# 2009 The Authors

Journal compilation # 2009 Blackwell Munksgaard

doi: 10.1111/j.1600-0854.2008.00858.xTraffic 2009; 10: 235–245Blackwell Munksgaard

Epsin 1 is Involved in Recruitment of UbiquitinatedEGF Receptors into Clathrin-Coated Pits

Maja Kazazic1,†, Vibeke Bertelsen1,†,

Ketil Winther Pedersen1, Tram Thu Vuong1,

Michael Vibo Grandal1,2, Marianne Skeie

Rødland1, Linton M. Traub3, Espen Stang1,4

and Inger Helene Madshus1,4,*

1Institute of Pathology, University of Oslo,Rikshospitalet HF, N-0027 Oslo, Norway2Department of Cellular and Molecular Medicine,The Panum Institute, University of Copenhagen,DK-2200 Copenhagen, Denmark3Department of Cell Biology and Physiology,University of Pittsburgh School of Medicine, Pittsburgh,PA15261, USA4Division of Pathology, Rikshospitalet UniversityHospital, Oslo, Norway*Corresponding author: Inger Helene Madshus,i.h.madshus@medisin.uio.no†These authors contributed equally to this work.

Epsin consists of an epsin NH2-terminal homology

domain that promotes interaction with phospholipids,

several AP-2-binding sites, two clathrin-binding sequen-

ces and several Eps15 homology domain-binding motifs.

Epsin additionally possesses ubiquitin-interacting motifs

(UIMs) and has been demonstrated to bind ubiquitinated

cargo. We therefore investigated whether epsin pro-

moted clathrin-mediated endocytosis of the ubiquiti-

nated EGF receptor (EGFR). By immunoprecipitation, we

found that epsin 1 interacted with ubiquitinated EGFR

and that functional UIMs were essential for complex

formation. Furthermore, RNA interference-mediated

knockdown of epsin 1 was found to inhibit internalization

of the EGFR, while having no effect on endocytosis of the

transferrin receptor. Additionally, upon knockdown of

epsin 1, translocation of the EGFR to central parts of

clathrin-coated pits was inhibited. This supports the

contention that epsin 1 promotes endocytosis of the

ubiquitinated EGFR.

Keywords: AP-2, clathrin-coated pit, EGF receptor, epsin,

ubiquitin

Received 20 December 2007, revised and accepted for

publication 17 November 2008, uncorrected manuscript

published online 20 November 2008

Endocytosis of the EGF receptor (EGFR) has been dem-

onstrated to be clathrin dependent (1–5). Recent studies

have suggested that the clathrin adaptor protein complex,

AP-2, plays different roles in endocytosis of the transferrin

receptor (TfR) and EGFR (3,4,6,7). Whereas AP-2 is strictly

required for TfR internalization (3), small and nonstoichio-

metric amounts of AP-2 appear sufficient for endocytosis

of the EGFR in HeLa cells (7). This is, however, in contrast

to previous interpretations arguing that AP-2 has the same

general role in endocytosis of TfR and EGFR (4). If AP-2 is

not required for interaction with ligand-activated EGFR, but

rather in clathrin-coated vesicle formation, a different

adaptor protein must be required. In this study, we have

therefore addressed whether the clathrin-, AP-2- and

ubiquitin-binding protein epsin functions as the cargo-

binding adaptor in endocytosis of the EGFR. There are

currently at least three knownmammalian variants of epsin

proteins (epsin 1, 2 and 3) (8–10). All epsin proteins are

characterized by having an N-terminal epsin NH2-terminal

homology (ENTH) domain that binds to phosphatidylinosi-

tol-4,5-bisphosphate (PIP2) (11,12). Directly adjacent to the

ENTH domain are two or three (depending on splice

variations) tandem ubiquitin-interacting motifs (UIMs)

(10,13). The central region of epsin contains a clathrin interac-

tion motif as well as repeats of a DPWmotif, which binds to

AP-2 (14,15). The C-terminal region contains an LVDLD

sequence, which binds to clathrin (16–18), as well as repeats

of the NPF motif, which binds to Eps15 homology (EH)

domains in proteins like Eps15 and POB1 (18,19). Outside

the ENTH domain, epsin is an intrinsically unstructured poly-

peptide (20) that undergoes local disorder–order transitions.

Epsin has been demonstrated to act as adaptor protein in

clathrin-mediated endocytosis of ubiquitinated proteins (21–

25), and interaction of the EGFRwith epsin has further been

demonstrated (26). The EGFR is ubiquitinated upon activa-

tion, but it has beenunclearwhether it is polyubiquitinated or

multiply monoubiquitinated (27,28). Recently, however,

mass spectrometric analysis revealed that the EGFR is both

polyubiquitinatedandmultiubiquitinatedwithin5 minofEGF

addition (29). Epsinwas recently demonstrated to efficiently

bind polyubiquitinated cargo through its UIMs (21,22), and

epsin homologs in Drosophila and Caenorhabditis elegans

have been demonstrated to be involved in endocytosis of

ubiquitinated proteins in the Notch pathway (30,31). The

role of epsin as a dedicated ubiquitin adaptor specifically in

clathrin-mediated endocytosis has, however, been ques-

tioned (32). It was found that deletion of the central AP-2-

and clathrin-binding segment of epsin 1 facilitates colocal-

ization of this epsin mutant with a ubiquitin reporter

construct present on endosomes. Furthermore, clathrin

RNA interference causes a similar redistribution of epsin 1,

so it was suggested that ubiquitin binding impairs epsin’s

clathrin-binding ability and vice versa (32).

In this study, we have investigated whether epsin 1

interacts with the ubiquitinated EGFR and whether epsin

www.traffic.dk 235

1 is required for endocytosis of the EGFR. Our present data

suggest that epsin 1 interacts with ubiquitinated EGFR

through its UIMs. Short interfering RNA (siRNA)-mediated

downregulation of epsin 1 was found to modestly inhibit

internalization of the EGF-bound EGFR, while having no

effect on internalization of the TfR, suggesting that epsin 1

was specifically involved in endocytosis of the EGFR.

Interestingly, in cells where epsin 1 had been knocked

down by the use of siRNA, the activated EGFR was found

to localize to the rims and not to the central parts of

clathrin-coated pits, strongly suggesting that epsin 1 is

required for recruitment of the ubiquitinated EGFR into

clathrin-coated pits. Together, our findings argue that

clathrin-dependent endocytosis of ubiquitinated EGFR is

promoted by interaction with epsin 1.

Results

Epsin interacts with ubiquitinated EGFR

in a UIM-dependent manner

Because epsin contains UIMs capable of binding ubiquiti-

nated cargo (21,22,32,33), we investigated whether epsin

1 interacted with ligand-activated and ubiquitinated EGFR.

We did not manage to immunoprecipitate endogenous

epsin with available antibodies. HeLa cells were therefore

transfected with a plasmid encoding the Myc-tagged wild-

type (wt) epsin 1 or with plasmids encoding either a Myc-

tagged epsin 1 UIM mutant, unable to bind ubiquitinated

proteins, or a Myc-tagged truncated form of epsin 1

(ENTH-UIM) lacking AP-2- and clathrin-binding sequences

(see Figure 1 for an overview of the epsin 1 constructs

used in this work). The cells were subsequently incubated

with or without EGF (60 ng/mL) for 3 min at 378C.Wt epsin 1 and the epsin 1 mutants were immunopreci-

pitated using anti-Myc antibody, and the immunoprecipi-

tated material was subjected to western blotting with an

antibody to the EGFR. As demonstrated in Figure 2A,

ubiquitinated EGFR coimmunoprecipitated with both wt

epsin 1 and ENTH-UIM part of epsin 1 upon activation of

the EGFR. Interestingly, the EGFR did not coimmunopre-

cipitate with the UIM mutant of epsin 1. These experi-

ments demonstrated that functional UIMs are required for

formation of an EGFR–epsin complex. However, as the

data do not clearly demonstrate that the epsin–EGFR

interaction is direct, we cannot rule out the possibility that

epsin 1 is recruited through another ubiquitinated protein

bound to the EGFR. Because the ENTH-UIM part of epsin

1 interacted more efficiently with the EGFR than did full-

length epsin 1, we took advantage of this truncated epsin

in immunoprecipitation experiments.

Human Sprouty2 (hSpry2) has previously been demon-

strated to inhibit both ubiquitination and internalization of

the EGFR by interacting with Cbl and thereby attenuate

Cbl’s ubiquitin ligase activity (34–36). To verify that the

interaction between epsin 1 and the EGFR was dependent

on ubiquitination of the EGFR, HeLa cells were transfected

with a plasmid encoding the FLAG-tagged ENTH-UIM part

of epsin 1 alone or together with a plasmid encoding Myc-

tagged hSpry2. In doubly transfected cells, reduced

amounts of the EGFR coimmunoprecipitated with the

ENTH-UIM part of epsin 1 (Figure 2B), suggesting that

epsin 1 interacts poorly with the EGFRwhen ubiquitination

of the EGFR is inhibited. It should be noted that the fraction

of EGFR coprecipitating with ENTH-UIM in the presence of

hSpry2 was clearly ubiquitinated (see ‘upsmearing’ of the

EGFR band compared with the band in lysate from cells

not incubated with EGF). To further verify this contention,

we made use of stably transfected PAE cells expressing

either wt-EGFR or Y1045F-EGFR. When phosphorylated,

pY1045 serves as a major docking site for Cbl.

The Y1045F-EGFR was previously demonstrated to have

reduced ubiquitination upon ligand-induced activation but

to be efficiently internalized (37,38). PAE cells stably

expressing wt-EGFR or Y1045F-EGFR were incubated

with and without EGF (60 ng/mL) for 3 min at 378C. EGFRwas then immunoprecipitated, and the precipitated mate-

rial was western blotted using anti-ubiquitin antibody.

As demonstrated (Figure 3A), ubiquitination of the Y1045F-

EGFR was in fact substantial and upon quantification was

estimated to be approximately 30% that of the wt-EGFR.

PAE cells stably expressing wt-EGFR or Y1045F-EGFR

were subsequently transiently transfected with a plasmid

encoding the Myc-tagged ENTH-UIM part of epsin 1. The

cells were then incubated with or without EGF (60 ng/mL)

for 3 min at 378C. The Myc-tagged ENTH-UIM part of

epsin 1 was immunoprecipitated using anti-Myc antibody,

and the immunoprecipitated material was subjected to

western blotting with an antibody to the EGFR. As

demonstrated in Figure 3B, comparable amounts of EGFR

coimmunoprecipitated with the ENTH-UIM part of epsin 1

when using lysate from cells expressing wt-EGFR or

Y1045F-EGFR. This was somewhat surprising but sug-

gests that although the amount of ubiquitin added to the

Y1045F-EGFR was reduced, the ubiquitin added wasFigure 1: Schematic overview of the rat epsin 1 constructs

used. ENTH domain binds to PIP2.

236 Traffic 2009; 10: 235–245

Kazazic et al.

quantitatively and qualitatively sufficient for efficient inter-

action with epsin 1 to take place (see Discussion). Alto-

gether, our data argue that ubiquitination of the EGFR

promotes formation of epsin–EGFR complexes and further

that the ubiquitinated EGFR binds to epsin 1 through

epsin’s UIMs. The results with the Y1045F mutant show

additionally that low levels of EGFR ubiquitination are

sufficient to engage epsin 1 through the UIMs.

siRNA-mediated knockdown of epsin 1 specifically

inhibited endocytosis of the EGFR by inhibiting

translocation of the EGFR into clathrin-coated pits

To confirm that epsin has a functional role in endocytosis

of the EGFR, we knocked down epsin 1 by siRNA. In HeLa

cells, two different target sequences were used, and both

duplexes (Dup1 and Dup3) (4) downregulated epsin 1

efficiently (Figure 4A). We then measured the rate of

EGF internalization using 1 ng/mL 125I-EGF in cells trans-

fected with or without siRNA to epsin 1. Downregulation

of epsin 1, using either of the two siRNA duplexes,

inhibited internalization of EGF (the ratio of internalized to

surface localized EGF was reduced by 45% upon 5-min

incubation) (Figure 4B), while knockdown of epsin 1 had no

effect on endocytosis of Tf (Figure 4C). We also trans-

fected cells with siRNA to green fluorescent protein (GFP)

to rule out that transfection with siRNA as such affected

EGF internalization (data not shown). In conclusion, our

results support the contention that epsin 1 is specifically

involved in endocytosis of the EGFR as cargo selective

adaptor. It should be noted that in contrast to data reported

by Sigismund et al. (26), our present data clearly demon-

strated that even 1 ng/mL EGF induced relatively efficient

ubiquitination of the EGFR (Figure S1). The fact that

efficient depletion of epsin 1 did not affect endocytosis

of Tf demonstrated that depletion of epsin 1 did not affect

formation of clathrin-coated pits and vesicles in general

[formation of clathrin-coated pits was also confirmed by

electron microscopy (see below)]. This argues that epsin 1

does not have an important universal role in curving and

tubulation of membranes (12,39).

We further wanted to investigate to what extent knock-

down of epsin 1 inhibited endocytosis of the less effi-

ciently ubiquitinated Y1045F-EGFR compared with

wt-EGFR. In PAE cells stably expressing either wt-EGFR or

Y1045F-EGFR, epsin 1was targeted usingDup3 (Figure 5A).

Figure 2: Epsin 1 interacts with ubiquitinated EGFR through its UIMs. A) HeLa cells were transfected with plasmids encoding wt

epsin 1, the N-terminal ENTH-UIM part of epsin 1 or the UIM mutant of epsin 1 incapable of binding ubiquitin. All epsin variants were Myc

tagged. Twenty-four hours after transfection the cells were incubated with or without 60 ng/mL EGF for 3 min at 378C. The cells were then

subjected to immunoprecipitation using antibody toMyc and analyzed bywestern blotting using antibodies to EGFR andMyc. B) HeLa cells

transfected with plasmids encoding the FLAG-tagged ENTH-UIM part of epsin 1 either alone or together with a plasmid encoding Myc-

tagged hSpry2 were incubated with or without 60 ng/mL EGF for 3 min at 378C. The ENTH-UIM part of epsin was immunoprecipitated

with antibody to FLAG, and the precipitated material was subjected to western blotting with antibodies to EGFR and FLAG.

Overexpression of hSpry2 in transfected cells was demonstrated using antibody to Myc.

Figure 3: Ubiquitinated wt- and Y1045F-EGFR interact with the ENTH-UIM part of epsin 1. A) PAE cells expressing wt-EGFR or

Y1045F-EGFR were incubated with or without 60 ng/mL EGF for 3 min at 378C as indicated. The cells were then subjected to

immunoprecipitation under denaturating conditions using antibody to EGFR and analyzed by western blotting using antibodies to ubiquitin

and EGFR. B) PAE cells expressingwt-EGFR or Y1045F-EGFRwere transiently transfectedwith theMyc-tagged ENTH-UIM part of epsin 1.

Twenty-four hours after transfection the cells were incubated with or without 60 ng/mL EGF for 3 min at 378C. The cells were then

subjected to coimmunoprecipitation analysis using antibody to Myc and analyzed by western blotting using antibodies to EGFR and Myc.

Traffic 2009; 10: 235–245 237

Epsin Localizes Ubiquitinated EGFR to Coated Pits

Wemeasured the rate of EGF internalization using 1 ng/mL125I-EGF in cells transfected with or without siRNA to epsin

1. The siRNA-mediated knockdown of epsin 1 inhibited the

endocytosis rate of EGF by approximately 40% (upon

5-min incubation) whether the cells expressed wt-EGFR

or Y1045F-EGFR (Figure 5B,C). This is consistent with our

finding that wt-EGFR and Y1045F-EGFR both efficiently

interacted with the ENTH-UIM part of epsin 1 and that this

facilitated endocytosis.

It should be noted that the rate of EGF internalization in

HeLa cells endogenously expressing EGFR and in PAE

cells expressing wt-EGFR or Y1045F-EGFR upon stable

transfection varies. The reason for this is most likely that

Figure 4: Epsin 1 is involved in EGFR but not in TfR endocytosis. A) Lysates from HeLa cells transfected with or without siRNA to

epsin 1 (Dup1 or Dup3) were subjected to western blot analysis using antibodies to epsin 1 and tubulin (loading control). Proteins were

detected using enhanced chemiluminescence. B) HeLa cells were transfected with or without siRNA as indicated and described in

Materials and Methods. The cells were then incubated with 1 ng/mL 125I-EGF for the times indicated, and the rate of EGF internalization

was measured as described inMaterials and Methods. The data represent one independent experiment (of three) with four parallels�SD.

C) HeLa cells were transfectedwith or without siRNA as indicated and described inMaterials andMethods. Cells were then incubatedwith125I-Tf (70 ng/mL) at 378C for 5 min, and the internalized 125I-Tf was plotted as percentage of total cell-associated 125I-Tf. The data

represent one independent experiment (of three) with four parallels �SD.

Figure 5: Knockdown of epsin 1 inhibits

endocytosis of wt-EGFR and Y1045F-

EGFR. PAE cells stably expressing wt-

EGFR or Y1045F-EGFR were depleted of

epsin 1 by siRNA-mediated knockdown

using Dup3 according to the procedure

described inMaterials and Methods. A) Cell

lysates were subjected to western blotting

using antibody to epsin 1 and tubulin (load-

ing control). B and C) Cells transfected with

or without siRNA to epsin 1 (as indicated)

were incubated with 1 ng/mL 125I-EGF for

the times indicated, and the rate of EGF

internalization was measured as described

in Materials and Methods. The data repre-

sent one independent experiment (of three)

with four parallels �SD.

238 Traffic 2009; 10: 235–245

Kazazic et al.

the stably transfected PAE cells express significantly more

EGFR than do HeLa cells.

To investigate at what step of clathrin-dependent endocy-

tosis epsin 1 is required, we used immunolabeling of

thawed cryosections and electron microscopy (immuno-

EM) and stereological analysis to examine the effect of

siRNA-mediated knockdown of epsin 1 in HeLa cells or

PAE cells expressing wt-EGFR. The cells were incubated

with or without EGF (60 ng/mL), and to avoid that EGFRs

within interior parts of coated pits escaped quantification

because of endocytic uptake, we incubated the cells on ice

to prevent membrane budding. We have previously dem-

onstrated that the EGFR is ubiquitinated on ice (38).

Incubation with EGF on ice was previously also demon-

strated to cause colocalization of EGFR and AP-2 (36,40),

and at the same time, endocytosis was inhibited. This

allowed a precise quantification of EGFRs in coated pits.

High concentrations of EGF were used to maximize

activation-dependent recruitment of EGFRs into coated

pits. As demonstrated in Figure S2, downregulation of

epsin 1 inhibited endocytosis of the EGFR also at 60 ng/mL.

In nontransfected HeLa cells, 1.3 � 0.9% of the EGFR at

the plasma membrane localized inside coated pits in the

absence of EGF, while in the presence of EGF, 9.5 � 2.5%

of the EGFR localized inside coated pits (Table 1 and

Figure 6A). In HeLa cells where epsin 1 had been knocked

down, 0.4 � 0.3% of the EGFR localized inside coated pits

in the absence of EGF, while upon activation of the EGFR,

only 5.7 � 0.5% of the EGFR showed this localization

(Table 1). This decrease is consistent with the diminished

uptake of EGF seen in epsin 1 knockdown cells (Figure 4).

Interestingly, in HeLa cells where epsin 1 was knocked

down, a significant fraction (6.7 � 0.5%) of the EGFR

localized to the rim of coated pits in the presence of EGF

(Figure 6B), while in nontransfected cells, only 1.0 � 0.8%

of the EGFR localized to the rim of the coats (Table 1). This

could indicate a role of epsin 1 in recruitment of cargo into

clathrin-coated pits. The number of coated pits at the

plasma membrane in epsin 1 knockdown cells was found

to be as in control cells (data not shown), and immuno-EM

experiments with labeling for a-adaptin demonstrated that

the amount of AP-2 in coated pits was comparable with

that of nontransfected cells (data not shown). In principle,

the same inhibitory effect of knocking down epsin 1 was

found on recruitment of EGFR to clathrin-coated pits in

PAE cells stably expressing wt-EGFR (Figure 7 and

Table 1). However, these cells showed more intense

antibody labeling for EGFR at the plasma membrane than

did HeLa cells because of higher expression. It has been

reported that epsin 1 is a driving force in the curvature of

clathrin-coated pits (12). Figure 7, however, clearly dem-

onstrates coated pits at all stages of invagination also in

epsin 1 knockdown cells. Furthermore, as demonstrated in

Figure 4, siRNA to epsin 1 did not affect TfR endocytosis,

confirming that epsin 1 knockdown did not affect forma-

tion of clathrin-coated pits.

Interestingly, recruitment of activated EGFR to clathrin-

coated pits was additionally strongly inhibited upon over-

expression of the ENTH-UIM part of epsin 1 (Table 2). This

argues that the clathrin- and AP-2-binding parts of epsin are

required to translocate the UIM-bound ubiquitinated EGFR

to clathrin-coated pits.

It should be noted that it has previously been reported that

knockdown of epsin 1 did not affect endocytosis of the

EGFR (4,26). In initial experiments, we could also not

observe inhibitory effects on endocytosis of EGF by

knocking down epsin 1. However, as demonstrated in

Figure S3, using the same siRNA duplexes as used by

Huang et al. (4), the knockdown of epsin 1 was more

efficient when cells were transfected twice with a 48-h

interval than when transfected once. In both cases, epsin 1

knockdown was evaluated 48 h after the last siRNA

transfection. We therefore believe that our more efficient

knockdown of epsin 1 explains the observed inhibitory

effect on endocytosis of EGF. It should further be noted

that we were not successful in rescuing the inhibitory

effect on endocytosisbyexpressingsiRNA-resistantepsin1.

This is probably because of our finding that overexpression

of epsin 1 sequestered AP-2 and clathrin and therefore

Table 1: Knockdown of epsin 1 causes localization of activated EGFR to the rim of clathrin-coated pitsa

HeLa PAE

% EGFR in coated pits % EGFR in coated pits

EGF Inside Rim Number of Au Inside Rim Number of Au

Control � 1.3 � 0.9 0 358 0.4 � 0.2 0.2 � 0.2 1089

Control þ 9.5 � 2.5 1 � 0.8 379 4.4 � 0.9 2.3 � 0.6 1084

Epsin 1 siRNA � 0.4 � 0.3 0.5 � 0.7 437 0.3 � 0.1 0.1 � 0.1 1320

Epsin 1 siRNA þ 5.7 � 0.5 6.7 � 0.5 450 1.5 � 0.4 2.4 � 0.6 1427

aHeLa cells or PAE cells expressing wt-EGFR, treated with Lipofectamine 2000 without (control) or with siRNA to epsin 1 [Dup1 (HeLa) or

Dup3 (PAE)] were incubated with or without EGF (60 ng/mL) for 1 h on ice and processed for immuno-EM and labeled with antibodies to

the EGFR. The plasmamembrane distribution of the EGFRwas quantified, and EGFR localizing to the interior or to the rim of coated pits are

presented as the percentage of the total labeling for EGFR at the plasma membrane. Each quantification represents the mean � SD of

three parallel labeling experiments. Number of Au indicates the total number of gold particles counted for each condition.

Traffic 2009; 10: 235–245 239

Epsin Localizes Ubiquitinated EGFR to Coated Pits

non-specifically inhibited clathrin-mediated endocytosis

(Figure S4).

Discussion

It has been reported that epsin can act as adaptor protein

for the endocytosis of ubiquitinated proteins (21–25,32), as

well as other uncharacterized cargo receptors (41) in

mammalian cells, and we now report that siRNA-mediated

knockdown of epsin 1 mildly inhibited EGF-induced endo-

cytosis of the EGFR. This is similar to the mild effect of

siRNA-mediated knockdown of epsin 1 on KSHV-mediated

internalization of major histocompatibility complex I (23).

The remaining endocytosis may be because of existence

of other yet unidentified ubiquitin-binding proteins. One

candidate could be epsin 2. However, our unpublished

results have demonstrated that siRNA-mediated knock-

down of epsin 2 non-specifically inhibited clathrin-

dependent endocytosis.

We further report that in the absence of epsin 1, the EGFR

was inefficiently recruited to the interior regions of clathrin-

coated pits but was instead found to localize to the rim of

such coats. The UIM-containing protein Eps15 has also

been reported to localize to the rim of clathrin-coated pits

(42–44), and the edge-like localization of EGFR observed

could thus potentially be explained by the ubiquitinated

EGFR binding to Eps15. In contrast to Eps15, epsin 1 was

found localized to all parts of clathrin-coated pits (36,45).

Our present data thus suggest that under normal condi-

tions, the EGF-activated and ubiquitinated EGFR initially

interacts with Eps15 in a transient manner but is rapidly

handed over to epsin 1 to enter central regions of clathrin-

coated pits. Indeed, we demonstrated coimmunoprecipita-

tion of epsin 1 with ubiquitinated EGFR.We also confirmed

that intact UIMs were essential for binding the EGFR to

epsin 1. Our data thus suggest that epsin’s ability to bind

ubiquitinated EGFR is important in clathrin-dependent en-

docytosis of the EGFR by recruiting the EGFR into clathrin-

coated pits. Transient binding of the EGFR to Eps15 and

subsequent handing off to epsin 1 localized inside the

Eps15-positive edge of clathrin-coated pits would ensure

packaging of the ubiquitinated EGFR in the invaginating

section of the pit. This would be very similar to the des-

cribed co-operation of Golgi-localized, g-ear-containing, Arf

binding proteins (GGAs) and AP-1 in packaging of mannose

6-phosphate receptors at the trans Golgi network (46).

It should be noted that the ENTH-UIM part of epsin 1 binds

ubiquitinated EGFR more efficiently than does full-length

epsin 1. This is partly consistent with the notion that clathrin

binding negatively regulates epsin’s binding of ubiquitinated

cargo (32). However, it should further be noticed that

overexpression of the ENTH-UIM part of epsin 1 not

containing AP-2- and clathrin-binding sequences was found

to efficiently exclude the EGFR from clathrin-coated pits. In

this regard, the ENTH-UIM part in fact acts in a dominant-

negative fashion. Because the EGFR was excluded from

coated pits under these conditions, the ubiquitinated EGFR

coimmunoprecipitating with the tagged ENTH-UIMwas not

localized to coated pits. These data clearly show that

ubiquitinated EGFR interacts with UIMs in epsin 1, and

we suggest that when the N-terminal part of epsin 1 is

positioned within the clathrin lattice by means of the C-

terminal clathrin-, AP-2- and Eps15-binding information, it

will use the UIMs for cargo recognition. This was in fact

suggested in direct biochemical experiments (21). Also, the

dominant-negative effect of the ENTH-UIM part of epsin 1 is

very reminiscent of what was observed when the tandem

Dab2 PTB domain was overexpressed. Like the ENTH-UIM,

the 2xPTB domain couples a PIP2-binding site with a cargo

recognition interface, and overexpression blocked LDL

receptors from entering clathrin-coated pits (47).

The endocytosis rates of wt-EGFR and Y1045F-EGFRwere

identical and so was the inhibitory effect of epsin 1 siRNA

on endocytosis. As the interaction between EGFR and

epsin 1 depends on ubiquitination (shown by inhibition of

Cbl’s ubiquitin ligase activity by hSpry2 overexpression)

and on intact UIMs, we believe that it is reasonable to

suggest that the ubiquitination of the Y1045F-EGFR,

although reduced, is sufficient for interaction with epsin 1

and thus for efficient endocytosis of EGFR. There are

different possible reasons why this reduced ubiquitination

could be sufficient. (i) The required stoichiometry of the

epsin–EGFR interaction is not known, but because of, for

Figure 6: Knockdown of epsin 1 causes localization of acti-

vated EGFR to the rim of clathrin-coated pits in HeLa cells.

HeLa cells were incubated with Lipofectamine 2000 without (A) or

with (B) siRNA to epsin 1 (Dup1) as described in Materials and

Methods. The cells were then incubated with EGF (60 ng/mL) for

1 h on ice and subsequently processed for immuno-EM and

labeled with antibodies to the EGFR. Arrowheads indicate the

clathrin coat, and arrows indicate EGFR labeling. Bar, 100 nm.

240 Traffic 2009; 10: 235–245

Kazazic et al.

example, sterical hindrance, it could be that only one epsin

molecule or a limited number of epsin molecules may at

any given time interact with each ubiquitinated EGFR.

Increased ubiquitination will therefore not necessarily

increase the number of epsin molecules interacting with

each EGFR, which will explain why the same amount of

Y1045F-EGFR and wt-EGFR was precipitated along with

epsin ENTH-UIM. (ii) wt-EGFR has been shown to be both

mono- and polyubiquitinated, and the polyubiquitin chains

were demonstrated to be largely K63 linked (29). Epsin has

also been demonstrated to efficiently interact with K63-

linked polyubiquitin chains in biochemical experiments

(21). Interestingly, Y1045F-EGFRwas found to have a simi-

lar ubiquitin linkage profile as wt-EGFR with mainly K63

linkages (29), and this could explain the efficiency of

endocytosis because of the capacity of K63-linked poly-

ubiquitin to interact with epsin.

Based on the observation that EGFRmutants with minimal

ubiquitination were endocytosed at the same rate as was

wt-EGFR, it was recently stated that ubiquitination of

EGFR is not required for its endocytosis (48). However,

in that paper, it was clearly demonstrated that in kinase-

deficient EGFR mutants, endocytosis was in fact rescued

by ubiquitination. This is consistent with our present

results, which seem to indicate that relatively small

amounts of ubiquitin are sufficient for endocytosis of

EGFR. Such low amounts of ubiquitin could in many

cases be possibly undetectable by standard biochemical

methods (49).

Our results demonstrate that epsin 1 is involved in

recruitment of ubiquitinated EGFR to coated pits because

siRNA-mediated knockdown of epsin 1 inhibited EGF-

induced recruitment of the EGFR to clathrin-coated pits.

Figure 7: Knockdown of epsin 1 causes localization of activated EGFR to the rim of clathrin-coated pits in PAE cells. PAE cells

stably expressing wt-EGFR were incubated with Lipofectamine 2000 without (A–C) or with (D–I) siRNA to epsin 1 (Dup3) as described in

Materials and Methods. The cells were then incubated without (A and D) or with EGF (60 ng/mL) (B, C and E–I) for 1 h on ice and

subsequently processed for immuno-EM and labeled with antibody to EGFR (arrowheads). Note the EGF-induced relocalization of EGFR

into coated pits (c.p.) in control cells and to the rim of coated pits in epsin siRNA-treated cells. Bar, 100 nm.

Traffic 2009; 10: 235–245 241

Epsin Localizes Ubiquitinated EGFR to Coated Pits

One possibility for the requirement of epsin 1 could be that

extensive modification of the EGFR tail with formation of

polyubiquitin chains as well as multiple monoubiquitination

(29) could interfere with the direct EGFR–AP-2 interaction

(50) either because of masking of dileucine and tyrosine

internalization sequences or because of conformational

changes in the EGFR tail. We further speculate that in the

absence of EGFR ubiquitination, other adaptor proteins,

such as AP-2, could recruit the EGFR to coated pits direc-

tly. But failure to detect large amounts of EGFR in clathrin-

coated regions of the plasma membrane in the absence of

added EGF (Tables 1 and 2) indicates that these signals are

not constitutively very active or that the EGFR is restricted

to surface domains that prevent entry into clathrin-coated

pits in the absence of activation. In conclusion, we favor

the idea that in case of ubiquitinated EGFR at the plasma

membrane, epsin 1 can act as cargo-associated sorting

protein, participating in cargo capture and concentration of

ubiquitinated EGFR in clathrin-coated pits.

Materials and Methods

MaterialsAll reagents were from Sigma Chemical Co., unless otherwise noted.

Na125I was from PerkinElmer Life And Analytical Services, Inc., and 125I-

EGF was from GE Healthcare UK Limited. Dako fluorescent mounting

medium was from Dako. FuGENE 6 was from Roche Diagnostics GmbH,

and Lipofectamine� 2000 was from Invitrogen Ltd. Human recombinant

EGF was from Bachem AG.

Cell cultureHeLa cells were grown in DMEMwith 0.5� penicillin–streptomycinmixture

and L-glutamine (2 mM), all from Lonza Group Ltd. PAE cells stably

expressing wt-EGFR and Y1045F-EGFRwere grown in Ham’s F-12medium

(Lonza Group Ltd) with 0.5� penicillin–streptomycin mixture and 400 mg/mL

G418 sulfate (PAA Laboratories). Ten percent (v/v) FBS was routinely used

for all cells. The cells were plated at a density of 15 000 cells/cm2 48 h prior

to experiments. EGF was added to cells in MEM (Invitrogen) without

bicarbonate and with 0.1% BSA.

PlasmidsXpress-tagged full-length rat epsin 1 and the FLAG-tagged ENTH-UIM epsin 1

deletionmutant were kind gifts from Pietro De Camilli (Yale University School

of Medicine, New Haven, CT, USA) (see Figure 1 for an overview over the

epsin structure). Wt epsin 1 was cloned into a pcDNA3-Myc vector, which

was a gift from Harald Stenmark (Rikshospitalet HF). The Myc-tagged ENTH-

UIM part of epsin 1was obtained by inserting a stop codon after the third UIM

(aa 252) of the wt epsin 1 by site-directed mutagenesis. The Xpress-tagged

full-length epsin 1 UIM mutant was obtained using QuikChange XL Site-

Directed Mutagenesis Kit (Stratagene). Two point mutations (D234A D235A)

in the third UIMweremade, while the secondUIM (aa 202–226)was deleted.

This UIM mutant was then cloned into a pcDNA3-Myc vector. GFP-tagged

hSpry2 was obtained from Graeme Guy (Institute of Molecular and Cell

Biology, Singapore). hSpry2 was then cloned into the pRK5-Myc plasmid

(kindly provided by Alan Hall, University College, London, UK).

AntibodiesMouse anti-a-adaptin, mouse anti-EGFR (sc-120), rabbit anti-Erk, mouse

anti-ubiquitin and rabbit anti-epsin 1 were from Santa Cruz Biotechnology,

Inc. Mouse anti-EGFR (Ab-3) was from Neomarkers, and sheep anti-EGFR

antibody used for western blotting was from Fitzgerald. Rabbit anti-FLAG

antibody was from Sigma. Mouse anti-clathrin heavy chain (ab2731), rabbit

anti-tubulin and rabbit anti-Myc antibodies were from Abcam. Mouse anti-

Myc antibody was a gift from Harald Stenmark. Alexa 488-conjugated goat

anti-mouse immunoglobulin G (IgG) was from Invitrogen. Rhodamine Red-

X-conjugated donkey anti-rabbit IgG and peroxidase-conjugated donkey

anti-goat IgG, donkey anti-mouse IgG and donkey anti-rabbit IgG antibodies

were from Jackson ImmunoResearch Laboratories. The rabbit anti-epsin

antibody used for immuno-EM has previously been described (51).

Internalization of 125I-EGF and 125I-TfInternalization of 125I-EGF and 125I-Tf was performed as previously

described (5).

Western blottingCells were treated as described in legends to figures 2, 3, 4, 5, S1 and S3

before being analyzed by western blotting as described (52). Chemilumi-

nescence detection kit from Pierce was used for protein visualization, and

the signal was detected and quantified using a Kodak Image Station 400R or

by autoradiography.

ImmunoprecipitationFor coimmunoprecipitation analysis and analysis of EGFR ubiquitination

(immunoprecipitation under denaturating conditions), cells were treated as

described in legends to figures 2 and 3 before proteins were immunopre-

cipitated as described (53).

siRNA-mediated knockdown of epsin 1HeLa cells and PAE cells were transfected twice with a 48-h interval

using siRNA directed against epsin 1. Target sequences used were GGAAGA

CGCCGGAGTCATT (Dup1) andGGACCTTGCTGACGTCTTC (Dup3) (4). siRNAs

usedwere synthesized and annealed by Invitrogen. 37.5 mL of 20 mM siRNA

duplex and 11.25 mL of Lipofectamine 2000 reagent were used for trans-

fection of 1 � 106 cells. Control cells were incubated with Lipofectamine

2000 only. As an additional control, HeLa cells were transfected with an

siRNA targeting GFP (54). Cells were immediately plated in mediumwithout

antibiotics and treated as indicated in legends to figures 4, 5, 6, S2 and S3.

Immuno-EMCells treated as described in legends to figures 6 and 7 and to table 2 were

prepared for immuno-EM as previously described (36). Immunocytochemical

labeling of thawed cryosections was performed essentially as described by

Griffiths et al. (55) using protein A gold (purchased fromG. Posthuma, Utrecht,

theNetherlands). Sectionswere examined using a Philips Tecnai 12 or a Philips

CM120 transmissionelectronmicroscope, bothequippedwith aMegaView III;

Soft Imaging System TEM camera (Olympus Soft Imaging Solutions GmbH).

Table 2: Overexpression of the ENTH-UIM part of epsin 1 inhibits

recruitment of the activated EGFR into clathrin-coated pitsa

% EGFR in coated pits

EGF Inside Rim Number of Au

Control cells � 1.7 � 0.5 0 300

Control cells þ 11.9 � 1.5 1.7 � 1.2 304

ENTH-UIM � 0 0 303

ENTH-UIM þ 0.9 � 0.7 0.9 � 0.7 341

aBy immuno-EM, the plasma membrane distribution of the EGFR

was quantified when nontransfected HeLa cells (control), and HeLa

cells overexpressing theMyc-tagged ENTH-UIM part of epsin 1 had

been incubated with or without EGF (60 ng/mL) for 1 h on ice.

Transfected cells were identified using rabbit anti-Myc antibody.

The EGFR localizing to the interior or to the rim of coated pits are

presented as the percentage of the total labeling for EGFR found at

the plasma membrane. The quantification represents the mean �SD of three parallel labeling experiments. Number of Au indicates

the total number of gold particles counted for each condition.

242 Traffic 2009; 10: 235–245

Kazazic et al.

To estimate the plasma membrane distribution of the EGFR, no less than 100

gold particles at the plasma membrane on randomly chosen HeLa cells or the

total number of gold particles (a minimum of 250 particles) on 20 profiles of

PAE cells were counted. As the anti-EGFR antibodies used for immuno-EM

(sc-120 and Ab-3) are directed against the extracellular part of the EGFR, the

labeling is not affected by interactions of the EGFR with intracellular proteins.

Labeling efficiency can therefore be ignored when only quantifying changes in

labeling distribution.

Acknowledgments

We thank Pietro De Camilli, Harald Stenmark, Graeme Guy and Alan Hall for

generously providing reagents. This study was supported by The Research

Council of Norway, including the functional genomics programme (FUGE),

The Norwegian Cancer Society, South-Eastern Norway Regional Health

Authority, Medinnova, NOVO Nordic Foundation, Anders Jahre’s Founda-

tion for the Promotion of Sciences, Torsted’s Legacy, Blix Legacy, Odd

Fellow’s Legacy and Bruun’s Legacy.

Supporting Information

Additional Supporting Information may be found in the online version of

this article:

Figure S1: EGFR is ubiquitinated both at low and at high concen-

trations of EGF.HeLa cells incubatedwith orwithout EGF (1 or 60 ng/mL, as

indicated) for 3 min at 378C were subjected to immunoprecipitation under

denaturating conditions as described in Materials and Methods using an

EGFR antibody and subsequently western blotting with antibodies to EGFR

and ubiquitin. Proteins were detected using enhanced chemiluminescence.

Figure S2: Downregulation of epsin 1 affects internalization of EGF at

high concentrations. HeLa cells transfected with or without siRNA to

epsin 1 (Dup1 or Dup3) were incubated with 60 ng/mL EGF for the times

indicated. 125I-EGF was diluted 1:60 with nonlabeled EGF. The internaliza-

tion rate was measured as described in Materials and Methods.

The experiment was carried out in parallel with the experiment in Figure 4,

where also the downregulation of epsin 1 is demonstrated (Figure 4A).

Figure S3: Epsin 1 is downregulated efficiently in HeLa cells by

transfection twice with siRNA. Cells were lysed either after one trans-

fection with siRNA to espin 1 (Dup1 or Dup3) (2 days after initial trans-

fection) or after two transfections with siRNA with a 48-h interval (4 days

after initial transfection). Cells lysates were then subjected to western

blotting analysis using antibodies to epsin 1 and Erk. Proteins were

detected using enhanced chemiluminescence.

Figure S4: Effect of overexpression of wt epsin on endocytosis of EGF

and Tf. A) Upon transfecting HeLa cells with an empty vector (control), or

with a plasmid encoding wt epsin 1, the cells were incubated with 1 ng/mL125I-EGF at 378C for the times indicated, and the rate of EGF internalization

was measured as described in Materials and Methods. The data represent

one independent experiment (of three) with four parallels �SD. B) HeLa

cells transfected as in (A) were incubated with 125I-Tf (70 ng/mL) at 378C for

5 min, and the internalized 125I-Tf was plotted as percentage of total cell-

associated 125I-Tf. The data represent one independent experiment (of

three) with four parallels �SD. C) HeLa cells were grown on coverslips and

transfected with the plasmid encoding Myc-tagged wt epsin 1. The cells

were then fixed and processed for fluorescence microscopy and examined

using a Leica TCS XP confocal microscope (Leica Microsystems AG). Cells

were labeled with antibody to either a-adaptin or clathrin heavy chain.

Additionally, the cells were labeled with rabbit anti-Myc antibody recogni-

zing tagged epsin 1. Bar, 10 mm.

Please note: Wiley-Blackwell are not responsible for the content or

functionality of any supporting materials supplied by the authors. Any

queries (other than missing material) should be directed to the correspond-

ing author for the article.

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