Endocytic

downregulation

of

ErbB

receptors

mechanisms

and

relevance

in

cancer

ErbB

receptors

-LRB-

EGFR

-LRB-

-RRB-

and

-RRB-

are

important

regulators

of

normal

growth

and

differentiation

and

they

are

involved

in

the

pathogenesis

of

cancer

Following

ligand

binding

and

receptor

activation

EGFR

is

endocytosed

and

transported

to

lysosomes

where

the

receptor

is

degraded

This

downregulation

of

EGFR

is

a

complex

and

tightly

regulated

process

The

functions

of

and

are

also

regulated

by

endocytosis

to

some

extent

although

the

current

knowledge

of

these

processes

is

sparse

Impaired

endocytic

downregulation

of

signaling

receptors

is

frequently

associated

with

cancer

since

it

can

lead

to

increased

and

uncontrolled

receptor

signaling

In

this

review

we

describe

the

current

knowledge

of

ErbB

receptor

endocytic

downregulation

In

addition

we

outline

how

ErbB

receptors

can

escape

endocytic

downregulation

in

cancer

and

we

discuss

how

targeted

anti-cancer

therapy

may

induce

endocytic

downregulation

of

ErbB

receptors

Introduction

Proper

endocytic

uptake

and

endosomal

sorting

of

signaling

receptors

are

crucial

mechanisms

for

the

regulation

of

signaling

activity

involved

in

cellular

growth

development

and

differentiation

-LRB-

Crosetto

et

al.

Fischer

et

al.

Giebel

and

Wodarz

Le

Borgne

Polo

and

Di

Fiore

Sorkin

and

von

Zastrow

-RRB-

This

is

not

least

true

for

the

epidermal

growth

factor

receptor

-LRB-

EGFR

-RRB-

a

member

of

the

ErbB

family

of

receptor

tyrosine

kinases

which

contains

four

members

EGFR

-LRB-

-RRB-

and

In

addition

to

playing

an

important

role

in

normal

cellular

functions

these

receptors

are

involved

in

the

pathogenesis

of

human

cancers

and

receive

much

attention

as

targets

for

development

of

new

anti-cancer

drugs

-LRB-

Hynes

and

Lane

-RRB-

There

are

several

mechanisms

whereby

cancer

cells

can

obtain

uncontrolled

ErbB

receptor

signaling

including

increased

receptor

expression

activating

mutations

and

escape

of

endocytic

receptor

downregulation

-LRB-

Bache

et

al.

Citri

and

Yarden

Normanno

et

al.

Polo

et

al.

Warren

and

Landgraf

-RRB-

Our

current

understanding

of

endocytic

downregulation

of

ErbB

receptors

is

largely

based

on

knowledge

of

EGFR

behavior

upon

binding

of

epidermal

growth

factor

-LRB-

EGF

-RRB-

-LRB-

Bache

et

al.

Dikic

Gruenberg

and

Stenmark

-RRB-

EGFR

is

among

the

most

well

studied

receptors

and

it

is

regarded

as

a

prototype

of

a

signaling

receptor

that

is

internalized

and

degraded

following

ligand

binding

In

contrast

surprisingly

little

is

known

about

endocytosis

of

as

well

as

about

EGFR

endocytosis

following

binding

of

ligands

other

than

EGF

However

it

is

becoming

increasingly

clear

that

the

other

ErbB

receptors

do

not

behave

like

EGFR

regarding

endocytosis

In

this

review

we

outline

the

molecular

mechanisms

of

EGF-induced

endocytic

downregulation

of

EGFR

In

addition

we

describe

the

current

knowledge

of

internalization

and

downregulation

of

the

other

ErbB

receptors

and

discuss

how

the

various

ErbB

ligands

differ

in

their

potential

to

induce

receptor

downregulation

Finally

we

focus

on

internalization

of

these

receptors

as

a

promising

target

in

anti-cancer

therapy

The

ErbB

receptor

system

A

thorough

description

of

the

structure

and

biological

functions

of

the

four

ErbB

receptors

and

their

ligands

is

beyond

the

scope

of

this

review

and

can

be

found

elsewhere

-LRB-

Citri

and

Yarden

Hynes

and

Lane

Jones

et

al.

-RRB-

In

brief

the

ErbB

receptors

are

transmembrane

receptor

tyrosine

kinases

consisting

of

a

glycosylated

extracellular

N-terminal

part

containing

a

ligand

binding

site

and

a

dimerization

arm

a

transmembrane

segment

and

an

intracellular

part

containing

a

tyrosine

kinase

domain

and

a

C-terminal

tail

with

several

phosphorylation

sites

-LRB-

Fig.

-RRB-

-LRB-

Normanno

et

al.

-RRB-

A

large

number

of

ErbB

ligands

have

been

identified

some

of

which

can

bind

to

more

than

one

of

the

ErbB

receptors

with

high

affinity

-LRB-

Table

-RRB-

-LRB-

Breuleux

Harris

et

al.

-RRB-

The

active

ligand-bound

ErbB

receptors

function

as

homo

or

heterodimers

which

can

activate

a

multitude

of

signaling

pathways

involved

in

proliferation

differentiation

cell

survival

and

migration

-LRB-

Normanno

et

al.

-RRB-

Fig.

of

EGFR

In

the

extracellular

part

of

the

receptor

EGFR

harbours

two

domains

-LRB-

and

-RRB-

that

upon

folding

form

the

ligand-binding

pocket

Between

and

is

another

domain

-LRB-

-RRB-

that

includes

the

dimerization

arm

Intracellularly

EGFR

has

a

kinase

domain

and

a

C-terminal

tail

with

several

amino

acid

residues

that

can

be

phosphorylated

The

tyrosine

residues

-LRB-

Y

-RRB-

that

are

involved

in

Cbl

binding

are

shown

The

parts

of

wild-type

EGFR

that

are

deleted

in

EGFRvIII

EGFRvIV

and

EGFRvV

are

indicatedTable

receptors

and

their

ligandsReceptorRemarksLigandsEGFR

-LRB-

-RRB-

Epidermal

growth

factor

-LRB-

EGF

-RRB-

Transforming

growth

factor

α

-LRB-

TGFα

-RRB-

Heparin-binding

EGF

-LRB-

HB-EGF

-RRB-

β-CellulinAmphiregulinEpiregulinEpigenErbB2Constitutively

exposed

dimerization

EGF

-LRB-

HB-EGF

-RRB-

β-CellulinEpiregulin

ErbB

receptor

activation

The

initial

steps

of

ligand-mediated

ErbB

receptor

activation

have

been

well

described

upon

solving

the

crystal

structure

of

the

EGFR

extracellular

domain

bound

to

EGF

-LRB-

Garrett

et

al.

Ogiso

et

al.

-RRB-

EGF

binding

to

EGFR

has

been

proposed

to

induce

a

conformational

change

exposing

the

dimerization

arm

in

the

receptor

extracellular

domain

-LRB-

Burgess

et

al.

Ferguson

et

al.

Ogiso

et

al.

-RRB-

Upon

dimerization

the

receptor

's

intrinsic

kinase

activity

cross-phosphorylates

specific

residues

in

the

C-terminal

tail

of

the

partnering

receptor

allowing

for

the

recruitment

of

phosphotyrosine-binding

proteins

to

EGFR

and

dimerize

in

response

to

ligand

binding

in

a

manner

similar

to

EGFR

whereas

differs

from

the

other

ErbB

receptors

by

not

binding

to

any

ligand

with

high

affinity

-LRB-

Bouyain

et

al.

Burgess

et

al.

Kani

et

al.

-RRB-

Instead

is

in

a

constitutively

active

conformation

with

an

exposed

dimerization

arm

even

in

the

absence

of

ligand

-LRB-

Burgess

et

al.

-RRB-

Its

ability

to

homodimerize

is

limited

due

to

the

electronegativity

of

its

extracellular

part

but

is

the

preferred

heterodimerization

partner

for

the

other

ErbB

receptors

-LRB-

Garrett

et

al.

Graus-Porta

et

al.

Tzahar

et

al.

-RRB-

The

signal

transduction

pathways

activated

by

ErbB

receptors

include

well-known

signaling

cascades

such

as

the

pathway

the

phospholipase

Cγ-protein

kinase

C

pathway

the

phosphatidyl

inositol

-LRB-

PI

-RRB-

kinase-Akt

pathway

and

STAT

signaling

Endocytic

downregulation

of

signaling

receptors

has

for

many

years

been

regarded

solely

as

a

means

of

attenuating

receptor

signaling

However

during

the

last

decade

an

increasing

amount

of

evidence

has

suggested

that

some

receptors

including

EGFR

may

continue

their

signaling

activity

from

endocytic

compartments

and

that

the

signaling

occurring

from

here

is

qualitatively

different

from

the

signaling

taking

place

at

the

plasma

membrane

-LRB-

Miaczynska

et

al.

-RRB-

Thus

ErbB

receptor

endocytosis

is

not

merely

a

means

of

turning

off

activated

receptors

it

may

also

be

a

regulatory

mechanism

altering

the

signaling

outcome

Endocytic

downregulation

of

EGFR

Regarding

ligand-induced

endocytic

receptor

downregulation

EGFR

has

been

the

most

popular

model

system

for

many

years

and

the

mechanisms

of

EGFR

endocytosis

and

intracellular

trafficking

are

therefore

relatively

well

understood

EGFR

is

activated

upon

EGF

binding

leading

to

signaling

and

relocation

to

invaginating

clathrin-coated

pits

-LRB-

CCPs

-RRB-

on

the

plasma

membrane

These

pits

give

rise

to

clathrin-coated

endocytic

vesicles

and

after

the

coat

has

been

released

from

the

membrane

the

vesicles

fuse

with

early

endosomes

and

thereby

deliver

the

receptor

to

this

compartment

Here

the

receptor

is

sorted

for

further

transport

either

back

to

the

cell

surface

by

recycling

or

to

intraluminal

vesicles

-LRB-

ILVs

-RRB-

a

pathway

that

eventually

leads

to

delivery

of

EGFR

to

lysosomes

for

degradation

-LRB-

Fig.

-RRB-

Some

of

these

steps

are

now

well

established

but

others

remain

elusive

Below

we

describe

both

what

is

known

and

what

challenges

we

are

facing

in

understanding

the

crucial

pathway

of

clathrin-mediated

EGFR

endocytosis

and

endosomal

sorting

Fig.

and

endosomal

sorting

of

EGFR

Main

figure

upon

activation

EGFR

-LRB-

green

-RRB-

is

translocated

to

clathrin

coated

pits

-LRB-

CCP

-RRB-

on

the

plasma

membrane

and

internalized

-LRB-

grey

arrows

-RRB-

After

transport

to

early

endosomes

-LRB-

EE

-RRB-

EGFR

is

either

recycled

back

to

the

plasma

membrane

-LRB-

white

arrows

-RRB-

or

taken

up

into

intraluminal

vesicles

-LRB-

ILVs

-RRB-

EE

will

mature

to

late

endosomes

-LRB-

LE

-RRB-

and

EGFR

in

ILVs

will

eventually

be

degraded

in

lysosomes

-LRB-

Lys

-RRB-

-LRB-

black

arrows

-RRB-

Upper

insert

at

the

plasma

membrane

EGF-activated

EGFR

dimerize

and

the

kinase

activity

of

the

receptors

phosphorylates

tyrosine

residues

-LRB-

P

-RRB-

in

EGFR

This

creates

docking

sites

for

intracellular

proteins

such

as

mediates

binding

of

the

ubiquitin

ligase

Cbl

that

adds

mono

or

polyubiquitins

-LRB-

Ub

-RRB-

to

EGFR

Activated

EGFR

is

transported

to

clathrin

coated

pits

that

in

addition

to

clathrin

also

consists

of

and

other

proteins

Lower

insert

At

the

EE

vacuolar

membrane

EGFR

destined

for

degradation

still

binds

Cbl

and

is

continuously

phosphorylated

and

ubiquitinated

The

EGFR

ubiquitins

are

bound

by

Hrs

that

resides

in

at

Hrs/STAM/clathrin

coat

and

this

is

followed

by

binding

of

ESCRT

complexes

to

the

ubiquitinated

EGFR

leading

to

uptake

into

ILVs

Sorting

of

EGFR

to

clathrin

coated

pits

ubiquitination

or

not

One

of

the

first

steps

in

clathrin-mediated

endocytic

downregulation

is

the

transport

of

cargo

into

CCPs

Despite

the

importance

of

regulating

whether

EGFR

is

endocytosed

the

molecular

machinery

controlling

this

is

poorly

understood

The

constitutively

endocytosed

receptors

the

transferrin

receptor

and

the

low-density

lipoprotein

receptor

are

sorted

into

CCPs

by

interaction

with

the

clathrin-binding

complex

through

conserved

tyrosine-based

motifs

in

the

receptor

's

intracellular

tail

EGFR

also

interacts

with

indicating

a

similar

role

of

in

EGFR

endocytosis

-LRB-

Sorkin

et

al.

Sorkin

and

Carpenter

-RRB-

However

EGFR

is

not

sorted

into

CCPs

in

its

resting

state

and

the

direct

interaction

of

with

EGFR

does

not

seem

to

be

necessary

for

EGFR

internalization

-LRB-

Nesterov

et

al.

-RRB-

Thus

additional

interaction

partners

in

the

CCPs

are

required

for

EGFR

to

be

sorted

into

these

plasma

membrane

microdomains

Since

the

kinase

activity

of

EGFR

has

been

shown

to

be

important

for

this

sorting

-LRB-

Lamaze

and

Schmid

-RRB-

such

interaction

partners

should

bind

modifications

of

EGFR

that

are

only

present

in

the

activated

state

e.g.

phosphorylated

residues

and/or

ubiquitinated

residues

Ubiquitination

-LRB-

Text

box

-RRB-

has

been

known

to

mediate

endocytosis

of

membrane

receptors

for

vacuolar

degradation

in

yeast

-LRB-

Hicke

and

Riezman

Kolling

and

Hollenberg

-RRB-

and

ubiquitination

has

also

been

found

to

be

involved

in

endocytic

downregulation

of

mammalian

receptors

such

as

EGFR

-LRB-

reviewed

in

-LRB-

Hicke

-RRB-

-RRB-

As

a

result

ubiquitination

has

long

been

considered

a

likely

candidate

as

a

CCP

targeting

signal

at

the

plasma

membrane.Text

box

Upon

activation

of

EGFR

it

is

heavily

ubiquitinated

by

the

attachment

of

both

monoubiquitin

and

polyubiquitin

-LRB-

Text

box

-RRB-

As

will

be

discussed

below

this

ubiquitination

is

a

prerequisite

for

endosomal

sorting

of

EGFR

to

ILVs

in

multivesicular

bodies

-LRB-

MVBs

-RRB-

but

its

importance

in

the

initial

sorting

to

CCPs

and

internalization

from

the

plasma

membrane

has

been

the

subject

of

controversy

for

some

time

The

ubiquitin

ligase

responsible

for

EGFR

ubiquitination

is

Cbl

a

ring-finger

domain

ubiquitin

ligase

-LRB-

Levkowitz

et

al.

-RRB-

Cbl

is

indeed

necessary

for

EGFR

endocytosis

-LRB-

Jiang

and

Sorkin

-RRB-

Cbl

can

bind

EGFR

either

directly

by

binding

to

phosphorylated

or

indirectly

via

the

adaptor

protein

which

binds

to

phosphorylated

and

-LRB-

Fig.

-RRB-

-LRB-

Levkowitz

et

al.

Waterman

et

al.

-RRB-

Binding

of

Cbl

via

phosphorylated

is

not

necessary

for

EGFR

endocytosis

since

the

EGFR

mutant

that

can

not

bind

Cbl

directly

is

internalized

almost

as

efficiently

as

wild-type

EGFR

-LRB-

Grøvdal

et

al.

Jiang

et

al.

-RRB-

In

contrast

Cbl

binding

to

EGFR

via

is

necessary

for

receptor

internalization

-LRB-

Huang

and

Sorkin

-RRB-

Thus

knockdown

inhibits

EGFR

endocytosis

but

a

chimeric

protein

consisting

of

the

domain

of

fused

to

Cbl

can

rescue

EGFR

internalization

in

depleted

cells

showing

that

the

prime

role

of

in

EGFR

internalization

is

recruitment

of

Cbl

-LRB-

Huang

and

Sorkin

-RRB-

Since

functional

Cbl

is

a

prerequisite

for

EGFR

internalization

-LRB-

Jiang

and

Sorkin

-RRB-

and

Cbl

ubiquitinates

EGFR

-LRB-

Levkowitz

et

al.

-RRB-

it

has

long

been

assumed

that

Cbl-mediated

ubiquitination

of

EGFR

is

the

prime

signal

for

EGFR

translocation

to

CCPs

In

accordance

with

this

overexpression

of

the

Cbl

mutant

with

impaired

ubiquitin

ligase

activity

inhibits

EGFR

internalization

-LRB-

Jiang

and

Sorkin

-RRB-

To

further

investigate

the

role

of

EGFR

ubiquitination

in

internalization

an

EGFR

mutated

on

lysine

residues

was

recently

constructed

This

mutant

had

an

ubiquitination

level

corresponding

to

of

that

seen

for

wild-type

EGFR

after

EGF

stimulation

Interestingly

the

mutated

EGFR

still

retained

full

kinase

activity

and

displayed

normal

internalization

-LRB-

Huang

et

al.

-RRB-

This

could

imply

that

EGFR

ubiquitination

is

not

a

signal

for

internalization

Alternatively

the

endocytic

machinery

recognizing

ubiquitinated

EGFR

is

highly

sensitive

so

that

even

a

residual

ubiquitination

is

sufficient

for

proper

targeting

of

EGFR

to

CCPs

Importantly

Cbl

may

serve

other

functions

in

addition

to

ubiquitin

ligase

activity

It

has

been

reported

that

Cbl

binds

endophilin

via

-LRB-

Soubeyran

et

al.

-RRB-

Endophilin

is

a

known

regulator

of

clathrin-mediated

endocytosis

-LRB-

Reutens

and

Begley

-RRB-

and

this

recruitment

of

and

endophilin

to

EGFR

by

Cbl

has

been

shown

to

be

important

for

EGFR

internalization

-LRB-

Soubeyran

et

al.

-RRB-

Although

the

role

of

EGFR

ubiquitination

as

an

internalization

signal

is

controversial

several

candidates

including

have

been

suggested

as

adaptors

for

sorting

ubiquitinated

EGFR

into

CCPs

is

found

in

CCPs

and

has

an

ubiquitin-binding

domain

-LRB-

Hawryluk

et

al.

Stang

et

al.

-RRB-

Furthermore

is

recruited

to

the

plasma

membrane

upon

EGF

stimulation

of

EGFR

-LRB-

Stang

et

al.

Torrisi

et

al.

-RRB-

and

expression

of

dominant-negative

as

well

as

siRNA-mediated

knockdown

of

has

been

shown

to

inhibit

EGFR

endocytosis

-LRB-

Bakowska

et

al.

Benmerah

et

al.

Fallon

et

al.

-RRB-

Phosphorylation

of

has

also

been

shown

to

be

necessary

for

EGFR

internalization

-LRB-

Confalonieri

et

al.

-RRB-

However

a

direct

evidence

of

as

an

adaptor

protein

recognizing

ubiquitinated

EGFR

via

interactions

with

EGFR-attached

ubiquitin

has

so

far

not

been

presented

At

present

it

remains

obscure

which

mechanisms

are

responsible

for

sorting

of

the

activated

EGFR

to

coated

pits

although

it

is

clear

that

Cbl

and

are

central

and

may

have

important

functions

One

possible

explanation

for

the

apparent

contradictory

results

regarding

ubiquitin

as

an

endocytic

signal

for

EGFR

is

that

it

is

not

ubiquitination

of

EGFR

itself

but

rather

ubiquitination

of

accessory

proteins

by

Cbl

that

is

necessary

for

EGFR

internalization

Endosomal

sorting

of

EGFR

a

key

role

of

ubiquitination

Upon

internalization

by

clathrin-mediated

endocytosis

EGFR

is

transported

to

early

endosomes

-LRB-

Fig.

-RRB-

Both

cargo

destined

for

lysosomal

degradation

and

for

recycling

back

to

the

plasma

membrane

is

delivered

here

and

the

compartment

is

sometimes

seen

in

the

electron

microscope

as

a

vacuole

with

tubular

elongations

The

tubular

elongated

parts

of

the

early

endosomes

are

believed

to

be

involved

in

recycling

whereas

sorting

for

degradation

initiates

at

the

vacuolar

membrane

of

the

endosomes

by

inward

budding

giving

rise

to

ILVs

containing

membrane

proteins

destined

for

lysosomal

degradation

-LRB-

Fig.

-RRB-

The

internalized

EGFR

can

be

sorted

both

for

recycling

and

for

lysosomal

degradation

Whereas

recycling

seems

to

be

the

default

pathway

from

early

endosomes

sorting

for

lysosomal

degradation

is

mediated

by

Hrs/STAM

and

by

the

endosomal

sorting

complex

required

for

transport

-LRB-

ESCRT

-RRB-

complexes

-LRB-

ESCRT-I

to

III

-RRB-

that

are

believed

to

mediate

receptor

translocation

into

ILVs

of

MVBs

-LRB-

for

recent

review

see

-LRB-

Williams

and

Urbe

-RRB-

-RRB-

The

EGF-activated

EGFR

is

also

associated

with

Cbl

in

early

endosomes

resulting

in

continued

ubiquitination

of

EGFR

-LRB-

Duan

et

al.

Longva

et

al.

-RRB-

Contrary

to

its

uncertain

role

in

receptor

internalization

-LRB-

see

above

-RRB-

ubiquitination

has

been

convincingly

demonstrated

to

be

important

for

endosomal

sorting

of

EGFR

to

lysosomes

EGFR

mutated

at

intracellular

lysine

residues

to

give

a

receptor

with

decreased

ubiquitination

is

severely

degradation

impaired

following

EGF

stimulation

-LRB-

Huang

-RRB-

Likewise

the

EGFR

mutant

that

does

not

bind

directly

to

Cbl

and

displays

reduced

ubiquitination

is

degradation

impaired

-LRB-

Grøvdal

et

al.

Jiang

and

Sorkin

Levkowitz

et

al.

-RRB-

This

mutant

does

not

translocate

to

ILVs

-LRB-

Grøvdal

et

al.

-RRB-

giving

further

support

to

a

model

where

ubiquitin

is

the

signal

for

EGFR

sorting

for

degradation

Ubiquitin

chains

are

recognized

by

ESCRT

complexes

which

retain

ubiquitinated

cargo

in

the

membrane

of

early

endosomes

thereby

hindering

receptor

recycling

-LRB-

Fig.

-RRB-

-LRB-

Raiborg

and

Stenmark

-RRB-

The

first

described

point

of

recognition

is

Hrs

and

STAM

also

known

as

the

complex

On

early

endosomes

Hrs

and

clathrin

form

dynamic

microdomains

which

can

be

seen

by

EM

as

flat

electron-dense

areas

on

the

limiting

membrane

-LRB-

Raiborg

et

al.

Sachse

et

al.

-RRB-

Hrs

and

STAM

interact

with

ubiquitinated

cargo

via

their

ubiquitin-interacting

motif

-LRB-

UIM

-RRB-

domains

The

proteins

preferentially

bind

polyubiquitin

-LRB-

Polo

et

al.

-RRB-

and

the

UIM

of

Hrs

has

recently

been

shown

to

bind

two

ubiquitin

molecules

simultaneously

-LRB-

Hirano

et

al.

-RRB-

Conceivably

cargo

must

be

either

polyubiquitinated

by

polyubiquitin

chains

or

multiply

monoubiquitinated

for

efficient

sorting

towards

the

lysosome

for

degradation

-LRB-

Barriere

et

al.

-RRB-

This

is

in

concordance

with

more

than

of

EGFR

ubiquitination

being

in

the

form

of

poly-ubiquitin

chains

primarily

via

-LRB-

Text

box

-RRB-

-LRB-

Huang

et

al.

-RRB-

After

Hrs/STAM

has

retained

ubiquitinated

cargo

within

microdomains

in

the

vacuolar

membrane

of

the

early

endosome

ESCRT

complexes

-LRB-

ESCRT-I

ESCRT-III

-RRB-

are

sequentially

recruited

eventually

leading

to

sorting

of

EGFR

into

ILVs

-LRB-

reviewed

in

-LRB-

Williams

and

Urbe

-RRB-

-RRB-

Recently

siRNA

studies

showed

that

knock-down

of

Hrs

or

ESCRT-I

components

caused

increased

recycling

of

EGFR

In

contrast

knockdown

of

ESCRT-II

and

ESCRT-III

components

did

not

result

in

increased

recycling

of

EGFR

but

did

impair

degradation

-LRB-

Raiborg

et

al.

-RRB-

This

indicates

that

the

fate

of

EGFR

-LRB-

recycling

versus

degradation

-RRB-

is

determined

upstream

of

ESCRT-II

ESCRT-II

appears

to

be

necessary

for

sorting

of

EGFR

to

ILVs

In

contrast

the

exact

function

of

the

ESCRT-III

complex

in

EGFR

sorting

seems

to

be

more

complex

The

original

model

was

that

ESCRT-III

is

important

for

formation

of

ILVs

-LRB-

Williams

and

Urbe

-RRB-

However

it

was

recently

reported

that

upon

depletion

of

the

ESCRT-III

component

EGFR

is

still

sorted

into

ILVs

in

MVBs

and

silenced

-LRB-

Bache

et

al.

-RRB-

Thus

the

ESCRT-III

complex

might

function

downstream

of

internalization

to

ILVs

perhaps

as

late

as

in

MVB

fusion

with

lysosomes

-LRB-

Bache

et

al.

-RRB-

Differential

effects

of

EGFR

ligands

on

EGFR

degradation

The

model

for

EGFR

sorting

to

lysosomes

described

above

has

been

developed

based

on

studies

of

EGF-stimulated

cells

Six

other

ligands

for

EGFR

have

been

described

-LRB-

Table

-RRB-

all

of

which

play

important

roles

both

during

embryogenesis

and

in

adult

life

In

addition

EGFR

ligands

have

been

found

in

high

concentrations

in

human

cancers

-LRB-

Normanno

et

al.

Normanno

et

al.

Revillion

et

al.

-RRB-

It

is

therefore

important

to

establish

whether

the

additional

ligands

have

the

same

effects

on

EGFR

downregulation

as

EGF

The

only

other

EGFR

ligand

that

has

been

well

investigated

with

respect

to

EGFR

trafficking

is

transforming

growth

factor

α

-LRB-

TGFα

-RRB-

Whereas

EGF

targets

EGFR

to

the

degradative

pathway

TGFα

allows

receptor

recycling

-LRB-

Decker

-RRB-

The

differential

intracellular

sorting

of

EGFR

after

stimulation

with

TGFα

compared

to

EGF

is

caused

by

differences

in

the

pH-dependence

of

receptor-ligand

binding

Whereas

EGF

binding

to

EGFR

is

relatively

stable

at

the

lower

pH

in

endosomes

TGFα

dissociates

from

the

receptor

at

endosomal

pH

-LRB-

Ebner

and

Derynck

-RRB-

It

is

believed

that

continuous

ligand

binding

is

necessary

for

continuous

ubiquitination

and

thereby

for

translocation

of

EGFR

to

the

ILVs

In

case

of

TGFα

the

ligand

rapidly

dissociates

from

the

receptor

in

endosomes

leading

to

receptor

dephosphorylation

de-ubiquitination

and

recycling

to

the

cell

surface

-LRB-

Longva

et

al.

-RRB-

Interestingly

this

difference

in

their

potential

to

induce

EGFR

degradation

is

thought

to

be

the

main

reason

why

TGFα

is

a

stronger

mitogen

than

EGF

-LRB-

Waterman

et

al.

-RRB-

At

present

the

effects

of

heparin-binding

EGF

-LRB-

HB-EGF

-RRB-

β-cellulin

Epiregulin

and

Epigen

on

EGFR

trafficking

has

not

been

investigated

A

single

study

has

investigated

the

effect

of

Amphiregulin

on

endocytic

EGFR

downregulation

and

found

that

Amphiregulin

failed

to

induce

EGFR

degradation

This

was

the

case

even

in

cells

overexpressing

Cbl

although

in

these

cells

EGFR

was

highly

ubiquitinated

following

Amphiregulin

stimulation

-LRB-

Stern

et

al.

-RRB-

Interestingly

this

suggests

that

pronounced

receptor

ubiquitination

is

not

sufficient

to

target

receptors

for

lysosomal

degradation

More

studies

are

warranted

to

uncover

how

the

remaining

EGFR

ligands

affect

receptor

ubiquitination

endocytosis

and

lysosomal

degradation

Clathrin-independent

endocytic

mechanisms

involved

in

EGFR

uptake

Although

clathrin-dependent

endocytosis

is

generally

considered

to

be

the

major

mechanism

for

internalization

and

downregulation

of

EGFR

other

endocytic

pathways

have

also

been

proposed

during

recent

years

especially

following

stimulation

with

high

concentrations

of

EGF

-LRB-

Sigismund

et

al.

-RRB-

The

concentration

of

EGF

varies

greatly

throughout

the

human

body

Thus

while

the

EGF

concentration

in

tissue

fluid

is

about

ng/ml

it

is

much

higher

up

to

ng/ml

or

more

in

tubular

duct

lumens

of

e.g.

the

kidney

salivary

glands

and

the

mammary

gland

-LRB-

Carpenter

and

Cohen

Hayashi

and

Sakamoto

-RRB-

Normally

EGFR

is

not

reached

by

the

high

luminal

concentrations

of

EGF

in

these

systems

since

the

receptor

is

present

at

the

basolateral

site

of

the

epithelial

cells

However

during

wound

healing

or

malignant

transformation

the

tight

junctions

disappear

and

allow

the

high

concentrations

of

EGF

to

get

access

to

the

receptor

-LRB-

Mullin

-RRB-

Very

high

EGF

concentration

can

also

be

found

in

solid

tumors

-LRB-

Salomon

et

al.

-RRB-

It

is

therefore

relevant

to

study

whether

the

mechanisms

of

endocytic

downregulation

of

EGFR

are

affected

by

the

concentration

of

stimulating

ligand

Sigismund

et

al.

-LRB-

-RRB-

have

reported

a

role

of

caveolae

in

EGFR

uptake

They

found

that

at

low

concentrations

of

EGF

-LRB-

ng/ml

-RRB-

EGFR

was

not

ubiquitinated

and

became

internalized

by

clathrin-dependent

endocytosis

In

contrast

at

high

concentrations

of

EGF

-LRB-

ng/ml

-RRB-

the

receptor

became

ubiquitinated

and

was

to

a

high

degree

internalized

by

caveolae

This

conclusion

was

based

upon

immunogold

labeling

electron

microscopy

showing

that

at

ng/ml

of

EGF

about

half

of

the

receptors

were

localized

to

caveolae

and

to

caveolae-like

structures

or

caveosomes

apparently

not

connected

to

the

plasma

membrane

-LRB-

Sigismund

et

al.

-RRB-

However

it

is

likely

that

such

caveolae-like

structures

that

appear

to

be

freely

located

in

the

cytoplasm

are

actually

surface-connected

in

another

plane

of

sectioning

-LRB-

Sandvig

et

al.

-RRB-

In

another

study

Orlichenko

et

al.

-LRB-

-RRB-

showed

that

incubation

of

epithelial

cells

with

ng/ml

of

EGF

for

min

resulted

in

an

eight

to

tenfold

increase

in

the

number

of

plasma

membrane

caveolae

due

to

EGF-induced

tyrosine

phosphorylation

of

Moreover

live

cell

imaging

revealed

increased

dynamics

of

green

fluorescent

protein

-LRB-

GFP

-RRB-

tagged

caveolin

upon

stimulation

of

cells

with

ng/ml

EGF

After

min

of

treatment

fluorescent

caveolae

were

seen

to

move

from

the

plasma

membrane

deeper

into

the

cytoplasm

-LRB-

Orlichenko

et

al.

-RRB-

Thus

some

studies

suggest

a

role

of

caveolae

in

EGFR

endocytosis

Caveolae

are

quite

immobile

under

normal

nonstimulated

conditions

as

revealed

by

FRAP

-LRB-

Fluorescence

Recovery

After

Photobleaching

-RRB-

analysis

of

cells

transfected

with

GFP-tagged

caveolin

and

by

other

approaches

-LRB-

Hommelgaard

et

al.

Kirkham

et

al.

Thomsen

et

al.

-RRB-

If

caveolae

were

involved

in

EGFR-internalization

stimulated

by

high

concentrations

of

EGF

to

any

significant

degree

two

possible

mechanisms

can

be

envisioned

either

the

EGFR-containing

caveolae

become

internalized

in

a

single-wave

process

leading

to

depletion

of

the

plasma

membrane

for

caveolae

as

has

been

reported

for

virus

-LRB-

Pelkmans

et

al.

-RRB-

or

activation

of

EGFR

stimulates

an

increased

mobility

or

turnover

of

caveolae

so

that

internalized

caveolae

become

replaced

by

new

caveolae

at

the

plasma

membrane

These

scenarios

have

been

tested

by

live

cell

imaging

of

cells

expressing

GFP

tagged

caveolin

-LRB-

Kazazic

et

al.

-RRB-

It

was

shown

that

even

ng/ml

of

EGF

did

not

cause

any

wave

of

internalized

caveolae

from

the

plasma

membrane

or

any

increased

turnover

of

caveolae

at

the

plasma

membrane

Moreover

it

was

found

that

inhibition

of

clathrin-mediated

endocytosis

by

siRNA

against

clathrin

heavy

chain

strongly

inhibited

internalization

of

EGF

both

at

and

ng/ml

EGF

It

was

therefore

concluded

that

clathrin-dependent

endocytosis

is

the

major

pathway

for

EGFR

uptake

also

at

high

EGF

concentrations

and

that

caveolae

do

not

contribute

significantly

to

endocytosis

of

EGFR

at

any

EGF

concentrations

-LRB-

Kazazic

et

al.

-RRB-

At

high

concentrations

of

EGF

-LRB-

ng/ml

-RRB-

induction

of

ruffling

and

macropinocytosis

can

be

observed

in

some

cells

-LRB-

Fig.

K.

Roepstorff

unpublished

observations

-RRB-

However

whether

ligand

and

receptor

internalization

in

this

way

leads

to

lysosomal

degradation

is

uncertain

Furthermore

Orth

et

al.

-LRB-

-RRB-

found

that

EGF

-LRB-

ng/ml

-RRB-

stimulated

the

formation

of

dorsal

circular

waves

which

progressed

inward

and

resulted

in

sequestration

of

about

of

the

activated

EGFR

from

the

plasma

membrane

The

process

resulted

in

the

formation

of

EGFR-positive

tubular

structures

and

it

was

dependent

on

actin

reorganization

receptor

phosphorylation

PI

activity

and

dynamin

The

functional

consequences

of

the

process

remain

to

be

determined

but

the

dorsal

wave

formation

seems

to

be

less

frequent

in

tumor

cells

than

in

normal

cells

-LRB-

Orth

et

al.

-RRB-

Fig.

induces

membrane

ruffling

and

macropinocytosis

cells

expressing

GPI-GFP

as

a

marker

of

the

plasma

membrane

were

followed

by

confocal

microscopy

over

time

The

upper

panel

shows

two

cells

that

were

imaged

before

and

min

after

stimulation

with

ng/ml

EGF

Arrows

indicate

membrane

ruffles

formed

after

EGF

stimulation

The

lower

panel

shows

that

ruffling

leads

to

formation

of

macropinosomes

-LRB-

arrows

-RRB-

The

large

image

to

the

left

shows

a

cell

min

after

stimulation

with

ng/ml

EGF

and

the

small

images

show

a

time

sequence

during

which

macropinosomes

are

formed

Bars

μm

In

conclusion

although

alternative

clathrin-independent

mechanisms

of

EGFR

endocytosis

have

been

suggested

clathrin-mediated

endocytosis

still

appear

to

be

by

far

the

most

important

mechanism

for

EGFR

downregulation

Endocytic

downregulation

of

Interestingly

the

behaviour

of

regarding

endocytic

downregulation

differs

significantly

from

that

of

EGFR

and

it

is

generally

accepted

that

avoids

efficient

endocytic

downregulation

-LRB-

Austin

et

al.

Baulida

et

al.

Hommelgaard

et

al.

Longva

et

al.

Sorkin

and

Carpenter

Wang

et

al.

-RRB-

Several

studies

have

investigated

why

is

less

susceptible

to

endocytic

downregulation

than

EGFR

after

activation

and

two

fundamentally

different

cellular

mechanisms

could

explain

this

inefficient

internalization

-LRB-

Baulida

et

al.

Hommelgaard

et

al.

Lerdrup

et

al.

Longva

et

al.

Sorkin

and

Carpenter

Wang

et

al.

-RRB-

or

efficient

recycling

of

endocytosed

back

to

the

plasma

membrane

-LRB-

Austin

et

al.

Citri

et

al.

Hendriks

et

al.

Klapper

et

al.

Lenferink

et

al.

Yarden

-RRB-

Using

antibodies

labeled

with

fluorophores

or

gold-particles

to

monitor

trafficking

Austin

and

co-workers

found

that

is

constantly

internalized

and

recycled

-LRB-

Austin

et

al.

-RRB-

In

contrast

two

studies

found

no

observable

intracellular

in

unstimulated

cells

and

that

treatment

with

monensin

a

compound

known

to

inhibit

recycling

led

to

very

little

intracellular

accumulation

of

within

two

hours

-LRB-

Lerdrup

et

al.

Longva

et

al.

-RRB-

Importantly

immunogold

labeling

electron

microscopy

has

demonstrated

that

is

very

infrequently

found

in

clathrin

coated

pits

as

little

as

of

labeled

molecules

localize

to

these

structures

in

unstimulated

cells

or

in

cells

treated

with

ErbB

ligands

-LRB-

Fig.

-RRB-

-LRB-

Hommelgaard

et

al.

-RRB-

This

suggests

that

the

inefficient

downregulation

of

is

due

to

a

low

basal

rate

of

internalization

although

it

is

possible

that

could

be

taken

up

by

clathrin

independent

endocytosis

Fig.

of

induces

receptor

internalization

a

Fixed

breast

cancer

cells

have

been

incubated

with

a

mouse

monoclonal

antibody

against

the

extracellular

N-terminal

part

of

and

subsequently

with

a

gold-conjugated

anti-mouse

antibody

Thereafter

the

cells

were

embedded

in

Epon

sectioned

and

examined

in

the

electron

microscope

It

is

seen

that

is

mainly

associated

with

membrane

protrusions

An

empty

clathrin-coated

pit

-LRB-

CCP

-RRB-

is

also

seen

b

c

cells

have

been

incubated

for

h

at

°C

with

the

antibody

followed

by

the

gold-conjugated

antibody

before

fixation

and

embedding

Note

how

the

antibody

crosslinking

drives

down

from

the

protrusions

to

the

bulk

membrane

where

it

is

seen

in

clathrin-coated

pits

-LRB-

CCP

-RRB-

Labeling

of

a

multivesicular

body

-LRB-

MVB

-RRB-

is

also

seen

in

c

showing

that

crosslinked

is

internalized

For

details

see

-LRB-

Hommelgaard

et

al.

-RRB-

Bar

μm

A

chimeric

EGFR

where

the

C-terminal

tail

has

been

replaced

by

that

of

is

as

endocytosis

impaired

as

a

chimeric

EGFR

where

the

entire

intracellular

part

has

been

replaced

by

that

of

-LRB-

Sorkin

and

Carpenter

-RRB-

This

suggests

that

the

C-terminal

tail

of

is

responsible

for

its

endocytic

impairment

and

an

interesting

hypothesis

is

that

this

is

caused

by

an

inhibitory

signal

rather

than

a

lack

of

endocytic

stimuli

-LRB-

Sorkin

and

Carpenter

-RRB-

This

is

supported

by

the

lower

stability

and

increased

endocytic

downregulation

of

deletion

mutants

which

lack

the

C-terminal

tail

compared

to

full-length

-LRB-

Lerdrup

et

al.

Tikhomirov

and

Carpenter

-RRB-

As

mentioned

previously

phosphorylation

of

EGFR

on

is

important

for

recruitment

of

Cbl

and

endocytic

downregulation

of

EGFR

A

Cbl

docking

site

is

also

present

in

-LRB-

Klapper

et

al.

Levkowitz

et

al.

-RRB-

and

Cbl

can

be

recruited

to

-LRB-

Klapper

et

al.

Levkowitz

et

al.

-RRB-

However

the

actual

extent

of

Cbl

binding

is

debated

and

inefficient

binding

of

Cbl

could

also

explain

the

low

lysosomal

degradation

of

-LRB-

Chan

et

al.

Dankort

et

al.

Levkowitz

et

al.

Olayioye

et

al.

-RRB-

Several

groups

have

reported

that

can

transfer

its

endocytic

reluctance

to

EGFR

since

overexpression

protects

EGFR

from

endocytic

downregulation

and

can

increase

EGFR

levels

-LRB-

Haslekas

et

al.

Muthuswamy

et

al.

Wang

et

al.

Worthylake

et

al.

-RRB-

However

the

studies

do

not

reach

similar

conclusions

regarding

the

underlying

mechanism

and

different

models

have

been

presented

including

lack

of

Cbl

recruitment

to

activated

EGFR

-LRB-

Muthuswamy

et

al.

-RRB-

inhibited

formation

of

clathrin

coated

pits

after

EGFR

stimulation

-LRB-

Haslekas

et

al.

-RRB-

or

reduced

targeting

of

internalized

EGFR

to

lysosomes

-LRB-

Worthylake

et

al.

-RRB-

Although

it

is

well

established

that

is

severely

endocytosis

impaired

compared

to

EGFR

there

is

currently

no

consensus

model

of

how

this

is

achieved

Taking

into

account

the

major

focus

that

receive

as

an

oncogene

and

as

a

drug

target

a

better

understanding

of

the

mechanisms

hindering

endocytosis

and

lysosomal

degradation

is

warranted

Endocytic

downregulation

of

and

At

present

remarkably

little

is

known

about

endocytosis

of

and

In

the

first

studies

of

their

endocytosis

and

were

reported

to

be

internalization

impaired

to

the

same

extent

as

-LRB-

Baulida

et

al.

-RRB-

In

accordance

with

this

the

and

ligand

was

found

to

be

internalized

less

efficient

and

much

slower

than

the

EGFR-specific

ligand

EGF

supporting

that

and

are

not

endocytosed

as

efficiently

as

EGFR

-LRB-

Baulida

and

Carpenter

Waterman

et

al.

-RRB-

Interestingly

the

endocytic

impairment

of

could

be

transferred

to

EGFR

by

replacing

the

EGFR

C-terminal

tail

with

the

C-terminal

tail

suggesting

that

there

are

regions

in

the

C-terminus

of

that

protect

the

receptor

against

internalization

-LRB-

Waterman

et

al.

-RRB-

Whereas

the

abovementioned

studies

suggest

that

and

are

endocytosis

impaired

both

of

the

receptors

can

be

ubiquitinated

-LRB-

Cao

et

al.

Omerovic

et

al.

Qiu

and

Goldberg

-RRB-

In

case

of

stimulation

leads

to

ubiquitination

of

the

receptor

by

the

ubiquitin

ligase

Knockdown

of

increases

the

steady-state

levels

of

suggesting

that

ubiquitination

may

target

to

degradation

-LRB-

Qiu

and

Goldberg

-RRB-

is

ubiquitinated

by

the

ubiquitin

ligase

Itch

which

induces

increased

degradation

of

when

overexpressed

-LRB-

Omerovic

et

al.

-RRB-

The

ubiquitination-stimulated

degradation

of

both

and

depends

upon

proteasomal

activity

-LRB-

Cao

et

al.

Omerovic

et

al.

Qiu

and

Goldberg

-RRB-

Interestingly

the

degradation

appears

to

involve

lysosomes

as

well

since

inhibition

of

lysosomal

function

retards

the

degradation

of

both

and

-LRB-

Cao

et

al.

Omerovic

et

al.

-RRB-

This

apparent

need

for

both

lysosomes

and

proteasomes

for

receptor

degradation

may

appear

as

a

paradox

but

it

has

been

shown

that

proteasomal

activity

is

required

for

lysosomal

degradation

of

EGFR

as

well

-LRB-

Alwan

et

al.

Longva

et

al.

-RRB-

Thus

if

this

is

also

the

case

for

and

the

block

of

receptor

degradation

seen

after

proteasomal

inhibition

could

be

due

to

an

effect

on

lysosomal

function

like

the

one

observed

for

EGFR

Obviously

this

is

in

discrepancy

with

the

studies

reporting

that

neither

nor

becomes

efficiently

endocytosed

and

more

studies

are

therefore

warranted

addressing

the

role

of

endocytosis

and

lysosomal

degradation

of

and

not

least

in

cancer

cells

Escape

of

endocytic

downregulation

in

cancer

Several

EGFR

mutants

have

been

found

in

tumors

indicating

an

oncogenic

role

of

these

mutants

The

mutations

can

be

in

the

form

of

small

or

large

deletions

point

mutations

or

amplifications

and

these

alterations

can

be

found

both

in

the

intra

and

extracellular

parts

of

EGFR

-LRB-

Fig.

-RRB-

Whether

the

mutations

are

the

cause

of

or

a

result

of

transformation

is

often

not

clear

but

some

EGFR

mutants

show

increased

signaling

which

will

contribute

to

oncogenesis

The

increased

signaling

can

be

the

result

of

constitutive

activation

and/or

impaired

downregulation

In

this

review

we

will

focus

on

examples

of

EGFR

mutants

with

impaired

downregulation

Proper

association

of

the

activated

receptor

with

the

downregulation

machinery

is

essential

for

stopping

the

signaling

of

EGFR

As

described

above

binding

of

the

ubiquitin

ligase

Cbl

to

EGFR

is

central

in

the

downregulation

EGFRvIV

and

EGFRvV

are

both

found

in

glioblastomas

-LRB-

Frederick

et

al.

Peschard

and

Park

Zandi

et

al.

-RRB-

Due

to

intracellular

deletions

these

mutants

lack

the

direct

binding

site

of

Cbl

and

in

addition

EGFRvV

also

lacks

the

indirect

binding

sites

-LRB-

Frederick

et

al.

Peschard

and

Park

Zandi

et

al.

-RRB-

The

viral

oncogene

v-ErbB

resembles

EGFR

but

lacks

certain

parts

including

the

direct

binding

site

of

Cbl

-LRB-

Peschard

and

Park

Zandi

et

al.

-RRB-

The

downregulation

of

EGFRvIV

EGFRvV

and

v-ErbB

has

not

been

investigated

but

they

would

be

expected

to

have

an

impaired

downregulation

The

best

characterized

EGFR

mutant

with

impaired

downregulation

is

EGFRvIII

Initially

found

in

glioblastomas

-LRB-

Sugawa

et

al.

-RRB-

EGFRvIII

has

later

been

found

in

a

range

of

other

types

of

tumors

-LRB-

Garcia

de

Palazzo

et

al.

Ge

et

al.

Moscatello

et

al.

Okamoto

et

al.

Olapade-Olaopa

et

al.

-RRB-

In

concordance

with

this

EGFRvIII

can

transform

fibroblasts

and

the

receptor

enhances

proliferation

and/or

tumorigenicity

of

cells

both

in

vivo

and

in

vitro

-LRB-

Batra

et

al.

Moscatello

et

al.

Nagane

et

al.

Nishikawa

et

al.

Pedersen

et

al.

Tang

et

al.

-RRB-

EGFRvIII

is

characterized

by

a

deletion

of

amino

acid

residues

found

in

the

extracellular

part

of

EGFR

This

gives

a

truncated

kDa

receptor

with

a

non-functional

ligand

binding

pocket

and

no

dimerization

arm

Thus

EGFRvIII

is

incapable

of

binding

any

ligands

the

receptor

is

however

constitutively

active

-LRB-

Kuan

et

al.

-RRB-

and

it

activates

and

PI

-LRB-

Antonyak

et

al.

Chu

et

al.

Montgomery

et

al.

Moscatello

et

al.

-RRB-

The

constitutive

activity

may

be

sufficient

for

tumorigenicity

but

impaired

downregulation

would

enhance

the

effect

EGFRvIII

can

associate

with

overexpressed

Cbl

and

this

leads

to

downregulation

-LRB-

Davies

et

al.

-RRB-

However

two

recent

reports

show

that

EGFRvIII

is

not

degraded

in

cells

with

endogenous

levels

of

Cbl

and

that

this

can

be

due

to

virtually

no

phosphorylation

of

-LRB-

Grandal

et

al.

Han

et

al.

-RRB-

In

concordance

with

the

hypophosphorylation

at

the

direct

binding

site

Cbl

binds

primarily

through

the

adaptor

protein

-LRB-

Grandal

et

al.

-RRB-

This

gives

no

detectable

ubiquitination

and

instead

of

being

degraded

internalized

EGFRvIII

is

recycled

back

to

the

plasma

membrane

-LRB-

Grandal

et

al.

-RRB-

Thus

as

seen

for

the

EGFR

mutant

direct

binding

of

Cbl

to

the

receptor

appears

to

be

necessary

for

efficient

degradation

In

conclusion

endocytic

impairment

may

be

a

returning

theme

of

oncogenic

EGFR

mutants

ErbB

signaling

can

also

be

sustained

if

the

molecular

machinery

normally

involved

in

receptor

downregulation

does

not

function

optimally

Indeed

several

mutations

of

such

proteins

have

been

found

in

tumors

including

Cbl

-LRB-

an

ESCRT-I

subunit

-RRB-

and

-LRB-

an

ESCRT-II

subunit

-RRB-

-LRB-

recently

reviewed

in

-LRB-

Haglund

et

al.

-RRB-

-RRB-

Endocytic

downregulation

of

ErbB

receptors

as

a

therapeutic

target

in

cancer

treatment

Since

lack

of

endocytic

downregulation

is

an

emerging

theme

in

ErbB

cancer

biology

it

is

evident

that

stimulation

of

ErbB

endocytosis

and

lysosomal

degradation

is

an

attractive

means

to

inhibit

tumor

growth

Antibody-mediated

crosslinking

can

induce

internalization

of

ErbB

receptors

From

a

therapeutic

perspective

an

appealing

way

to

induce

endocytic

downregulation

of

ErbB

receptors

is

by

antibody-mediated

crosslinking

Indeed

an

efficient

way

to

induce

endocytosis

of

both

EGFR

and

is

crosslinking

with

polyvalent

antibody

conjugates

-LRB-

Fig.

b

c

-RRB-

-LRB-

Friedman

et

al.

Hommelgaard

et

al.

-RRB-

The

most

studied

antibody

with

an

anti-tumor

potential

is

Trastuzumab

which

is

currently

used

in

the

treatment

of

breast

cancer

-LRB-

Hudis

-RRB-

Although

the

dominating

opinion

has

been

that

Trastuzumab

causes

endocytic

downregulation

of

-LRB-

Menard

et

al.

Rubin

and

Yarden

Yarden

-RRB-

several

recent

studies

suggest

that

Trastuzumab

does

not

induce

endocytosis

of

to

a

significant

degree

-LRB-

Austin

et

al.

Hommelgaard

et

al.

Longva

et

al.

-RRB-

Cetuximab

is

an

antibody

targeting

EGFR

that

is

currently

used

in

treatment

of

colorectal

cancer

and

head

and

neck

cancer

-LRB-

Harari

et

al.

-RRB-

Several

studies

have

shown

that

Cetuximab

induces

internalization

of

EGFR

-LRB-

Jaramillo

et

al.

Sunada

et

al.

-RRB-

In

contrast

to

ligand-induced

endocytosis

Cetuximab-induced

EGFR

internalization

is

independent

of

receptor

tyrosine

kinase

activity

and

it

is

both

slower

and

less

efficient

in

terms

of

receptor

downregulation

than

the

ligand-induced

endocytosis

-LRB-

Jaramillo

et

al.

-RRB-

At

present

the

knowledge

of

mechanisms

underlying

antibody-mediated

endocytic

downregulation

is

relatively

sparse

It

is

clear

that

the

endocytic

potential

of

antibodies

vary

but

the

reason

for

this

remains

elusive

Furthermore

it

is

debated

whether

the

endocytic

potential

of

antibodies

is

correlated

to

their

anti-tumor

potential

-LRB-

Hurwitz

et

al.

Klapper

et

al.

Park

et

al.

-RRB-

Indeed

in

cases

where

the

anti-tumor

effect

of

therapeutic

antibodies

is

in

part

due

to

antibody-dependent

activation

of

an

immune

response

internalization

of

bound

antibodies

will

be

a

therapeutic

disadvantage

since

it

will

clear

the

surface

for

antibodies

that

will

otherwise

be

recognized

by

immune

cells

such

as

natural

killer

cells

However

antibody-mediated

receptor

endocytosis

followed

by

degradation

may

be

advantageous

in

the

treatment

of

tumors

that

rely

heavily

upon

ErbB

receptor

signaling

as

a

growth-promoting

signal

A

useful

observation

is

that

extensive

antibody-based

crosslinking

of

ErbB

receptors

is

far

more

efficient

at

inducing

ErbB

endocytic

downregulation

than

single

antibodies

are

-LRB-

Friedman

et

al.

Hommelgaard

et

al.

-RRB-

Crosslinking

can

either

be

done

using

antibodies

that

forms

multivalent

aggregates

via

secondary

antibodies

or

gold

particles

-LRB-

Hommelgaard

et

al.

-RRB-

or

by

a

more

clinically

relevant

approach

using

combinations

of

monoclonal

antibodies

against

distinct

epitopes

in

an

ErbB

receptor

-LRB-

Friedman

et

al.

-RRB-

Thus

whereas

administration

of

Trastuzumab

alone

did

not

induce

significant

endocytosis

the

combination

of

Trastuzumab

with

another

monoclonal

antibody

to

was

very

efficient

at

downregulating

In

addition

the

combination

of

two

antibodies

was

much

more

efficient

at

inhibiting

tumor

growth

in

a

mouse

model

compared

to

Trastuzumab

administered

alone

-LRB-

Friedman

et

al.

-RRB-

inhibition

stimulates

cleavage

and

internalization

Another

efficient

way

to

induce

endocytosis

and

lysosomal

degradation

of

is

inhibition

of

the

chaperone

has

gained

a

lot

of

attention

due

to

the

involvement

of

many

of

its

clients

in

cancers

and

several

inhibitors

are

being

tested

in

clinical

trials

including

the

Geldanamycin

derivative

-LRB-

Goetz

et

al.

Solit

and

Chiosis

Vastag

Whitesell

and

Lindquist

-RRB-

is

one

of

the

most

prominent

client

proteins

of

the

chaperone

-LRB-

Citri

and

Yarden

Linggi

and

Carpenter

-RRB-

and

this

interaction

is

considered

as

a

potentially

valuable

pharmacological

target

-LRB-

Citri

et

al.

Marmor

et

al.

Neckers

Neckers

and

Ivy

-RRB-

overexpressing

cancer

cells

often

binds

with

an

even

higher

affinity

than

other

cancers

cells

suggesting

that

overexpressing

cells

are

highly

dependent

on

activity

-LRB-

Kamal

et

al.

-RRB-

Inhibition

of

e.g.

by

using

Geldanamycin

leads

to

recruitment

of

the

cochaperone

and

ubiquitin

ligase

CHIP

to

and

ubiquitination

of

the

receptor

-LRB-

Xu

et

al.

Zhou

et

al.

-RRB-

Several

degradation

mechanisms

are

involved

in

downregulation

following

its

ubiquitination

Mimnaugh

and

coworkers

-LRB-

Mimnaugh

et

al.

-RRB-

found

that

led

to

degradation

of

in

a

proteasome-dependent

manner

and

this

has

been

interpreted

as

if

degradation

of

the

transmembrane

was

carried

out

by

the

proteasome

-LRB-

Citri

et

al.

Hong

et

al.

Way

et

al.

Xu

et

al.

Zheng

et

al.

-RRB-

However

later

studies

found

that

is

endocytosed

and

degraded

in

lysosomes

after

Geldanamycin

stimulation

-LRB-

Fig.

a

-RRB-

-LRB-

Austin

et

al.

Lerdrup

et

al.

Longva

et

al.

Tikhomirov

and

Carpenter

-RRB-

and

that

the

proteasomal

activity

is

actually

needed

for

endocytosis

and

lysosomal

degradation

of

after

Geldanamycin

stimulation

-LRB-

Lerdrup

et

al.

-RRB-

Finally

Tikhomirov

and

Carpenter

have

demonstrated

that

the

C-terminus

of

is

cleaved

by

endoproteases

into

a

transmembrane

and

several

cytosolic

fragments

in

response

to

Geldanamycin-treatment

-LRB-

Tikhomirov

and

Carpenter

-RRB-

Recent

work

from

our

group

showed

that

this

cleavage

also

promotes

endocytosis

and

lysosomal

degradation

of

-LRB-

Lerdrup

et

al.

-RRB-

Using

a

doubly

fluorescent

tagged

with

yellow

fluorescent

protein

-LRB-

YFP

-RRB-

and

cyan

fluorescent

protein

-LRB-

CFP

-RRB-

in

each

terminus

we

found

that

was

cleaved

at

the

plasma

membrane

after

Geldanamycin

stimulation

C-terminally

cleaved

as

well

as

an

deletion

mutant

lacking

its

C-terminal

tail

were

endocytosed

and

degraded

in

lysosomes

far

more

efficiently

than

full

length

-LRB-

Fig.

-RRB-

-LRB-

Lerdrup

et

al.

-RRB-

Fig.

inhibition

stimulates

cleavage

and

internalization

a

is

internalized

after

h

of

Geldanamycin-stimulation

-LRB-

μM

-RRB-

of

breast

cancer

cells

Sequential

immunocytochemistry

was

used

to

distinguish

between

internalized

and

surface

associated

Fixed

cells

were

stained

with

antibodies

against

before

permeabilization

-LRB-

green

-RRB-

After

permeabilization

cells

were

once

again

stained

with

antibodies

against

-LRB-

red

-RRB-

Only

the

latter

antibody

staining

-LRB-

red

-RRB-

gain

access

to

intracellular

which

therefore

appears

red

whereas

surface

is

stained

both

red

and

green

and

therefore

appears

yellow

For

details

see

-LRB-

Lerdrup

et

al.

-RRB-

b

is

cleaved

after

h

of

Geldanamycin-stimulation

of

cells

A

construct

with

YFP

fused

to

the

extracellular

N-terminus

and

CFP

fused

to

the

intracellular

C-terminus

of

was

expressed

in

cells

for

h

and

followed

by

incubation

with

μM

Geldanamycin

for

h

as

indicated

where

the

C-terminal

tail

has

been

cleaved

off

appears

green

Note

the

increased

amount

of

such

cleavage

in

vesicles

compared

to

the

plasma

membrane

after

Geldanamycin

stimulation

For

details

see

-LRB-

Lerdrup

et

al.

-RRB-

Bars

μm

As

opposed

to

wild-type

EGFR

is

relatively

insensitive

to

inhibition

However

the

Geldanamycin

derivative

stimulates

degradation

of

constitutively

active

EGFR

mutants

commonly

found

in

lung

cancer

-LRB-

Sawai

et

al.

-RRB-

Whether

induces

degradation

of

mutant

EGFRs

by

stimulating

endocytosis

and

lysosomal

degradation

remains

to

be

determined

Perspectives

In

spite

of

its

crucial

importance

both

in

health

and

disease

the

ErbB

receptor

system

still

has

hidden

secrets

regarding

its

regulation

It

is

evident

that

we

need

to

know

much

more

about

the

molecular

mechanisms

underlying

endocytic

downregulation

of

the

otherwise

heavily

investigated

ErbB

receptors

One

important

area

that

deserves

future

attention

is

further

mapping

of

the

mechanisms

responsible

for

recruitment

of

EGFR

to

CCPs

Moreover

EGFR

endocytosis

does

not

seem

to

be

representative

of

the

other

ErbB

receptor

family

members

which

all

appear

to

be

endocytosis

impaired

to

some

extent

compared

to

EGFR

It

is

especially

of

interest

to

decipher

how

formation

of

heterodimers

between

the

different

ErbB

receptors

affects

their

endocytic

downregulation

In

addition

the

large

number

of

ErbB

ligands

adds

further

complexity

to

the

system

since

they

may

vary

in

the

capability

to

stimulate

endocytosis

and

lysosomal

degradation

of

their

receptors

Interestingly

the

few

ligands

that

have

been

investigated

so

far

differ

significantly

in

their

ability

to

induce

endocytic

downregulation

of

ErbB

receptors

More

insight

into

endocytic

downregulation

of

ErbB

receptors

is

indeed

relevant

for

understanding

the

role

of

these

receptors

in

cancer

and

it

could

lead

to

identification

of

novel

targets

for

cancer

therapy

directed

at

downregulating

signaling

receptors

Although

still

in

the

making

the

clinical

as

well

as

preclinical

results

show

that

targeted

therapy

stimulating

endocytic

downregulating

of

ErbB

receptors

is

a

promising

tool

in

cancer

treatment

O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I I O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B I O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O B I O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O B O O B I O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B B O B I O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O B I O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B I O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B I O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

epidermal growth factor receptor
endocytosis
lysosomal degradation
erbb2
ubiquitin
endosomal sorting