding to the higher level of EGFR tyrosine phosphorylation, we also found that in response to EGF, the recruitment of Grb2 and Shc to DRMs was greater than that in whole endosomes, suggesting that this pool of receptor is competent for signaling. In further support of a role for this distinctive pool of intracellular EGFR in signaling, we found that multiple forms of the {alpha}-subunit of heterotrimeric G proteins were selectively increased in endosomal DRMs after EGF stimulation. This agrees with previous work assigning a role for heterotrimeric G proteins in mediating aspects of EGFR signaling in liver (58). More recently, a study carried out in yeast demonstrated that Gp{alpha}1 (a G protein {alpha}-subunit) can activate the phosphatidylinositol 3-kinase Vps34 in endosomes rather than at the PM (59). This recent work and our results identify endosomes as a compartment where G protein signaling likely occurs.

A recent study demonstrated that in addition to the classical clathrin-mediated internalization pathway, EGFR was also internalized through a clathrin-independent, lipid raft/caveolae-dependent pathway but only when high doses of EGF were used (25). This previous study also showed that ubiquitinated EGFR is internalized exclusively through lipid rafts/caveolae and suggested that this route of internalization is associated only with receptor degradation (25). Our results are in apparent discrepancy with those of Sigismund et al. (25). We should note that the dose of EGF used (1 µg/100 g BW) in our study saturates 50% of the hepatic PM receptors and has a negligible effect on EGFR degradation (3). Thus, at this dose of EGF, the EGFR would likely preferentially sort to signaling compartments.

However, in accordance with Sigismund et al. (25), we found that DRMs contained a higher proportion of ubiquitinated EGFR relative to those in the soluble fraction, suggesting that EGFRs in rafts are efficiently sorted to late endosomes. In further support of this hypothesis, EGFRs are detected in DRMs derived from late endosomes as soon as 5 min after EGF. However, although the bulk of receptor is dephosphorylated before being sorted to late endosomes, EGFR in late endosomal DRMs maintain a level of Tyr phosphorylation comparable to that in early endosomal DRMs. This suggests that EGFRs in late endosomal DRMs retain signaling competence. This finding also suggests that lipid rafts in endosomes may constitute a specialized compartment shielding the activated receptor from dephosphorylation by endosome-associated phosphotyrosine phosphatases. This hypothesis would explain why the EGFR in DRMs is more Tyr phosphorylated than the bulk of endosomal EGFR.

In relation to signaling from late endosomal compartments, previous work has shown that the adaptor protein p14 is essential for targeting the MP1-ERK1/2 complex to late endosomes during EGF-mediated ERK activation (60). The formation of a complex of p14-MP1-ERK1/2 appears to be necessary for the late but not the early (PM associated) phase of EGF-induced ERK activation (60). Thus, it seems likely that intracellular rafts efficiently target a complex constituted of ubiquitinated PY-EGFR, Grb2, and Shc to the cytoplasmic face of the late endosomes for specific activation of ERK1/2. Subsequently, ubiquitinated EGFRs would be sorted into the intralumenal vesicles of late endosomes (multivesicular bodies) before their degradation (61) or may be recycled back to the PM as suggested by the observation of Lai et al. (3).

Fivaz et al. (62) showed that trafficking of glycosyl-phosphatidylinositol-anchored proteins to late endosomes correlated with their association to DRMs, and speculated that signaling proteins resident in rafts might also follow this sorting route. This is consistent with the evidence that TGF-ß receptors, located in caveolae/rafts, are preferentially targeted to a degradative compartment (41). It might also be possible that the residence of proteins in rafts is a requisite sufficient for their efficient targeting to late endosomes.

It is interesting to note that in our study, IRK appeared to be preferentially excluded from endosomal DRMs. This is compatible with the observation that internalization of the insulin-IRK complex into endosomes is followed by rapid dissociation of insulin from the IRK with recycling of the latter to the PM (63, 64). Furthermore, in contrast to the EGFR, sorting of the IRK to late endosomes has yet to be demonstrated.

Interestingly, highly tyrosine-phosphorylated EGFR together with elevated levels of Grb2 and Shc were found in the pellet derived from Triton X-100-treated endosomes. This fraction was devoid of raft markers, Rab proteins, ubiquitinated proteins, and indeed any membrane structure as determined by electron microscopy (data not shown). These receptors seem to have been originally associated, directly or indirectly, with microtubules but not with the actin cytoskeleton. Additional work is necessary to determine what role(s) this Triton-insoluble fraction, which appears to contain a distinctive pool of EGFRs, might play in EGFR signaling and/or trafficking.

In summary, the present study demonstrates EGF-induced compartmentalization of hyperphosphorylated EGFRs in endosomes. The concentration of signaling-competent molecules in a detergent-resistant domain supports the emerging view that spatial compartmentalization within organelles plays an important role in the regulation of the signaling cascade.


   Acknowledgments
 
We thank Robert E. Kearney and Florence Servant (Réseau Protéomique de Montréal/Montreal Proteomics Network) for assistance with developing bioinformatics tools for comparing proteomic data. We also thank Drs. Simon Wing and Craig Mandato for their critical reading of the manuscript.


   Footnotes
 
First Published Online March 15, 2007

1 Morales F, Kearney R, Bencsath-Makkai Z, Bergeron J, Human Proteome Organization (HUPO) 2nd Annual and International Union of Biochemistry and Molecular Biology (IUBMB) XIX Joint World Con-gress, Molecular and Cellular Proteomics, Montreal, Canada, 2003, 2(9) 832. Back

2 Kearney RE, Blondeau F, McPherson P, Bell A, Servant F, Drapeau M, Grandpre SD, Bergeron J, 27th Annual International Conference of the IEEE Engineering in Medicine and Biology Society, Shanghai, 2005. Back

Abbreviations: BW, Body weight; DRM, detergent-resistant membrane; EEA1, early endosome antigen 1; EGF, epidermal growth factor; EGFR, EGF receptor; IRK, insulin receptor kinase; LAMP-1, lysosomal-associated membrane protein-1; MS/MS, tandem mass spectrometry; m/z, mass-to-charge ratio; PM, plasma membrane; PY, phosphotyrosine.

We acknowledge the financial support from Genome Canada/Genome Québec (The Réseau Protéomique de Montréal/Montreal Proteomics Network and the Type 2 Diabetes Gene Discovery program), the Canadian Institutes of Health Research, the Valorisation Recherche Québec (VRQ), and the Canadian Foundation for Innovation (CFI) for their invaluable financial support.

Disclosure Statement: The authors have nothing to disclose.

Received December 13, 2006.

Accepted for publication March 8, 2007.


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