Dimerization drives PDGF receptor endocytosis through a C-terminal hydrophobic motif shared by EGF receptor

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Research Article

Dimerization drives PDGF receptor endocytosis through a C-terminal hydrophobic motif shared by EGF receptor Justin Pahara, Huaiping Shi, Xinmei Chen, Zhixiang Wang⁎ Department of Cell Biology and Signal Transduction Research Group, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7

A R T I C L E I N F O R M A T I O N

AB S TR AC T

Article Chronology:

Like many other receptor tyrosine kinases (RTKs), platelet-derived growth factor (PDGF) receptor β

Received 18 August 2009

(PDGFR-β) is internalized and degraded in lysosomes in response to PDGF stimulation, which

Revised version received 11 May 2010

regulates many aspects of cell signalling. However, little is known about the regulation of PDGFR-β

Accepted 11 May 2010

endocytosis. Given that ligand binding is essential for the rapid internalization of RTKs, the events

Available online 16 May 2010

induced by the ligand binding likely contribute to the regulation of ligand-induced RTK internalization. These events include receptor dimerization, activation of intrinsic tyrosine kinase activity and

Keywords:

autophosphorylation. In this communication, we examined the role of PDGFR-β kinase activity,

PDGF receptor-beta

PDGFR-β dimerization and PDGFR-β C-terminal motifs in PDGF-induced PDGFR-β internalization. We

Dimerization

showed that inhibition of PDGFR-β kinase activity by chemical inhibitor or mutation did not block

Kinase activation

PDGF-induced PDGFR-β endocytosis, suggesting that the kinase activity is not essential. We further

Endocytosis

showed that dimerization of PDGFR-β is essential and sufficient to drive PDGFR-β internalization

EGF receptor

independent of PDGFR-β kinase activation. Moreover, we showed that the previously reported 14

Internalization motif

amino acid sequence 952–965 is required for PDGF-induced PDGFR-β internalization. Most importantly, we showed that this PDGFR-β internalization motif is exchangeable with the EGFR internalization motif (1005–1017) in mediating ligand-induced internalization of both PDGFR-β and EGFR. This indicates a common mechanism for the internalization of both PDGFR-β and EGFR. © 2010 Elsevier Inc. All rights reserved.

Introduction Internalization of cell surface receptors into cytoplasmic compartments is a characteristic of many receptors. The internalization process may be a means for down-regulating surface receptors for cellular desensitization or receptor degradation [1–3]. Recent evidence has reinforced the notion that the initiation and transduction of receptor signals does not occur only at the plasma membrane, but also occurs in endosomes; a phenomenon referred to as the ‘signalling endosome hypothesis’ [4–6]. This has led to the hypothesis that the transition from surface to endosomal signal transduction is a means for modulating the intensity or directionality of cellular outcomes [7]. ⁎ Corresponding author. Fax: +1 780 492 0450. E-mail address: [email protected] (Z. Wang). 0014-4827/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2010.05.012

Moreover, mutant receptors strongly associated with cancer and fibrotic diseases have been shown to remain catalytically active on the plasma membrane rather than internalizing upon ligand binding [3]. In support of this hypothesis, several viral proteins have been shown to stabilize signalling receptors on the plasma membrane, thereby increasing the host cell's mitogenic potential and ultimately leading to aberrant cell growth and metastasis [2]. Being able to understand and resolve these pathogenic mechanisms demands that we understand the regulation of receptor internalization. Like many other RTKs, binding of platelet growth factor (PDGF) to PDGF receptor-β (PDGFR-β) induces receptor dimerization, followed by the receptor kinase activation and phosphorylation of C-terminal

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tyrosine residues. This leads to both the internalization of PDGFR-β and activation of various signalling pathways downstream of PDGFRβ [8–12]. Work from the 1980's and 1990's suggested that RTK endocytosis is largely dependent on intrinsic kinase activity [10,13– 15]. Studies have also implicated importance of PDGFR-β kinase in PDGFR-β internalization. Mutation of a juxtamembrane tyrosine residue decreased the efficiency of PDGFR-β internalization [16]. This residue is phosphorylated upon kinase activation and thus, kinase activity is suggested to be required in PDGFR-β internalization. PI3K has also been shown to have an effect on PDGFR-β internalization. This was suggested when decreased PDGFR-β internalization was detected when PDGFR-β amino acids residues important for the interaction with PI3K were mutated [12]. It has also been shown that the relative rate of receptor internalization of kinase-dead (K634A) PDGFR-β was slower than wild-type PDGFR-β [10]. Moreover, studies have been conducted to understand the internalization motif(s) within PDGFR-β for PDGF-induced PDGFR-β internalization. Early work by Mori et al. [17] raised the possibility that a short 14 amino acid sequences in the C-terminus of PDGFR-β is involved in PDGFR-β internalization. Analysis of truncation mutants with the 14 amino acids removed suggested that the sequence is important for PDGFR-β internalization [17]. More recent evidence suggests that kinase activity may not be required for RTK internalization [18,19]. Inhibition EGFR kinase activation by chemical inhibitor AG1478 does not block the internalization of EGFR into endosomes [19]. It was further shown that EGF-induced EGFR internalization is driven by receptor dimerization, rather than receptor kinase activation [19]. Moreover, the dimerization-driven EGFR internalization requires a C-terminal hydrophobic amino acid motif from 1005 to 1017 [20]. Similarly, inhibition PDGFR-β kinase activation by chemical inhibitor AG1296 does not block the internalization of PDGFR-β into endosomes [21]. It is not clear whether PDGFR-β internalization is also driven by receptor dimerization as EGFR internalization. More importantly, whether there is a common mechanism for both PDGFR-β and EGFR internalization. In this communication, we examined the role of PDGFR-β kinase activity, PDGFR-β dimerization and PDGFR-β Cterminal motifs in PDGF-induced PDGFR-β internalization. We showed that kinase-dead PDGFR-β mutant is internalized robustly following addition of PDGF, suggesting that the kinase activity is not essential. We further showed that dimerization of PDGFR-β is essential and sufficient to drive PDGFR-β internalization independent of PDGFR-β kinase activation. Moreover, we showed that the previously suggested short 14 hydrophobic amino acid sequences 952–965 is required for PDGF-induced PDGFR-β internalization, acting as an internalization motif. Most importantly, we showed that this PDGFR-β internalization motif is exchangeable with the EGFR internalization motif (1005–1017) in mediating ligand-induced internalization of both PDGFR-β and EGFR. This indicates a common mechanism for the internalization of both PDGFR-β and EGFR.

Materials and methods

AG1296 was from Calbiochem (La Jolla, CA). PDGF-βB was from Upstate Biotechnology. Alexa Fluor 647-labelled EGF and Texas Red (TR)-labelled EGF were from (Molecular Probes Inc.). Unless otherwise specified, all the chemicals were purchased from Sigma.

Cell culture and treatment The cell lines that were used include mouse NIH-3 T3-F442 (F442) fibroblast cell line expressing wild-type endogenous PDGFR-β (expressing 50,000–100,000 receptors/cell) (from David Brindley, University of Alberta), human HepG2 cell lines stably transfected with wild-type PDGFR-β (HepG-PDGFR-β) or kinase-dead K634R PDGFR-β (HepG-K634R) (expressing ∼500,000 receptors/cell) (from Andrius Kazlauskus, Harvard University), wild-type HepG2 (HepG) cell lines with no detectable endogenous PDGFR-β expression (from Richard Lehner, University of Alberta), and a human embryonic kidney cell line containing simian virus 40T-antigen (293T). Cells were grown at 37 °C in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, and were maintained in a 5% CO2 atmosphere. For HepG cells, 100 μM non-essential amino acids (Invitrogen Co., Grand Island, NY) were added. To activate PDGFR-β, cells were treated with PDGF-BB at 10 ng/ml. To inhibit PDGFR-β kinase activation cells were treated with 0.05 µM AG1296 for 15 min. For treatment with monosialotetrahexosylganglioside (GM1) to block receptor dimerization, cells were treated with 100 µM GM1 for 1 h. To dimerize FKBP, cells were treated with AP20187 (50 nM, ARIAD) for 30 min.

Indirect immunofluorescence Indirect immunofluorescence was performed as described previously [22]. Briefly, cells were grown on glass coverslips and serum starved for 24 h. After treatment, the cells were fixed by ice cold methanol and permeabilized with 0.02% Triton X-100. Next, the cells were incubated with indicated primary antibodies at room temperature for 1 h followed by fluorescence-labelled secondary antibodies for 1 h. For both indirect immunofluorescence and intrinsic fluorescence, fluorescence images were photographed using a DeltaVision deconvolution Olympus IX71 microscope with a CoolSnap HQ2 CCD camera using a 60×, 1.42 NA UplanSApo60XO oil objective lens (Applied Precision). The data was analyzed using Delta Vision softWoRx software.

Immunoblotting Following treatments, cells were lysed using Mammalian Protein Extraction Reagent (M-PER, Pierce) and Nonidet P40 (NP-40, BDH) with protease inhibitor cocktail (Table 2.2.8). Lysates were centrifuged at 4 °C for 30 min at 17,000 ×g. Protein concentration from the supernatant was quantified using the Bradford protein dye assay. Protein concentration was calculated using the 595 nm absorbance value as measured by a Beckman DU 640 spectrophotometer (Beckman Instrument, Fullerton, CA). The lysate was boiled for 5 min in loading buffer and analyzed with PAGE and Western blot using anti-PDGFR-β, anti-pPDGFR-β and anti-GFP antibodies (Luc Berthiaume, University of Alberta).

Antibodies and chemicals Mouse anti-PDGFR-β (sc-6252), rabbit anti-PDGFR-β (sc-339) and rabbit anti-phosphorylated PDGFR-β (pPDGFR-β) (sc-12909) antibodies were from Santa Cruz Biotech (Santa Cruz, CA). Rabbit anti-GFP/YFP was from Luc Berthiaume (University of Alberta).

Cell surface biotinylation assay to examine PDGFR internalization To examine PDGFR internalization cell surface biotinylation assay was modified from Nishimura and Sasaki [23] and used in this

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study. To achieve the selective labelling and removal of only the cell surface proteins, the cleavable, water-soluble, and membraneimpermeable biotin analog sulfo-NHSSS-biotin was used. Then, free sulfo-NHS-SS-biotin was quenched by NH4Cl. Briefly, HepGPDGFR, HepG-K634R and F442 cells were incubated with 0.5 mg/ ml sulfo-NHS-SS-biotin in PBS/CM (pH7.2: 10 mM Na2HPO4, 2 mM KH2PO4) on ice for 30 min with rocking. Then the cells were incubated three times with 50 mM NH4Cl in PBS/CM on ice for 5 min to quench free biotin. The cells were then stimulated with PDGF for 0, 5’, 15’, and 30’ with or without pre-incubation with AG1296. Following incubation with 100 mM MESNA at 4 °C for 10 min to remove biotin from the sulfo-NHS-SS-biotin-labelled proteins on the cell surface, cells were treated with 5 mg/ml iodoacetamide in PBS/CM on ice for 5 min to quench free SH groups. Endocytosis was detected by the accumulation of the sulfo-NHS-SS-biotin-labelled cargo proteins within the cells, which were protected from reduction by MESNA. The cells those were biotinylated but not treated with MESNA were used as a control to show the total biotinylation PDGFR. Cells were then lysed and the biotinylated proteins were pulled down by NeutrAvidin beads. The internalized and total biotinylated PDGFR was determined by immunoblotting with antibody to PDGFR. For the quantification, PDGFR internalization was determined by the ratio between internalized biotinylated PDGFR and total biotinylated PDGFR and expressed as percentages.

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mutants including PDGFR-β truncated from C-terminus to 952 (Ct952) and to 965 (Ct965), polymerase chain reaction (PCR) primers were designed complementary to the 5’ end of PDGFR-β template as well as the 3’ end at the designated truncation point. PCR was used to amplify the coding region beginning at the 5’ end of PDGFR-β coding sequence to the 3’ end of PDGFR-β coding region as dictated by the complementation point of the PCR primer. For the construction of PDGFR-β mutant with deletion of 952–965 (Δ952–965), PDGFR-β mutant with 952–965 replaced by EGFR 1005–1017 (PDGFR-βe), or EGFR mutant with 1005–1017 replaced by PDGFR-β 952–965 (EGFRp), primers were designed so that they would anneal at the junction of interest (952–965 or 1005–1017) and in place of the wild-type junction of interest sequence, one-half of the exogenous sequence was tagged onto each primer. Following PCR with these primers, the resultant product was the entire vector excluding the junction of interest, which was now exchanged for the desired sequence. To allow for blunt end ligation of PCR products, 5’ phosphorylation modification was incorporated into the oligonucleotides. The deletion mutant oligonucleotides simply lacked an exogenous sequence. The methylated template strands were degraded using DpnI and XL-10 Gold competent bacterial cells were transformed. PDGFR-ββ with FKBP at the carboxyl terminus was generated using the ARGENT™ Regulated Homodimerization Kit 2.0 plasmid pC4-Fv1E vector (ARIAD).

Cross-linking assays

Results After treatment, intact cells were collected and treated with 0.01– 1 mM di-succinimidyl suberate (DSS, Calbiochem) at 4 °C. After 1h, 1 M Tris–HCl, pH 7.5, was added to the cells to a final concentration of 20 mM in order to quench the cross-linking reaction. This was followed by addition of Triton X-100 (BDH) and NP-40 to a concentration of 1% and extensive vortexing. The lysate was analyzed via PAGE and Western blot.

Flow cytometry The internalization of EGFR and mutants was assessed using flow cytometry as described previously [19]. Following various treatments, cells were incubated with Alexa 647-EGF at 4 °C for 45 min to allow for binding of the ligands without induction of internalization. The cells were washed once with cold PBS and once with room temperature PBS and 37 °C DMEM was added to the culture. The cells were incubated at 37 °C for the indicated times. Residual membrane-bound ligand was stripped from the sample using acid stripping buffer for 3 min at 4 °C and washed with 4 °C PBS. The total fluorescence sample was only washed after pre-incubation while the background fluorescence negative control sample was washed and acid stripped in a similar manner to the experimental samples. The cells were suspended in FACS buffer and FACS fixation buffer and 10,000 cells were analyzed using a BD Bioscience FACScanto. Mean and standard deviation was calculated using 3 independent experiments.

Constructs The pEYFP-N1 expression plasmids containing subcloned wildtype PDGFR-β or wild-type EGFR were used as a template for the construction of mutants employed in this study. For truncation

PDGFR-β kinase activity is not necessary for PDGFR-β internalization We first determined whether PDGFR-β kinase activity is required for PDGF-induced PDGFR-β internalization in HepG cells that stably expressing wild-type human PDGFR-β (HepG-PDGFR-β). The chemical inhibitor AG1296 is used to inhibit PDGFR-β kinase activation and the effect of AG1296 on PDGFR-β kinase activation was measured by examining the phosphorylation of PDGFR-β in immunoblotting. HepG-PDGFR-β cells were treated with AG1296 for 15 min and then stimulated with PDGF for indicated time. As shown in Fig. 1A, in the absence of AG1296, PDGF stimulated strong phosphorylation of PDGFR-β; however, treatment of the cells with AG1296 completely inhibited PDGF-induced PDGFR-β phosphorylation. We then determined effect of AG1296 on PDGFinduced PDGFR-β internalization by indirect immunofluorescence. The localization and the phosphorylation of PDGFR-β were revealed by double indirect immunofluorescence with antibodies to PDGFR-β and phosphorylated PDGFR-β (pPDGFR-β). As shown in Fig. 1B, without PDGF stimulation, PDGFR-β is not phosphorylated and the majority of PDGFR-β is localized to the plasma membrane with or without treatment with AG1296. Following PDGF stimulation for 5, 15, and 30 min, PDGFR-β was phosphorylated and gradually internalized in the absence of AG1296. In the presence of AG1296, PDGFR-β was not phosphorylated but was internalized in response to PDGF. We next examined the effects of AG1296 on endogenous PDGFR-β in F442 cells. As shown in Fig. 1C and D, similar to HepG-PDGFR-β cells, treatment with AG1296 significantly inhibited PDGF-induced PDGFR-β phosphorylation by inhibiting PDGFR-β kinase activation, but did not block PDGFinduced PDGFR-β internalization. This strongly indicates that

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Fig. 2 – PDGF-induced internalization of PDGFR-β in the presence or absence of PDGFR-β kinase activity. (A) The PDGF-induced internalization of PDGFR-β in HepG-PDGFR, F442 and HepG-K634R cells was determined by cell surface biotinylation with or without the treatment with AG1296 as described in Materials and methods. (B) Quantification of the data from (A). Each value is the average of at least three independent experiments. The error bar is standard error.

kinase activation of PDGFR-β is not essential for PDGF-induced PDGFR-β internalization. To confirm that PDGFR-β kinase activation is not required for PDGF-induced PDGFR-β internalization, we examined the internalization of a kinase-dead PDGFR-β mutant K634R with a single mutation of K634 to R in HepG cells stably expressing K634R (HepG-K634R). We showed by immunoblotting that K634R is not

phosphorylated in response to PDGF treatment (Fig. 1E), indicating that K634R is indeed a kinase-dead mutant. The internalization and phosphorylation of K634R were examined by double indirect immunofluorescence with antibodies to PDGFP and pPDGFR-β as described above. As shown in Fig. 1F, K634R is not phosphorylated in response to PDGF with or without AG1296 treatment, but robustly internalized similarly to wild-type PDGFR-β shown in

Fig. 1 – PDGF-induced phosphorylation and internalization of PDGFR-β in the presence or absence of PDGFR-β kinase activity. (A–D) HepG-PDGFR (A and B) and F442 (C and D) cells were treated with AG1296 and stimulated with PDGF for indicated time. The phosphorylation of PDGFR-β was examined by immunoblotting (A and C). The phosphorylation and internalization of PDGFR-β were examined by double indirect immunofluorescence (B and D). (E and F) HepG-K634R cells were stimulated with PDGF for indicated time. The phosphorylation of PDGFR-β was examined by immunoblotting (E). The phosphorylation and internalization of PDGFR-β were examined by double indirect immunofluorescence (F). (G) Co-localization of PDGFR and EEA 1. F442, HepG-PDGFR, and HepG-K634R cells with or without AG1296 treatment was stimulated with PDDG-BB for indicated time. The colocalization (yellow) of PDGFR (green) and EEA 1 (red) was determined by double indirect immunofluorescence as described in Materials and methods. Size bar = 15 μm.

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Fig. 1A. To confirm that the observed intracellular punctuated stain of PDGFR-β is indeed the indication of internalized PDGFR-β in endosomes, we co-stained PDGFR-β and EEA1 by double indirect immunofluorescence in F442, HepG-PDGFR, and HepG-K634R cells. As shown in Fig. 1G, with or without inhibition of PDGFR-β kinase activation, PDGFR-β and EEA1 co-localized in endosomes following PDGF stimulation. Together, we showed that PDGFR-β kinase activation is not essential for PDGF-induced PDGFR-β internalization. We further determine the effects of PDGFR kinase inhibition on PDGFR internalization by surface biotinylation experiments in above cell lines (Fig. 2). Similar to above indirect immunofluorescence experiments, we showed that inhibition of PDGFR kinase activity by AG1296 did not block the internalization of PDGFR in both HepG and F442 cells. Inhibition of PDGFR kinase by mutation of K634 to R also did not block PDGF-induced PDGFR internaliza-

tion. However, quantification of the data showed that inhibition of PDGFR kinase slightly reduced/delayed PDGF-induced PDGFR internalization. Together, our data indicate that PDGFR kinase activation may have minor effects, but is not essential for PDGFinduced PDGFR internalization.

The effects of PDGF-induced PDGFR-β dimerization on PDGFR-β internalization Since the only well defined event between ligand binding and kinase activation is the receptor dimerization, our results therefore suggest that the receptor dimerization may be the critical post-ligand binding event in regulating PDGFR-β internalization. To test this possibility, we first examined whether PDGF-induced dimerization of PDGFR-β is sufficient to stimulate PDGFR-β internalization. We showed in Fig. 1 that inhibition of PDGFR-β kinase activation did not

Fig. 3 – PDGF-induced dimerization of PDGFR-β with or without kinase inhibition. F442 (A and B), HepG-PDGFR-β (C and D) and HepG-K634R (E and F) cells were stimulated with PDGF for indicated time in the presence and absence of AG1296. Cells were cross-linked with DSS. The cell lysates were immunoblotted with anti-PDGFR-β antibody (A, C and E). The dimer formation was then quantitated by densitometry (B, D and F). Each value is the average of at least three experiments and the error bar is the standard error.

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block PDGF-induced PDGFR-β internalization. We further determined whether PDGFR-β is dimerized under these conditions. PDGFR-β dimerization was examined by cross-linking with DSS followed by immunoblotting. Our results showed that inhibition of PDGFR-β kinase activity in HepG-EGFR and F442 cells by AG1296 did not block PDGF-induced PDGGFR dimerization (Fig. 3A and B). We also showed that kinase-dead PDGFR-β K634R is dimerized as wildtype PDGFR-β following PDGF stimulation for 15 min (Fig. 3C). Thus, binding of PDGF to PDGFR-β followed by PDGF-induced PDGFR-β dimerization without subsequent kinase activation is sufficient to stimulate PDGFR-β internalization.

The effects of non-ligand-induced PDGFR-β dimerization on PDGFR-β internalization We next examined whether PDGFR-β dimerization itself without ligand binding is sufficient to stimulate PDGFR-β internalization. PDGFR-β was artificially dimerized by using the receptor dimerization kit “ARGENT™ Regulated Homodimerization kit” from ARIAD Pharmaceuticals, Inc. We first constructed the fusion protein by fusing the full-length PDGFR-β with FKBP (PDGFR-β-FKBP) according to the instructions provided by the company. The fusion proteins were expressed in 293T cells. Cross-linking experiments showed that this fusion protein is indeed dimerized following the addition of AP20187 or PDGF and dimerization is not affected by inhibition of PDGFR-β kinase activation with AG1296 (Fig. 4A). The internaliza-

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tion of PDGFR-β-FKBP is examined by indirect immunofluorescence. As shown in Fig. 3B, treatment of PDGFR-β-FKBP with PDGF or AP20187 stimulates PDGFR-β internalization with or without inhibition of kinase activation by AG1296 (Fig. 4B). These data indicate that PDGF binding is not directly necessary for the initiation of PDGFR-β internalization and non-ligand-induced dimerization is sufficient to stimulate PDGFR-β internalization.

Inhibition of PDGFR-β dimerization and the effects on PDGFR-β internalization We then examined whether PDGF-induced PDGFR-β dimerization is essential for PDGF-induced internalization of PDGFR-β. It has been previously shown by several groups that ganglioside GM1 inhibits PDGFR-β dimerization [24]. The current understanding for ganglioside GM1 inhibition of PDGFR-β dimerization involves only the transmembrane domain or the extracellular domain of PDGFR-β [24]. To determine whether GM1 inhibits PDGFR-β dimerization in our experimental system, we treated HepG-EGFR, HepG-K634R and F442 cells with GM1 and examined the effects on PDGFR-β dimerization. As shown in Fig. 5A, treatment with GM1 at 100 μM blocked PDGFinduced PDGFR-β dimerization. We then examined the effects of GM1 on PDGF-induced PDGFR-β phosphorylation and internalization by double indirect immunofluorescence in F442 cells. We showed that treatment with GM1 greatly inhibited PDGFR-β phosphorylation (red) (Fig. 5B), which further confirms that PDGFR-β does not

Fig. 4 – PDGFR-β internalization following non-ligand-induced dimerization. (A) non-ligand-induced dimerization of PDGFR-β. PDGFR-β was fused with FKBP (PDGFR-β-FKBP) by using “ARGENT™ Regulated Homodimerization kit” from ARIAD Pharmaceuticals, Inc. PDGFR-β-FKBP was expressed in 293T cells by transient transfection. Following treatment with either AP20187 (100 nM) or PDGF (20 ng/ml) in the presence or absence of AG1296, the proteins were cross-linked with DSS. The cells were then lysed and the protein samples were immunoblotted with anti-PDGFR-β antibody. (B) Internalization of PDGFR-β-FKBP by indirect immunofluorescence. PDGFR-β-FKBP was expressed in both 293T and HepG cells by transient transfection. The cells were grown in coverslips. Following treatment with AP20187 (100nM) or PDGF (20 ng/ml) in the presence or absence of AG1296, the localization of PDGFR-β-FKBP was examined by anti-PDGFR-β antibody followed by FITC-conjugated secondary antibody as described in Materials and methods. Size bar = 20 μm.

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undergo PDGF-induced dimerization in the presence of GM1 because receptor phosphorylation is a direct result of receptor dimerization. Moreover, GM1 treatment significantly inhibited PDGF-induced PDGFR-β internalization (green). This phenomenon is also seen in both HepG-PDGFR-β and HepG-K634R cells (Fig. 4B). Although in these experiments, HepG-PDGFR-β and HepG-K634R cells cannot be serum starved to the point where PDGFR-β is completely absent from punctate structures, PDGFR-β remains on the plasma membrane after PDGF treatment. Additionally, at all PDGF treatment time points in the presence and absence of AG1296, accumulation of PDGFR-β in punctate or sorting endosomal structures is absent (Fig. 5B). We further showed by surface biotinylation assay that GM1 treatment blocked PDGF-induced internalization of PDGFR in HepG-EGFR, HepG-K634R and F442 cells (Fig. 5C and D). In conclusion, these data suggest that inhibition of PDGFR-β dimerization results in inhibition of PDGF-mediated PDGFR-β endocytosis. This is strong evidence that dimerization is an essential event that must occur for PDGFR-β internalization to take place. It is important to note that the inhibition of PDGFR-β dimerization in the presence of GM1 is not due to inhibition of PDGF binding to the receptor [24], ruling out the possibility that the absence of receptor dimerization, phosphorylation, and internalization was due to a lack of ligand binding. To confirm that the effects of GM1 on PDGFR-β internalization are due to its inhibitory effects on PDGFR-β dimerization, we examined the effects of GM1 on the internalization of PDGFR-β-FKBP. It is known that GM1 prevents dimerization of PDGFR-β in a cytoplasmic domain independent mechanism [25]. Thus, GM1 will only block PDGF-induced dimerization of PDGFR-β-FKBP through the extracellular domain, but not AP20187-induced dimerization of PDGFR-βFKBP through the C-terminal FKBP domain. Consequently, GM1 should not block AP20187-induced PDGFR-β-FKBP internalization. 293T cells expressing PDGFR-β-FKBP were treated with GM1. The cells were then stimulated with PDGF or AP20187 for 30 min and the effects on PDGFR-β internalization were measured by the percentage of cells displaying receptor internalization. Indeed, treatment with GM1 blocked PDGF-stimulated PDGFR-β-FKBP internalization, but did not block AP20187-stimulated PDGFR-β-FKBP internalization (Fig. 6). This further indicates that dimerization is the driving force for PDGFR-β internalization. Together, our data demonstrate that dimerization is essential for PDGFR-β internalization.

The PDGFR-β C-terminal sequences required for PDGFR-β internalization, but not receptor dimerization The next question needs to be answered is how PDGF-induced PDGFR-β dimerization stimulates the internalization of PDGFR-β in

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Fig. 6 – The effects of GM1 on PDGF- and AP20187-induced internalization of PDGFR-β-FKBP. 293T cells expressing FKBP-PDGFR-β were pre-incubated with GM1 (100 µM) for 1 h and then treated for 30 min with either PDGF (20 ng/ml) or AP20187 (50 nM). The internalization of PDGFR-β was examined by indirect immunofluorescence and presented by the percentage of cells displaying receptor internalization. Data is a result of three independent experiments and the error bar is standard error.

the absence of PDGFR-β kinase activation. It is possible that PDGFinduced PDGFR-β dimerization causes necessary conformational changes of the receptor to expose the cryptic internalization codes at its C-terminus. It was suggested that a portion of the C-terminus of PDGFR-β, especially the 14 amino acid sequences 952–965, play important role in the internalization of PDGFR-β [17]. Based on these previous data, we constructed two YFP-tagged PDGFR-β truncation mutants, PDGFR965 and PDGFR952. The PDGFR965 and PDGFR952 mutants are PDGFR-β truncated from the C-terminus to 965 and 952, respectively. In addition, we generated a PDGFR-β deletion mutant Δ952–965 which has the amino acids between 952 and 965 removed. Each mutant was expressed in 293T cells by transient transfection. The cells were then treated with PDGF (10 ng/ml) for up to 60 min and the internalization of these mutants was examined by fluorescence microscopy (Fig. 7). Untreated serum-free cells exhibited little endocytosis in each of the wild-type PDGFR-β, PDGFR965, PDGFR952, and Δ952–965 expressing cells. At 5 min post-PDGF stimulation, PDGFR-β internalization was visually detected in the wild-type and PDGFR965 expressing cells (arrows), while PDGFR952and Δ952-65 expressing cells appeared devoid of PDGFR-β endocytosis (Fig. 7A). The trend continued as punctate

Fig. 5 – Inhibition of PDGFR-β dimerization by GM1 and the effects on PDGFR-β internalization. (A) Inhibition of PDGFR-β dimerization by GM1. HepG-PDGFR, HepG-K634R and F442 cells were treated with GM1 and then stimulated with PDGF for 30 min. The cells were then treated with DSS to cross-link the dimers. The formation of PDGFR-β dimers was then examined by immunoblotting with anti-PDGFR-β antibody. (B) Indirect immunofluorescence to show the inhibition of PDGFR-β internalization by GM1. F442, HepG-PDGFR and HepG-K634R cells grown on coverslips were treated with GM1 to block PDGFR-β dimerization. The cells were then stimulated with PDGF for indicated time in the presence and absence of AG1296. The localization (green) and phosphorylation (red) of PDGFR-β were revealed by double indirect immunofluorescence with antibodies to PDGFR-β and pPDGFR-β followed by FITC and Rhodamine-conjugated secondary antibody. Size bar = 20 μm. (C) Cell surface biotinylation to show the inhibition of PDGFR-β internalization by GM1. F442, HepG-PDGFR and HepG-K634R cells were treated with GM1 to block PDGFR-β dimerization. The cells were then stimulated with PDGF for indicated time. The internalization of PDGFR-β was determined by cell surface biotinylation as described in Materials and methods. (D) Quantification of the data from (C). Each value is the average of at least three independent experiments. The error bar is standard error.

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Fig. 7 – The role of PDGFR-β C-terminal sequences 952–965 in PDGFR-β internalization and dimerization. YFP-tagged PDGFR-β and the mutants including PDGFR965, PDGFR952 and Δ952–965 were expressed in 293T cells by transient expression as described in Materials and methods. (A) The internalization of PDGFR-β and the mutants was examined by the intrinsic fluorescence of YFP in fixed cells as described in Materials and methods. Arrow showed the internalized PDGFR-β. Size bar = 20 μm. (B) The dimerization of PDGFR-β and the mutants was examined by cross-linking experiments as described in Materials and methods. The cells were stimulated with PDGF for 30 min followed by DSS treatment to cross-link the proteins. The PDGFR-β dimer formation was examined by immunoblotting with antibody to PDGFR-β.

intracellular accumulation of PDGFR-β occurred in wild-type PDGFR-β and PDGFR965 expressing cells up to the 60 min time point (arrows), but not in the PDGFR952 and Δ952-65 expressing cells treated with PDGF for 15, 30, and 60min (Fig. 7A). These data suggest that the 952–965 amino acid region of PDGFR-β is required for receptor internalization. We next determined whether the residues 952–965 function to facilitate PDGF-induced PDGFR-β dimerization that drives PDGFRβ internalization or function to mediate internalization directly. The extent of PDGFR-β mutant dimerization was evaluated by DSS cross-linking experiments (Fig. 7B). After 30 min of PDGF stimulation, increased receptor dimerization was detected in all PDGFR-β mutants (Fig. 7B). PDGFR-β dimer band intensity increased in the PDGFR952 and Δ952–965 mutant, illustrating that even though the receptors are able to dimerize, they are defective for internalization. This suggests that while dimerization of the receptor is likely a driving force for PDGFR-β internalization, it is equally important that the cytoplasmic domain retains an

internalization motif and the sequences 952–965 functions as an internalization motif for PDGFR-β.

EGFR and PDGFR-β share the same C-terminal endocytic motif While the PDGFR-β internalization motif 952–965 contains a dileucine motif (Fig. 8A) that has been implicated in the trafficking [20,26–29], the whole sequence between 952 and 965 is not a defined motif for endocytosis. Interestingly, we have shown recently that a short amino acid sequence between EGFR 1005 and 1017 also contains a dileucine motif and is essential for EGF-induced EGFR internalization (Fig. 8A) [20]. Beside both containing a dileucine motif, amino acids 1005–1017 in EGFR and amino acids 952–965 in PDGFR-β are also similar in that there are a high number of hydrophobic amino acid residues (Fig. 8A). The similar endocytosis pathway between EGFR and PDGFR-β suggests that they may both use a hydrophobic motif to mediate their endocytosis driven by ligand-induced dimerization. To test this possibility, we constructed

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Fig. 8 – Mediation of EGFR internalization by PDGFR-β internalization motif 952–965. (A) Amino acid sequence alignment of EGFR internalization motif 1005–1017 and PDGFR-β internalization motif 952–965. (B) A YFP-tagged mutant EGFR with its C-terminal internalization motif 1005–1017 replaced with PDGFR-β internalization motif 952–965 (EGFRp) was expressed in 293T cell. Following stimulation with TR-EGF for indicated time, the internalization of both EGFRp (green) and TR-EGF (red) was examined by following the intrinsic fluorescence of YFP and TR. 293T cells transfected with YFP-tagged wild-type EGFR and mutant EGFR lacking the internalization motif 1005–1017 (Δ1005–1017) were used as control. (C) EGFR and EGFRp were expressed in 293T cells and EGF-induced EGFR internalization was examined by live imaging. (D) EGFR and EGFRp were expressed in 293T cells and their internalization was examined by flow cytometry following stimulation with Alexa 647 conjugated EGF. Each datum is the average of at least three experiments and the error bar is the standard error. Size bar = 20 μm.

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Fig. 9 – Mediation of PDGFR-β internalization by EGFR internalization motif 1005–1017. A YFP-tagged mutant PDGFR-β with its C-terminal internalization motif 952–965 replaced with EGFR internalization motif 1005–1017 (PDGFR-βe) was expressed in 293T cell. Following stimulation with PDGF for indicated time, the internalization of PDGFR-βe was examined by following the intrinsic fluorescence of YFP. 293T cells transfected with YFP-tagged wild-type PDGFR-β was used as control. Size bar = 20 μm.

two YFP-tagged mutant PDGFR-β/EGFR receptors by exchanging the above hydrophobic internalization motifs: a mutant EGFR with the replacement of 1005–1017 by PDGFR-β 952–965 (EGFRp) and a mutant PDGFR-β with the replacement of 952–965 by EGFR 1005– 1017 (PDGFR-βe). These two mutants were expressed in 293T cells and the internalization of PDGFR-βe and EGFRp was examined by fluorescence microscopy. Analysis of images from fixed cells showed that EGFRp internalized upon treatment with Texas Red-labelled EGF (TR-EGF) (Fig. 8B). At all TR-EGF treatment time points, TR-EGF and YFP-tagged EGFRp were co-localized to endosomes (Fig. 8B). A control panel showing the impaired endocytosis of mutant EGFR Δ1005–1017 highlights the functionality of the PDGFR-β internalization motif with EGFR (Fig. 8B). The internalization of EGFRp was further examined by live imaging. As shown in Fig. 8C, following addition of TR-EGF, both EGFR and EGFRp co-internalized with TR-EGF in a similar pattern. Moreover, quantification of EGFR, EGFRp and EGFRΔ1005–1017 internalization by flow cytometry showed that both EGFR and EGFRp internalized by a very similar kinetics (Fig. 8D). Together, our data strongly indicate that the internalization motif of PDGFR-β is able to mediate EGF-stimulated EGFR internalization. Similarly, when expressed in 293T cells, PDGFR-βe was also internalized following PDGF stimulation in a pattern similar to PDGFR-β (Fig. 9). This indicates that the internalization motif of EGFR is able to mediate PDGF-stimulated PDGFR-β internalization. Thus, a universal internalization motif has been recognized in the Cterminus of two receptors.

Discussion It is well established that endocytosis of RTKs from the cell surface to lysosomes results in degradation of the receptor, which can attenuate receptor signalling and may even be conceived of as a tumor suppressor pathway [30]. The internalization of constitutively internalized receptors is largely mediated by sorting signals such as YXXΦ and NPXY [31]. However, for the RTKs that are internalized in response to ligand binding, there is likely some means of switching

their sorting signals on and off [32]. Given that ligand binding is essential for the rapid internalization of RTKs, the events induced by the ligand binding likely contribute to the regulation of ligandinduced RTK internalization. These events include receptor dimerization, activation of intrinsic tyrosine kinase and autophosphorylation. Indeed, all these events have been extensively studied and implicated in EGF-induced EGFR endocytosis [19,33]. However, very little is known about the regulation of PDGFR-β endocytosis. In this research, we not only examined the role of ligand binding, receptor dimerization and receptor kinase activation in PDGFR-β endocytosis, but also determined whether there is a common mechanism regulating both EGFR and PDGFR internalization. We showed by indirect immunofluorescence and cell surface biotinylation that inhibition of PDGFR-β kinase activation by chemical inhibitor AG1296 did not block PDGF-induced PDGFR-β internalization (Figs. 1 and 2), which confirmed our previous report [21]. We further showed that inhibition of PDGFR-β kinase activity by point mutation did not block PDGF-induced PDGFR-β internalization (Figs. 1 and 2). Our results indicate that PDGFR-β kinase activation is dispensable in PDGF-induced PDGFR-β internalization. It is reported that PDGFR-β kinase activity increase the rate of PDGFR-β internalization [10], however, no direct evidence reported to indicate that the kinase activity is essential for PDGFR-β internalization. Our data regarding PDGFR-β internalization are also consistent with our data regarding EGFR internalization. We showed previously that EGFR kinase activity is not required for EGF-induced EGFR internalization [4,19,20]. We then focused our research on the role of PDGFR-β dimerization in PDGFR-β internalization and demonstrated that proper PDGFR-β dimerization is essential and sufficient for PDGFRβ internalization (Figs. 3–6). We showed that with the inhibition of PDGFR-β kinase activation did not affect PDGF-induced PDGFR dimerization and internalization (Fig. 3). Moreover, by using a PDGFR-β fused with FKBP in its C-terminus we showed that nonligand-induced dimerization of PDGFR-β through its C-terminal FKBP domain in the absence of PDGF is sufficient to stimulate PDGFR-β internalization (Fig. 4). This suggests that proper dimerization itself is the driving force for PDGFR-β internalization

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and the ligand binding is not necessary. As previously suggested, the role of dimeric PDGF appears to be exclusively to bring PDGFRβs into close proximity to force receptor dimerization and does not extend to eliciting structural changes in PDGFR-β that enable the internalization of the receptor [34,35]. If this is the case, the physical act of receptor dimerization is an essential event in mediating receptor internalization. These results indicate that a proper dimerization itself in the absence of ligand binding and kinase activation is sufficient to stimulate PDGFR-β internalization. We also showed here that inhibition of PDGFR-β dimerization by treatment with GM1 inhibited PDGF-induced PDGFR-β internalization (Fig. 5). We further provided convincing evidence to show that the effects of GM1 on PDGFR-β internalization are specifically through its inhibitory effects on receptor dimerization. By using the PDGFR-β-FKBP fusion protein, we showed that PM1 only block PDGF-induced internalization of PDGFR-β-FKBP, but not AP20187induced internalization of PDGFR-β-FKBP (Fig. 6). This is consistent with previous finding that GM1 prevents dimerization of PDGFR-β in a cytoplasmic domain independent mechanism [25]. This indicates that PDGF binding by itself in the absence of PDGFR-β dimerization is not able to stimulate PDGFR-β endocytosis. Moreover, we also showed that non-ligand-induced dimerization of PDGFR-β in the absence of PDGF is sufficient to stimulate PDGFR-β internalization. This suggests that proper dimerization itself is the driving force for PDGFR-β internalization. Based on the previous report of the possible role of PDGFR-β Cterminal sequences between 952 and 965 in PDGFR-β internalization [17], we showed here that this 14 amino acid motif is essential for PDGF-induced PDGFR-β internalization (Fig. 7A). Truncation of PDGFR-β from C-terminus to 965 has no significant effects on PDGFR-β internalization, further truncation to 952 or deletion of this 14 amino acid sequence greatly inhibits PDGFR-β internalization (Fig. 7A). Furthermore, the role of this 14 amino acid sequence seems to mediate the internalization of PDGFR-β directly, rather than to alter receptor dimerization (Fig. 7B). Thus, this 14 amino acid sequence functions as an internalization motif for PDGFR-β internalization. This internalization motif does not contain tyrosine residues that are phosphorylated by PDGFR-β kinase, which further supports our finding that PDGFR-β-dimerization rather than kinase activation controls PDGFR-β internalization. While this 14 amino acid internalization motif is not an established motif for internalization, a similar short amino acid internalization motif EGFR 1005–1017 was identified previously as essential for EGFR internalization [20]. Beside both containing a dileucine motif, amino acids 1005–1017 in EGFR and amino acids 952–965 in PDGFR-β are also similar in that there are a high number of hydrophobic amino acid residues (Fig. 8A). The similar endocytosis pathway and regulation between EGFR and PDGFR-β suggests that they may share a common internalization motif. Indeed, we showed PDGFR-β internalization motif 952–965 is able to mediate EGFR internalization and EGFR internalization motif 1005–1017 is also able to mediate the internalization of PDGFR-β (Figs. 8 and 9). This finding is very significant. First, it suggests that RTKs may use a common mechanism to mediate their internalization. Previously, it was suggested that the kinase activation and phosphorylation of C-terminal residues may be the common mechanisms for RTK internalization. However, more evidence now suggests that kinase activity is dispensable for the internalization of both EGFR and PDGFR-β and no tyrosine phosphorylation sites have been identified as essential for RTK internalization

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[4,19,21]. Second, our data may provide a base for the establishment of a common internalization motif that contain an established dileucine motif and surrounding hydrophobic amino acids. Further research is needed to examine whether this motif exist in other RTKs and to precisely define and characterize this internalization motif. It would be also highly interesting to identify the proteins interacting with this motif.

Acknowledgments We thank ARIAD Pharmaceuticals, Inc. for providing “ARGENT™ Regulated Homodimerization kit.” This work was supported in part by grants from the Canadian Institutes of Health Research (CIHR) and the Alberta Heritage Foundation for Medical Research (AHFMR). Z.W. is an AHFMR Senior Scholar.

REFERENCES

[1] A.K. Samanta, J.J. Oppenheim, K. Matsushima, Interleukin 8 (monocyte-derived neutrophil chemotactic factor) dynamically regulates its own receptor expression on human neutrophils, J. Biol. Chem. 265 (1990) 183–189. [2] Y. Yarden, Sliwkowski, Untangling the ErbB signalling network, Nat. Rev. 2 (2001) 127–137. [3] M.V. Grandal, R. Zandi, M.W. Pedersen, B.M. Willumsen, D.B. van, H.S. Poulsen, EGFRvIII escapes down-regulation due to impaired internalization and sorting to lysosomes, Carcinogenesis 28 (2007) 1408–1417. [4] Y. Wang, S. Pennock, X. Chen, Z. Wang, Endosomal signaling of epidermal growth factor receptor stimulates signal transduction pathways leading to cell survival, Mol. Cell Biol. 22 (2002) 7279–7290. [5] S. Pennock, Z. Wang, Stimulation of cell proliferation by endosomal epidermal growth factor receptor as revealed through two distinct phases of signaling, Mol. Cell Biol. 23 (2003) 5803–5815. [6] Y. Shao, W. Akmentin, J.J. Toledo-Aral, J. Rosenbaum, G. Valdez, J.B. Cabot, B.S. Hilbush, S. Halegoua, Pincher, a pinocytic chaperone for nerve growth factor/TrkA signaling endosomes, J. Cell Biol. 157 (2002) 679–691. [7] P. Burke, K. Schooler, H.S. Wiley, Regulation of epidermal growth factor receptor signaling by endocytosis and intracellular trafficking, Mol. Biol. Cell 12 (2001) 1897–1910. [8] P. Liu, Y. Ying, Y.G. Ko, R.G. Anderson, Localization of platelet-derived growth factor-stimulated phosphorylation cascade to caveolae, J. Biol. Chem. 271 (1996) 10299–10303. [9] B.P. Liu, K. Burridge, Vav2 activates Rac1, Cdc42, and RhoA downstream from growth factor receptors but not beta1 integrins, Mol. Cell Biol. 20 (2000) 7160–7169. [10] A. Sorkin, C. Waters, K.A. Overholser, G. Carpenter, Multiple autophosphorylation site mutations of the epidermal growth factor receptor. Analysis of kinase activity and endocytosis, J. Biol. Chem. 266 (1991) 8355–8362. [11] R. Kapeller, R. Chakrabarti, L. Cantley, F. Fay, S. Corvera, Internalization of activated platelet-derived growth factor receptor- phosphatidylinositol-3' kinase complexes: potential interactions with the microtubule cytoskeleton, Mol. Cell Biol. 13 (1993) 6052–6063. [12] M. Joly, A. Kazlauskas, F.S. Fay, S. Corvera, Disruption of PDGF receptor trafficking by mutation of its PI-3 kinase binding sites, Science 263 (1994) 684–687. [13] M. Dougher, B.I. Terman, Autophosphorylation of KDR in the kinase domain is required for maximal VEGF-stimulated kinase

2250

[14]

[15]

[16]

[17]

[18]

[19]

[20]

[21]

[22]

E XP E RI ME N T AL C E L L R E SE A RC H 31 6 ( 20 1 0) 2 2 37 – 2 25 0

activity and receptor internalization, Oncogene 18 (1999) 1619–1627. J.R. Glenney Jr., W.S. Chen, C.S. Lazar, G.M. Walton, L.M. Zokas, M.G. Rosenfeld, G.N. Gill, Ligand-induced endocytosis of the EGF receptor is blocked by mutational inactivation and by microinjection of anti- phosphotyrosine antibodies, Cell 52 (1988) 675–684. J.B. Welsh, R. worthylake, H.S. Wiley, G.N. Gill, Specific factors are required for kinase-dependent endocytosis of insulin receptors, Mol. Biol. Cell 5 (1994) 539–547. S. Mori, L. Ronnstrand, K. Yokote, A. Engstrom, S.A. Courtneidge, L. Claesson-Welsh, C.H. Heldin, Identification of two juxtamembrane autophosphorylation sites in the PDGF beta-receptor; involvement in the interaction with Src family tyrosine kinases, EMBO J. 12 (1993) 2257–2264. S. Mori, L. Claesson-Welsh, C.H. Heldin, Identification of a hydrophobic region in the carboxyl terminus of the platelet-derived growth factor beta-receptor which is important for ligand-mediated endocytosis, J. Biol. Chem. 266 (1991) 21158–21164. Y. Wang, S. Pennock, X. Chen, Z. Wang, Internalization of inactive EGF receptor into endosomes and the subsequent activation of endosome-associated EGF receptors. Epidermal growth factor, Sci. STKE 2002 (2002) L17. Q. Wang, G. Villeneuve, Z. Wang, Control of epidermal growth factor receptor endocytosis by receptor dimerization, rather than receptor kinase activation, EMBO Rep. 6 (2005) 942–948. Q. Wang, F. Zhu, Z. Wang, Identification of EGF Receptor C-terminal Sequences 1005–1017 and Di-leucine Motif 1010LL1011 as Essential in EGF Receptor Endocytosis, Exp. Cell Res. 313 (2007) 3349–3363. Y. Wang, S.D. Pennock, X. Chen, A. Kazlauskas, Z. Wang, Platelet-derived growth factor receptor-mediated signal transduction from endosomes, J. Biol. Chem. 279 (2004) 8038–8046. Z. Wang, L. Zhang, T.K. Yeung, X. Chen, Endocytosis deficiency of epidermal growth factor (EGF) receptor-ErbB2 heterodimers in response to EGF stimulation, Mol. Biol. Cell 10 (1999) 1621–1636.

[23] N. Nishimura, T. Sasaki, Cell-surface biotinylation to study endocytosis and recycling of occludin, Methods Mol. Biol. 440 (2008) 89–96. [24] B.J. Van, E.G. Bremer, A.J. Yates, Gangliosides inhibit platelet-derived growth factor-stimulated receptor dimerization in human glioma U-1242MG and Swiss 3T3 cells, J. Neurochem. 61 (1993) 371–374. [25] J.L. Oblinger, C.L. Boardman, A.J. Yates, R.W. Burry, Domaindependent modulation of PDGFRbeta by ganglioside GM1, J. Mol. Neurosci. 20 (2003) 103–114. [26] J.S. Bonifacino, L.M. Traub, Signals for sorting of transmembrane proteins to endosomes and lysosomes, Annu. Rev. Biochem. 72 (2003) 395–447. [27] S.J. Kil, M. Hobert, C. Carlin, A leucine-based determinant in the epidermal growth factor receptor juxtamembrane domain is required for the efficient transport of ligand-receptor complexes to lysosomes, J. Biol. Chem. 274 (1999) 3141–3150. [28] A. Tsacoumangos, S.J. Kil, L. Ma, F.D. Sonnichsen, C. Carlin, A novel dileucine lysosomal-sorting-signal mediates intracellular EGF-receptor retention independently of protein ubiquitylation, J. Cell Sci. 118 (2005) 3959–3971. [29] F. Huang, X. Jiang, A. Sorkin, Tyrosine phosphorylation of the beta2 subunit of clathrin adaptor complex AP-2 reveals the role of a di-leucine motif in the epidermal growth factor receptor trafficking, J. Biol. Chem. 278 (2003) 43411–43417. [30] P.P. Di Fiore, G.N. Gill, Endocytosis and mitogenic signaling, Curr. Opin. Cell Biol. 11 (1999) 483–488. [31] J.S. Bonifacino, E.C. Dell'Angelica, Molecular bases for the recognition of tyrosine-based sorting signals, J. Cell Biol. 145 (1999) 923–926. [32] I.S. Trowbridge, J.F. Collawn, C.R. Hopkins, Signal-dependent membrane protein trafficking in the endocytic pathway, Annu. Rev. Cell Biol. 9 (129–61) (1993) 129–161. [33] A. Sorkin, L.K. Goh, Endocytosis and intracellular trafficking of ErbBs, Exp. Cell Res. 315 (2009) 683–696. [34] C.H. Heldin, Dimerization of cell surface receptors in signal transduction, Cell 80 (1995) 213–223. [35] J. Schlessinger, Cell signaling by receptor tyrosine kinases, Cell 103 (2000) 211–225.