Internalization

and

desensitization

of

adenosine

receptors

Until

now

more

than

distinct

G

protein-coupled

receptors

-LRB-

GPCRs

-RRB-

have

been

identified

in

the

human

genome

The

four

subtypes

of

the

adenosine

receptor

-LRB-

and

receptor

-RRB-

belong

to

this

large

family

of

GPCRs

that

represent

the

most

widely

targeted

pharmacological

protein

class

Since

adenosine

receptors

are

widespread

throughout

the

body

and

involved

in

a

variety

of

physiological

processes

and

diseases

there

is

great

interest

in

understanding

how

the

different

subtypes

are

regulated

as

a

basis

for

designing

therapeutic

drugs

that

either

avoid

or

make

use

of

this

regulation

The

major

GPCR

regulatory

pathway

involves

phosphorylation

of

activated

receptors

by

G

protein-coupled

receptor

kinases

-LRB-

GRKs

-RRB-

a

process

that

is

followed

by

binding

of

arrestin

proteins

This

prevents

receptors

from

activating

downstream

heterotrimeric

G

protein

pathways

but

at

the

same

time

allows

activation

of

arrestin-dependent

signalling

pathways

Upon

agonist

treatment

adenosine

receptor

subtypes

are

differently

regulated

For

instance

the

are

not

-LRB-

readily

-RRB-

phosphorylated

and

internalize

slowly

showing

a

typical

half-life

of

several

hours

whereas

the

and

undergo

much

faster

downregulation

usually

shorter

than

h

The

is

subject

to

even

faster

downregulation

often

a

matter

of

minutes

The

fast

desensitization

of

the

after

agonist

exposure

may

be

therapeutically

equivalent

to

antagonist

occupancy

of

the

receptor

This

review

describes

the

process

of

desensitization

and

internalization

of

the

different

adenosine

subtypes

in

cell

systems

tissues

and

in

vivo

studies

In

addition

molecular

mechanisms

involved

in

adenosine

receptor

desensitization

are

discussed

Introduction

Adenosine

is

an

important

neuromodulator

involved

in

a

variety

of

brain

activities

and

it

also

serves

many

different

functions

in

the

periphery

This

nucleoside

when

extracellular

exerts

its

action

via

specific

G

protein-coupled

receptors

-LRB-

GPCRs

-RRB-

of

the

class

divided

into

four

subtypes

and

-LSB-

-RSB-

GPCRs

consist

of

a

single

polypeptide

containing

seven

α-helices

which

are

oriented

perpendicular

to

the

membrane

The

N

terminus

is

located

at

the

extracellular

side

of

the

cell

and

often

contains

one

or

more

glycosylation

sites

The

C

terminus

is

located

intracellularly

and

contains

phosphorylation

and

palmitoylation

sites

which

are

involved

in

regulation

of

receptor

desensitization

and

internalization

-LSB-

-RSB-

All

adenosine

receptors

with

the

exception

of

the

contain

a

palmitoylation

site

near

the

C

terminus

The

is

the

only

subtype

with

an

extraordinary

long

C

terminus

amino

acids

versus

amino

acids

in

e.g.

the

-LSB-

-RSB-

All

the

adenosine

receptors

are

glycosylated

on

the

second

extracellular

loop

although

glycosylation

does

not

appear

to

influence

ligand

binding

The

third

intracellular

loop

and/or

the

C

terminus

are

involved

in

coupling

the

adenosine

receptors

to

G

proteins

Phosphorylation

of

in

particular

intracellular

loop

is

involved

in

desensitization

and

internalization

of

adenosine

receptors

-LSB-

-RSB-

Adenosine

and

analogues

Adenosine

consisting

of

a

purine

ring

connected

to

a

ribose

group

is

the

endogenous

ligand

for

the

adenosine

receptors

-LRB-

Fig.

-RRB-

Under

normal

conditions

adenosine

is

continuously

formed

extracellularly

by

dephosphorylation

of

ATP

ADP

and/or

AMP

to

adenosine

by

NTPDases

-LRB-

ectonucleoside

triphosphate

diphosphohydrolases

-RRB-

However

the

can

also

be

activated

by

inosine

a

breakdown

product

from

adenosine

-LSB-

-RSB-

Most

adenosine

receptor

agonists

are

analogues

of

adenosine

modified

by

and

substitutions

at

the

adenine

base

and

modifications

of

the

ribose

moiety

-LSB-

-RSB-

Antagonists

lack

the

ribose

group

and

usually

possess

a

mono

bi

or

tricyclic

core

structure

e.g.

caffeïne

which

contains

a

xanthine

as

basic

structure

-LRB-

Fig.

-RRB-

For

extended

reviews

on

high

affinity

agonists

and

antagonists

for

adenosine

receptors

and

their

structure-activity

relationships

see

Palmer

and

Stiles

-LSB-

-RSB-

Fredholm

et

al.

-LSB-

-RSB-

Jacobson

and

Gao

-LSB-

-RSB-

Beukers

et

al.

-LSB-

-RSB-

Müller

-LSB-

-RSB-

and

Klotz

-LSB-

-RSB-

Fig.

structures

of

the

endogenous

ligand

adenosine

and

the

antagonist

caffeine

Occurrence

and

physiological

functions

of

adenosine

receptors

The

adenosine

receptors

are

widespread

throughout

the

body

and

exert

many

different

functions

both

in

the

CNS

and

in

the

periphery

The

is

particularly

prevalent

in

the

central

nervous

system

with

high

levels

in

the

cerebral

cortex

hippocampus

cerebellum

thalamus

brain

stem

and

spinal

cord

Numerous

peripheral

tissues

also

express

the

including

vas

deferens

testis

white

adipose

tissue

stomach

spleen

pituitary

adrenal

gland

heart

aorta

liver

eye

and

bladder

Low

levels

are

found

in

the

lung

kidney

and

small

intestine

-LSB-

-RSB-

The

is

involved

in

cardiovascular

effects

-LRB-

e.g.

reducing

heart

rate

-RRB-

inhibition

of

lipolysis

and

stimulation

of

glucose

uptake

in

white

adipocytes

and

the

modulation

of

neurotransmitter

release

in

the

CNS

-LSB-

-RSB-

The

also

plays

a

role

in

anxiety

hyperalgesia

bronchoconstriction

and

the

glomerular

filtration

rate

and

renin

release

in

the

kidney

-LSB-

-RSB-

In

the

CNS

the

is

highly

expressed

in

the

striatum

and

olfactory

tubercle

-LSB-

-RSB-

In

the

periphery

it

is

highly

expressed

in

the

spleen

thymus

leucocytes

and

blood

platelets

and

intermediate

levels

are

found

in

the

heart

lung

and

blood

vessels

-LSB-

-RSB-

The

is

involved

in

the

onset

of

vasodilation

inhibition

of

platelet

aggregation

exploratory

activity

aggressiveness

and

hypoalgesia

-LSB-

-RSB-

In

addition

plays

a

role

in

Parkinson

's

disease

Huntington

's

disease

Alzheimer

's

disease

ischaemia

attenuation

of

inflammation

and

neuroprotection

particularly

in

peripheral

tissues

-LSB-

-RSB-

receptor

antagonists

slow

the

neurodegeneration

which

occurs

in

Parkinson

's

and

Huntington

's

disease

and

also

prevent

toxicity

induced

by

beta-amyloid

in

the

development

of

Alzheimer

's

disease

-LSB-

-RSB-

The

is

widely

expressed

in

the

brain

but

generally

at

very

low

levels

In

the

periphery

high

levels

of

were

detected

in

the

caecum

large

intestine

and

urinary

bladder

Lower

levels

were

observed

in

the

lung

spinal

cord

vas

deferens

pituitary

adipose

tissue

adrenal

gland

kidney

liver

and

ovaries

-LSB-

-RSB-

Since

there

is

a

lack

of

specific

agonists

for

the

little

is

known

about

the

functional

significance

of

this

receptor

However

the

plays

a

role

in

mediating

vasodilation

in

a.o.

the

aorta

the

renal

artery

and

the

coronary

artery

of

different

species

It

is

also

involved

in

allergic

and

inflammatory

disorders

-LSB-

-RSB-

The

is

expressed

in

the

CNS

but

at

relatively

low

levels

and

only

the

hypothalamus

and

the

thalamus

have

been

reported

to

contain

-LSB-

-RSB-

The

highest

levels

of

adenosine

have

been

found

in

the

lung

and

liver

and

somewhat

lower

levels

were

found

in

the

aorta

-LSB-

-RSB-

In

addition

the

was

found

in

eosinophils

mast

cells

testis

kidney

placenta

heart

spleen

uterus

bladder

jejunum

aorta

proximal

colon

and

eye

although

with

pronounced

differences

in

expression

level

between

species

-LSB-

-RSB-

The

has

been

implicated

in

mediating

allergic

responses

airway

inflammation

and

apoptotic

events

however

the

latter

is

dependent

on

the

cell

type

involved

and/or

the

type

of

activation

-LSB-

-RSB-

Furthermore

the

is

involved

in

the

control

of

the

cell

cycle

and

inhibition

of

tumour

growth

both

in

vitro

and

in

vivo

-LSB-

-RSB-

In

fact

adenosine

receptors

have

been

demonstrated

to

be

more

highly

expressed

in

tumours

than

in

healthy

cells

suggesting

a

role

for

as

a

tumour

marker

-LSB-

-RSB-

Signal

transduction

of

adenosine

receptors

G

protein-coupling

and

second

messengers

Heterotrimeric

G

proteins

are

guanine-nucleotide

regulatory

protein

complexes

composed

of

α

and

βγ

subunits

They

are

responsible

for

transmitting

signals

from

G

protein-coupled

receptors

to

effectors

e.g.

adenylyl

cyclase

Until

now

α

β

and

γ

isoforms

have

been

reported

-LSB-

-RSB-

G

proteins

are

divided

into

several

subclasses

with

a

specific

activity

profile

Gs

proteins

stimulate

adenylyl

cyclase

Gi

proteins

inhibit

adenylyl

cyclase

and

stimulate

GIRK

channels

proteins

stimulate

K

ion

channels

proteins

activate

phospholipase

C

proteins

activate

Rho

guanine-nucleotide

exchange

factors

-LRB-

GEFs

-RRB-

and

the

olfactory

G

protein

Golf

stimulates

adenylyl

cyclase

Upon

receptor

activation

both

the

α

subunit

and

the

βγ

subunit

can

signal

but

to

different

effectors

-LSB-

-RSB-

For

a

recent

review

on

G

proteins

see

Milligan

and

Kostenis

-LSB-

-RSB-

The

is

usually

coupled

to

a

pertussis

toxin-sensitive

protein

which

mediates

inhibition

of

adenylyl

cyclase

and

regulates

calcium

and

potassium

channels

-LSB-

-RSB-

Both

the

third

intracellular

loop

and

the

C-terminal

tail

of

the

are

involved

in

coupling

-LSB-

-RSB-

In

addition

it

has

been

reported

that

under

certain

conditions

the

couples

to

Gs

to

stimulate

adenylyl

cyclase

or

to

to

stimulate

inositol

phosphate

production

Apparently

the

specific

activity

state

of

the

receptor

or

the

nature

of

the

agonist

determine

which

G

protein

class

is

activated

by

the

-LSB-

-RSB-

The

in

the

periphery

is

coupled

to

cholera

toxin-sensitive

proteins

which

increase

adenylyl

cyclase

activity

upon

receptor

activation

The

in

the

striatum

is

presumably

coupled

to

Golf

-LSB-

-RSB-

The

third

intracellular

loop

but

not

the

C

terminus

of

the

is

involved

in

coupling

-LSB-

-RSB-

The

is

coupled

to

proteins

leading

to

stimulation

of

adenylyl

cyclase

upon

receptor

activation

-LSB-

-RSB-

There

is

quite

some

evidence

that

can

activate

phospholipase

C

as

well

via

proteins

-LSB-

-RSB-

The

is

coupled

to

pertussis

toxin-sensitive

proteins

which

mediate

inhibition

of

adenylyl

cyclase

In

addition

can

stimulate

phospholipase

C

via

proteins

-LSB-

-RSB-

For

an

extensive

overview

of

adenosine

receptor-G

protein

coupling

see

Fredholm

et

al.

-LSB-

-RSB-

Desensitization

and

internalization

general

principles

and

players

Mechanisms

to

dampen

GPCR

signalling

exist

at

every

level

in

the

cell

In

this

section

attention

will

be

paid

to

the

underlying

principles

of

desensitization

and

internalization

and

the

protein

partners

involved

in

these

processes

Receptor

localization

Depending

on

the

localization

signal

GPCRs

in

the

plasma

membrane

can

be

targeted

to

lipid

or

Different

regions

of

GPCRs

can

influence

not

only

the

targeting

to

either

lipid

rafts

or

caveolae

but

may

also

enable

an

interaction

of

the

receptor

with

constituents

of

these

rafts

and

caveolae

For

instance

the

extracellular

part

of

the

receptor

might

interact

with

gangliosides

-LRB-

glycosphingolipids

-RRB-

present

in

lipid

rafts/caveolae

In

addition

the

C-terminal

fatty

acid

acylation

or

palmitoylation

may

also

affect

targeting

of

GPCRs

to

either

lipid

rafts

or

caveolae

Finally

transmembrane

regions

may

interact

with

cholesterol

in

the

lipid

rafts

resulting

in

a

change

in

conformation

of

the

α-helices

Since

the

conformation

of

α-helices

depends

on

the

activation

state

of

GPCRs

it

may

well

be

that

agonist

binding

to

the

receptor

may

affect

its

localization

in

lipid

rafts

by

means

of

molecular

transitions

leading

to

receptor

activation

-LSB-

-RSB-

Desensitization

Desensitization

reduces

receptor

activity

and

plays

a

role

in

signal

duration

intensity

and

quality

Desensitization

is

initiated

by

phosphorylation

of

serine

and/or

threonine

residues

in

the

third

intracellular

loop

and

C

terminus

of

the

receptor

Two

types

of

desensitization

occur

heterologous

and

homologous

desensitization

and

both

are

the

result

of

receptor

phosphorylation

Heterologous

desensitization

is

induced

by

phosphorylation

of

the

receptor

by

protein

kinase

A

or

C

sometimes

even

without

agonist

occupancy

On

the

other

hand

homologous

desensitization

is

specific

for

agonist-occupied

receptors

and

consists

in

most

cases

of

two

steps

First

the

receptor

is

phosphorylated

by

one

of

the

G

protein-coupled

receptor

kinases

-LRB-

GRKs

-RRB-

then

it

binds

to

β-arrestin

of

which

two

subtypes

exist

which

exhibit

high

affinity

for

agonist-occupied

phosphorylated

receptors

β-Arrestin

serves

to

sterically

inhibit

G

protein

coupling

thereby

terminating

the

G

protein

activation

and

may

also

target

the

receptor

to

clathrin-coated

for

internalization

-LRB-

Fig.

-RRB-

-LSB-

-RSB-

Fig.

-LRB-

a

-RRB-

and

heterologous

-LRB-

b

-RRB-

desensitization

and

subsequent

internalization

of

GPCRs

subunit

subunit

L

ligand

GRK

G

protein-coupled

receptor

kinase

P

phosphorylated

amino

acids

β-arr

β-arrestin

adaptin

E

effector

second

messenger

PKA

protein

kinase

A

PKC

protein

kinase

C

Internalization

Receptor

desensitization

initiated

by

phosphorylation

of

the

receptor

by

different

protein

kinases

-LRB-

A

or

C

-RRB-

or

GRKs

can

be

subsequently

followed

by

receptor

internalization

Upon

phosphorylation

β-arrestin

or

is

attracted

to

the

receptor

-LSB-

-RSB-

β-Arrestins

not

only

interact

with

the

phosphorylated

receptor

but

also

bind

to

the

heavy

chain

of

clathrin

to

the

subunit

of

the

clathrin

adaptor

protein

and

to

phosphoinositides

These

interactions

direct

the

phosphorylated

receptor

to

punctate

clathrin-coated

pits

in

the

cell

membrane

which

are

internalized

by

action

of

the

GTPase

dynamin

Upon

internalization

receptors

can

either

be

rapidly

recycled

to

the

plasma

membrane

targeted

to

larger

endosomes

and

slowly

recycled

or

degraded

in

lysosomes

The

final

destination

of

the

internalized

receptors

largely

depends

on

the

β-arrestin

subtype

-LRB-

or

-RRB-

that

is

recruited

by

the

receptor

upon

phosphorylation

and

the

duration

of

β-arrestin

binding

In

this

way

internalization

may

regulate

receptor

resensitization

and

contributes

to

a

positive

regulation

of

receptor

signalling

-LSB-

-RSB-

Internalization

pathways

From

internalization

studies

with

several

receptors

it

appears

that

the

internalization

pathway

is

specific

for

receptor

type

cell

type

metabolic

state

of

the

cell

cell-specific

factors

etc.

Receptor

trafficking

can

be

regulated

in

different

ways

-LRB-

Fig.

-RRB-

-LRB-

a

-RRB-

the

receptor

resides

mainly

in

lipid

rafts/caveolae

and

enters

the

cell

via

this

pathway

by

default

-LRB-

b

-RRB-

the

receptor

is

in

lipid

rafts

but

leaves

these

upon

agonist

binding

to

be

internalized

via

clathrin-coated

pits

-LRB-

c

-RRB-

the

receptor

moves

into

lipid

rafts

upon

agonist

binding

and

is

internalized

via

this

pathway

-LRB-

d

-RRB-

the

receptor

moves

into

lipid

rafts

after

agonist

binding

to

activate

certain

signalling

events

but

is

eventually

moved

out

of

the

lipid

rafts

to

be

internalized

via

clathrin-coated

pits

Internalization

can

even

be

achieved

via

uncoated

vesicles

or

by

a

combination

of

two

or

more

of

the

aforementioned

pathways

For

example

is

internalized

via

both

lipid

rafts

and

clathrin-coated

pits

PKA

phosphorylation

directs

to

a

clathrin-coated

pit

whereas

GRK

phosphorylation

directs

the

receptor

to

lipid

raft-mediated

internalization

-LSB-

-RSB-

Fig.

internalization

pathways

adapted

from

Chini

and

Parenti

-LSB-

-RSB-

Internalization

via

-LRB-

a

-RRB-

lipid

rafts/caveolae

-LRB-

b

-RRB-

upon

agonist

binding

the

receptor

moves

to

clathrin-coated

pits

to

be

internalized

-LRB-

c

-RRB-

the

receptor

moves

into

lipid

rafts

upon

agonist

binding

and

is

internalized

-LRB-

d

-RRB-

the

receptor

moves

into

lipid

rafts

upon

agonist

binding

to

activate

certain

signalling

events

but

is

eventually

moved

out

to

be

internalized

via

clathrin-coated

pits

Ligand

-LRB-

L

green

triangle

-RRB-

clathrin-coated

pits

-LRB-

dotted

blue

lines

-RRB-

and

lipid

rafts

-LRB-

solid

pink

lines

-RRB-

are

indicated

Function

of

lipid

rafts/caveolae

The

existence

of

lipid

rafts/caveolae

serves

different

functions

First

of

all

lipid

rafts

act

as

stations

in

which

GPCRs

accomplish

specific

signalling

tasks

by

meeting

a

selected

set

of

signalling

molecules

e.g.

G

proteins

and

adenylyl

cyclases

Another

possible

function

of

lipid

rafts

is

the

protection

of

receptors

from

rapid

constitutive

or

agonist-induced

internalization

thus

allowing

their

coupling

to

specific

signalling

pathways

In

addition

caveolin

may

regulate

the

constitutive

activity

of

receptors

-LSB-

-RSB-

Finally

the

endocytic

pathways

that

GPCRs

choose

may

depend

on

cell-specific

factors

Switching

the

internalization

pathway

from

lipid

rafts/caveolae

to

clathrin-coated

pits

may

alter

the

final

receptor

destination

-LSB-

-RSB-

Research

on

adenosine

receptors

probably

provided

the

first

account

of

receptor

internalization

via

caveolae

and

lipid

rafts

as

an

alternative

to

the

well-described

β-arrestin

pathway

-LSB-

-RSB-

G

protein-coupled

receptor

kinases

-LRB-

GRKs

-RRB-

Upon

agonist

binding

to

GPCRs

not

only

G

proteins

are

recruited

to

the

activated

receptor

conformation

but

also

two

other

protein

families

namely

the

G

protein-coupled

receptor

kinases

-LRB-

GRKs

-RRB-

and

the

β-arrestins

-LSB-

-RSB-

The

GRK

family

consists

of

seven

different

genes

The

GRKs

have

been

divided

into

three

protein

subfamilies

based

on

sequence

similarity

and

belong

together

The

second

subfamily

consists

of

and

since

their

membrane

recruitment

depends

on

interaction

with

Gβγ

subunits

and

phosphatidylinositol

and

are

primarily

responsible

for

agonist-dependent

receptor

phosphorylation

β-arrestin

recruitment

and

functional

uncoupling

of

the

receptor

and

form

the

third

subfamily

and

are

constitutively

associated

with

the

membrane

and

are

expressed

mainly

in

the

retinal

rods

and

cones

whereas

expression

is

limited

to

the

cerebellum

testis

and

kidney

In

contrast

and

are

widely

expressed

in

mammalian

tissues

-LSB-

-RSB-

β-Arrestins

β-Arrestins

belong

to

the

arrestin

family

of

which

four

members

have

been

identified

Arrestin

and

arrestin

are

expressed

in

the

visual

system

in

retinal

photoreceptor

cells

arrestin

-LRB-

also

called

β-arrestin

-RRB-

and

arrestin

-LRB-

also

called

β-arrestin

-RRB-

are

more

widely

expressed

and

are

involved

in

the

regulation

of

nonvisual

GPCRs

The

β-arrestins

were

originally

discovered

as

molecules

that

bind

to

and

desensitize

the

activated

and

phosphorylated

form

of

G

protein-coupled

receptors

as

described

before

-LSB-

-RSB-

The

β-arrestin

protein

consists

of

an

N

and

a

C

domain

which

are

almost

entirely

composed

of

antiparallel

β

sheets

connected

by

a

linker

of

amino

acids

A

polar

core

is

embedded

between

those

two

domains

and

its

disruption

by

the

phosphorylated

C

terminus

of

the

activated

receptor

leads

to

conformational

changes

in

the

β-arrestin

The

C

tail

of

the

β-arrestin

is

then

released

exposing

both

the

clathrin

and

the

binding

domains

Recently

it

appeared

that

β-arrestins

not

only

are

involved

in

the

desensitization

of

G

protein-coupled

receptors

but

also

act

as

signal

transducers

on

their

own

As

a

consequence

they

emerge

as

multifunctional

adaptor/scaffold

proteins

which

mediate

cellular

processes

such

as

chemotaxis

apoptosis

and

metastasis

besides

receptor

signalling

and

trafficking

For

an

extensive

and

recent

review

on

the

interactions

of

β-arrestins

with

other

cellular

proteins

see

Lefkowitz

et

al.

-LSB-

-RSB-

GRKs

and

β-arrestins

orchestrate

GPCR

activities

at

three

different

levels

-LRB-

-RRB-

silencing

the

functional

uncoupling

of

the

receptor

from

its

G

protein

by

a

mechanism

known

as

homologous

desensitization

-LRB-

-RRB-

trafficking

which

involves

receptor

internalization

resensitization

and/or

degradation

and

-LRB-

-RRB-

cross-signalling

activation

or

inhibition

of

intracellular

signalling

pathways

independent

of

heterotrimeric

G

proteins

-LSB-

-RSB-

Dampening

adenosine

receptor

signals

In

this

section

we

will

discuss

the

evidence

for

and

mechanisms

of

desensitization

and

internalization

of

the

four

adenosine

receptor

subtypes

For

each

receptor

we

will

first

summarize

results

from

in

vitro

ex

vivo

and

in

vivo

studies

This

will

be

followed

by

a

more

biochemical

approach

in

which

we

will

focus

on

the

molecular

mechanisms

of

adenosine

receptor

desensitization

and

internalization

by

paying

attention

to

the

role

of

accessory

proteins

the

influence

of

receptor

mutations

etc.

We

will

refer

to

earlier

seminal

publications

but

mostly

focus

on

more

recent

evidence

for

adenosine

receptor

desensitization

and

internalization

receptor

Cellular

and

physiological

studies

The

early

evidence

for

adenosine

receptor

desensitization

was

largely

obtained

from

primary

cells

cell

lines

tissues

or

tissue

slices

and

intact

animals

that

were

exposed

to

varying

concentrations

of

adenosine

receptor

agonists

-LRB-

either

or

not

-RRB-

often

examined

over

several

time

periods

Stiles

and

co-workers

were

among

the

first

to

study

receptor

desensitization

in

detail

-LRB-

-RRB-

Ramkumar

et

al.

pretreated

cells

a

smooth

muscle

cell

line

expressing

both

and

receptors

with

R-phenylisopropyl

adenosine

-LRB-

R-PIA

-RRB-

for

up

to

h

after

which

the

adenylyl

cyclase

activity

was

reduced

by

approximately

This

was

associated

with

a

significant

decrease

in

cell

membrane-bound

receptors

and

a

concomitant

increase

of

receptor

number

in

intracellular

compartments

The

authors

also

showed

an

increase

in

receptor

phosphorylation

nicely

paralleling

the

time

course

of

adenylyl

cyclase

modulation

-LSB-

-RSB-

In

a

later

study

Nie

et

al.

reported

similar

findings

in

the

same

cell

line

although

desensitization

occurred

at

a

somewhat

faster

pace

-LRB-

h

-RRB-

-LSB-

-RSB-

Interestingly

Palmer

et

al.

were

unable

to

demonstrate

desensitization

in

CHO

cells

expressing

the

human

-LSB-

-RSB-

Klaasse

et

al.

however

were

able

to

show

internalization

of

the

human

tagged

with

a

C-terminal

yellow

fluorescent

protein

-LRB-

-RRB-

stably

expressed

in

CHO

cells

Exposure

of

these

cells

for

h

to

nM

or

μM

CPA

resulted

in

and

receptor

internalization

respectively

Addition

of

μM

of

the

allosteric

enhancer

did

not

accelerate

the

internalization

process

but

lowered

the

threshold

concentration

at

which

internalization

occurred

Under

those

conditions

a

small

degree

of

internalization

was

observed

already

at

a

concentration

of

nM

CPA

and

at

nM

CPA

of

the

receptors

internalized

-LSB-

-RSB-

Exposure

of

cerebellar

granule

cells

endogenously

expressing

to

nM

R-PIA

for

h

led

to

a

blunting

of

the

inhibition

of

adenylyl

cyclase

Along

with

this

observation

a

decrease

of

-LSB-

-RSB-

cyclohexyladenosine

-LRB-

-LSB-

-RSB-

CHA

-RRB-

binding

to

intact

cerebellar

granule

cells

and

an

increase

of

-LSB-

-RSB-

CHA

binding

in

microsomes

was

detected

Simultaneously

a

decrease

in

the

steady-state

level

of

in

plasma

membrane

and

microsomes

was

observed

These

findings

point

not

only

to

homologous

desensitization

but

also

to

subsequent

internalization

of

the

in

the

microsomal

fraction

of

cerebellar

granule

cells

upon

long-term

agonist

exposure

However

no

change

in

mRNA

level

was

observed

suggesting

that

post-transcriptional

regulation

underlies

receptor

desensitization

-LSB-

-RSB-

In

hippocampal

neurons

are

present

on

both

presynaptic

and

postsynaptic

terminals

as

well

as

on

the

cell

body

and

dendrites

where

they

exert

different

actions

To

address

the

question

of

whether

desensitization

was

influenced

by

subcellular

localization

neurons

were

exposed

to

the

agonist

-LRB-

CADO

μM

-RRB-

from

up

to

h

It

was

found

that

upon

agonist

exposure

in

cultured

hippocampal

neurons

the

presynaptic

desensitize

less

quickly

-LRB-

h

-RRB-

than

the

postsynaptic

-LRB-

h

-RRB-

In

accordance

the

recovery

of

desensitized

presynaptic

also

requires

more

time

-LRB-

h

vs

h

for

postsynaptic

-RRB-

Desensitization

of

the

postsynaptic

apparently

occurs

at

the

level

of

the

receptor

because

the

other

elements

of

the

signal

transduction

machinery

appeared

to

be

fully

functional

upon

receptor

desensitization

All

in

all

these

results

suggest

that

the

extent

and

the

kinetics

of

agonist-induced

desensitization

of

depend

on

the

subcellular

localization

of

the

receptors

-LSB-

-RSB-

Using

a

more

intact

preparation

Coelho

et

al.

found

that

the

density

of

in

rat

hippocampal

slices

was

decreased

by

upon

min

of

imposed

hypoxia

This

desensitization

could

be

mimicked

by

adding

the

agonist

CADO

-LRB-

μM

-RRB-

for

min

and

was

prevented

by

adding

the

antagonist

DPCPX

These

results

suggest

that

hypoxia

leads

to

an

increase

in

extracellular

adenosine

levels

and

a

subsequent

quite

rapid

-LRB-

min

-RRB-

desensitization

possibly

followed

by

subsequent

internalization

of

the

in

nerve

terminals

-LSB-

-RSB-

Rat

in

vivo

studies

also

demonstrated

desensitization

In

a

study

by

Parsons

and

Stiles

rats

were

chronically

-LRB-

days

-RRB-

infused

with

R-PIA

Examination

of

adipocyte

membranes

of

both

treated

and

control

rats

revealed

a

lower

inhibition

of

adenylyl

cyclase

in

the

treated

animals

coinciding

with

a

reduced

number

of

-LRB-

agonist-occupied

receptors

remaining

-RRB-

as

determined

by

radioligand

binding

-LSB-

-RSB-

Upon

further

study

it

was

noticed

that

the

effects

on

adenylyl

cyclase

were

not

specific

but

were

controlled

at

the

level

of

adipocyte

G

proteins

Gi

levels

appeared

downregulated

whereas

the

amount

of

Gs

in

the

preparation

was

increased

although

not

at

the

level

of

their

mRNA

suggesting

a

heterologous

form

of

desensitization

-LSB-

-RSB-

Chronic

exposure

of

rats

to

R-PIA

-LRB-

days

-RRB-

also

led

to

desensitization

in

the

brain

and

subsequent

reduced

inhibition

of

adenylyl

cyclase

This

loss

of

response

was

accompanied

by

a

significant

decrease

in

both

total

numbers

of

and

proteins

in

synaptic

plasma

membranes

in

the

brain

paralleling

the

finding

by

Stiles

and

co-workers

in

adipocytes

-LSB-

-RSB-

As

a

consequence

a

significant

increase

of

was

observed

in

microsomes

and

coated

vesicles

which

suggested

a

role

for

coated

vesicles

in

the

internalization

of

Similarly

chronic

agonist

exposure

of

rats

to

NECA

-LRB-

days

-RRB-

resulted

in

a

significant

decrease

of

in

the

high-affinity

state

in

the

rat

brain

however

without

changes

in

adenylyl

cyclase

activity

or

inhibition

of

the

proteins

-LSB-

-RSB-

Molecular

mechanisms

As

discussed

in

the

general

introduction

to

this

review

receptor

posttranslational

modifications

receptor

phosphorylation

recruitment

of

arrestins

and

the

formation

of

clathrin-coated

pits

are

elements

of

the

molecular

machinery

of

desensitization

and

internalization

In

addition

other

potential

protein

partners

in

the

two

processes

have

been

studied

for

the

various

adenosine

receptor

subtypes

which

will

also

be

discussed

Effect

of

receptor

posttranslational

modifications

on

desensitization

and/or

internalization

Gao

et

al.

studied

the

effects

of

preventing

palmitoylation

of

the

It

appeared

that

the

mutation

thus

removing

the

palmitoylation

site

of

the

human

had

no

effect

on

internalization

-LSB-

-RSB-

These

findings

were

later

confirmed

by

Ferguson

et

al.

-LSB-

-RSB-

GRKs

and

arrestins

phosphorylation

by

GRKs

has

been

subject

to

debate

Palmer

et

al.

and

Ferguson

et

al.

failed

to

demonstrate

phosphorylation

of

in

the

latter

study

it

was

found

that

nonphosphorylated

redistributes

arrestin

from

the

cytoplasm

into

punctate

clusters

at

the

plasma

membrane

-LSB-

-RSB-

Nie

et

al.

however

reported

that

within

h

of

exposure

of

cells

to

R-PIA

rapid

translocation

of

GRKs

was

observed

from

the

cytosol

to

the

cytoplasm

-LSB-

-RSB-

In

a

further

biochemical

approach

with

both

purified

receptor

and

a

phosphorylated

receptor

was

obtained

that

showed

enhanced

affinity

for

arrestins

over

G

proteins

The

same

receptor

kinase

upon

overexpression

in

cells

influenced

signalling

however

not

via

mediated

adenylyl

cyclase

but

through

mediated

MAP

kinase

activation

-LSB-

-RSB-

It

may

be

concluded

that

under

physiological

conditions

phosphorylation

does

not

-LRB-

readily

-RRB-

take

place

which

would

be

a

rationale

for

the

long

time

periods

required

for

receptor

internalization

Other

protein

partners

The

ectoenzyme

adenosine

deaminase

-LRB-

ADA

-RRB-

regulates

the

extracellular

adenosine

concentration

by

converting

excess

adenosine

into

inosine

However

ADA

also

plays

a

role

in

the

desensitization

and

internalization

of

in

smooth

muscle

cells

-LSB-

-RSB-

and

epithelial

cells

-LSB-

-RSB-

acting

as

a

receptor

activity-modifying

protein

-LRB-

RAMP

-RRB-

Upon

agonist

exposure

-LRB-

nM

R-PIA

h

-RRB-

ADA

and

formed

complexes

on

the

cell

surface

clustered

and

internalized

together

to

intracellular

compartments

Such

clustering

of

adenosine

receptors

prior

to

internalization

was

also

reported

by

Ciruela

et

al.

and

Saura

et

al.

-LSB-

-RSB-

The

intracellular

vesicles

contained

the

lipid

raft

marker

protein

caveolin

Filipin

an

agent

that

disrupts

rafts

or

caveolae

inhibited

internalization

In

contrast

acidic

treatment

disrupting

clathrin-coated

vesicles

did

not

inhibit

agonist-induced

internalization

of

These

results

indicate

that

ADA

and

form

a

stable

complex

in

the

cell

membrane

of

cells

internalizing

upon

agonist

exposure

via

lipid

rafts

in

a

clathrin-independent

pathway

Furthermore

a

direct

interaction

of

the

C

terminus

of

with

caveolin

was

demonstrated

-LSB-

-RSB-

These

and

other

data

suggest

that

the

mode

of

receptor

compartmentalization

in

response

to

agonist

stimulation

may

be

governed

by

both

receptor

subtype

and

cell

type

-LSB-

-RSB-

Another

accessory

protein

the

heat

shock

cognate

protein

-LRB-

-RRB-

a

member

of

the

family

was

identified

as

a

cytosolic

component

able

to

interact

with

the

third

intracellular

loop

of

the

The

interaction

between

-LRB-

nM

-RRB-

and

led

to

a

marked

reduction

in

affinity

of

ligands

for

the

and

also

prevented

activation

of

the

G

proteins

even

more

so

than

the

addition

of

GTP

analogues

or

GTP

itself

-LRB-

μM

-RRB-

These

effects

were

completely

prevented

by

the

addition

of

nM

ADA

A

high

percentage

of

was

coupled

to

in

cell

lysate

according

to

immunoprecipitation

experiments

A

remarkable

feature

upon

internalization

of

the

receptor

in

cells

was

found

internalized

via

two

different

vesicle

types

one

in

which

and

are

colocalized

and

another

in

which

was

absent

These

observations

open

the

possibility

that

signalling

via

GPCRs

is

regulated

at

least

to

some

extent

by

heat

shock

proteins

-LSB-

-RSB-

Receptor-receptor

interactions

Dunwiddie

et

al.

found

that

activation

of

with

a

selective

agonist

resulted

in

subsequent

heterologous

desensitization

of

the

as

observed

in

electrophysiological

experiments

in

the

region

of

rat

hippocampus

Similar

results

were

obtained

after

occupancy

via

a

brief

superfusion

with

a

high

concentration

of

adenosine

-LSB-

-RSB-

Lopes

et

al.

investigated

how

activated

influenced

function

and

whether

this

interaction

was

modified

in

aged

rats

In

hippocampal

and

cortical

nerve

terminals

the

agonist

CGS

-LRB-

nM

-RRB-

was

able

to

lower

the

binding

affinity

of

the

agonist

CPA

and

this

was

taken

as

proof

for

desensitization

The

effect

was

counteracted

by

the

addition

of

the

antagonist

ZM

-LRB-

nM

-RRB-

This

reduction

in

function

could

only

be

detected

in

young

adult

rats

-LRB-

weeks

-RRB-

but

not

in

old

rats

-LRB-

months

-RRB-

The

addition

of

a

PKC

inhibitor

chelerythrine

-LRB-

μM

-RRB-

also

prevented

the

desensitization

of

-LSB-

-RSB-

Similarly

Ciruela

et

al.

-LSB-

-RSB-

demonstrated

that

heteromers

were

able

to

modulate

the

glutamatergic

neurotransmission

in

the

rat

striatum

The

main

biochemical

characteristic

of

this

heteromer

is

the

ability

of

the

agonist-occupied

to

reduce

the

agonist

affinity

of

the

-LSB-

-RSB-

By

binding

to

cannabinoid

receptors

in

Purkinje

fibers

in

the

cerebellum

inhibits

adenylyl

cyclase

and

consequently

motor

coordination

Long-term

treatment

resulted

in

downregulation

desensitization

of

the

protein

and

desensitization

of

adenylyl

cyclase

G

protein

activation

by

however

was

unaffected

Surprisingly

heterologous

attenuation

of

inhibition

of

adenylyl

cyclase

was

observed

These

results

indicate

that

long-term

administration

produces

a

disruption

of

inhibitory

receptor

control

of

cerebellar

adenylyl

cyclase

and

suggest

a

potential

mechanism

of

cross-tolerance

to

the

motor

effects

of

cannabinoid

and

agonists

-LSB-

-RSB-

and

have

been

shown

to

form

functionally

interacting

heteromeric

complexes

in

engineered

cell

lines

and

in

cortical

neurons

in

culture

Pretreatment

with

an

agonist

caused

complex

formation

of

both

receptors

Combined

pretreatment

with

selective

agonists

for

both

receptors

-LRB-

but

not

one

agonist

alone

-RRB-

substantially

reduced

cAMP

accumulation

induced

via

the

indicative

for

this

receptor

's

desensitization

-LSB-

-RSB-

receptor

Cellular

and

physiological

studies

cells

expressing

both

and

were

exposed

to

an

agonist

which

resulted

in

a

rapid

loss

-LRB-

min

-RRB-

of

agonist-stimulated

cAMP

production

in

these

cells

This

receptor

desensitization

however

did

not

involve

a

reduction

in

cell

membrane

receptor

number

or

a

change

in

ligand

affinity

-LSB-

-RSB-

Prolonged

exposure

of

cells

expressing

both

and

to

AR

agonists

led

to

a

fast

-LRB-

min

-RRB-

and

significant

inhibition

of

stimulation

by

the

agonist

This

effect

appeared

to

occur

at

the

level

of

adenylyl

cyclase

since

no

change

was

observed

in

receptor

number

or

in

's

affinity

for

the

receptor

This

conclusion

was

corroborated

by

the

finding

of

reduced

activation

of

adenylyl

cyclase

by

forskolin

Longer

agonist

exposure

-LRB-

h

-RRB-

led

to

a

reduction

of

levels

whereas

no

changes

occurred

in

the

short-term

protocol

-LSB-

-RSB-

neuroblastoma

x

glioma

hybrid

cells

express

both

and

-LSB-

-RSB-

Treatment

of

the

cells

with

the

non-selective

agonist

NECA

followed

by

its

washout

led

to

a

rapid

-LRB-

min

-RRB-

and

pronounced

reduction

in

cAMP

production

by

the

agonist

when

compared

to

vehicle-treated

cells

Palmer

et

al.

-LSB-

-RSB-

generated

CHO

cells

solely

expressing

the

recombinant

-LRB-

canine

-RRB-

and

examined

the

desensitization

process

of

this

receptor

Cells

exposed

to

NECA

showed

a

rapid

desensitization

of

adenylyl

cyclase

activity

with

no

obvious

difference

between

pretreatment

of

min

or

h

This

was

associated

with

a

slightly

reduced

affinity

of

the

receptor

for

the

radiolabelled

agonist

-LSB-

-RSB-

Cell

surface

receptor

numbers

only

diminished

significantly

-LRB-

up

to

-RRB-

upon

longer-term

pretreatment

-LRB-

h

-RRB-

-LSB-

-RSB-

Using

a

tissue

preparation

Conti

et

al.

investigated

whether

prolonged

exposure

of

to

the

non-selective

agonist

NECA

or

to

the

selective

agonists

and

influenced

desensitization

The

authors

used

the

porcine

coronary

artery

as

a

sensitive

vascular

model

expressing

among

others

The

arteries

were

first

precontracted

by

adding

μM

PGFα

NECA

and

showed

high

affinities

for

the

-LRB-

's

of

and

nM

respectively

-RRB-

inducing

vasorelaxation

Next

coronary

arteries

were

pretreated

with

μM

NECA

for

min

or

h

After

a

washout

period

the

functional

response

was

assessed

It

appeared

that

preincubation

with

NECA

did

not

hamper

the

vasorelaxing

effects

of

and

However

NECA

response

curves

were

shifted

to

the

right

after

NECA

pretreatment

-LSB-

-RSB-

These

results

seem

inconclusive

since

NECA

pretreatment

might

hit

other

adenosine

receptor

subtypes

next

to

are

present

on

presynaptic

baroreceptor

afferent

terminals

within

the

nucleus

tractus

solitarius

-LRB-

NTS

-RRB-

in

the

brain

-LSB-

-RSB-

It

was

observed

that

these

modulate

release

as

a

mechanism

of

baroreflex

control

mediated

by

the

NTS

Low

concentrations

of

the

non-selective

agonist

NECA

-LRB-

nM

-RRB-

briefly

exposed

-LRB-

min

-RRB-

to

NTS

brain

slices

induced

the

release

of

caused

by

the

activation

of

the

present

at

presynaptic

nerves

This

effect

could

be

blocked

by

the

addition

of

the

adenosine

antagonist

-LRB-

-RRB-

caffeine

-LRB-

CSC

nM

-RRB-

Longer

exposure

of

NECA

-LRB-

min

-RRB-

to

the

NTS

slices

resulted

in

inhibition

of

the

release

probably

caused

by

quick

desensitization

of

the

-LRB-

min

-RRB-

and

subsequent

involvement

of

the

-LSB-

-RSB-

These

findings

were

corroborated

in

a

similar

study

with

an

agonist

-LSB-

-RSB-

The

effects

on

AR

fate

upon

chronic

agonist

exposure

were

studied

in

vivo

in

rat

brain

-LSB-

-RSB-

After

days

of

NECA

treatment

the

adenylyl

cyclase

activity

in

synaptic

plasma

membranes

was

decreased

suggesting

a

desensitization

of

although

the

authors

did

not

specify

or

study

which

subtype

was

involved

Gs

protein

levels

were

decreased

indicating

Gs

downregulation

as

the

mechanism

of

desensitization

Interestingly

Rekik

and

Mustafa

-LSB-

-RSB-

showed

that

chronic

antagonist

treatment

-LRB-

days

-RRB-

of

porcine

coronary

arteries

with

led

to

a

decreased

agonist

responsiveness

Although

receptor

expression

went

up

it

appeared

that

the

levels

of

Gs

had

decreased

altogether

leading

to

a

functional

desensitization

of

the

relaxing

response

by

e.g.

-LSB-

-RSB-

It

should

be

kept

in

mind

that

is

also

a

potent

antagonist

for

the

adenosine

receptor

Molecular

mechanisms

Effect

of

receptor

C

terminus

on

desensitization

and/or

internalization

To

investigate

the

importance

of

the

-LRB-

amino

acid

residues

-RRB-

long

C

terminus

of

the

receptor

in

inducing

desensitization

and

internalization

Palmer

and

Stiles

introduced

several

mutations

and

deletions

into

the

receptor

tail

It

appeared

that

deletion

of

the

last

amino

acids

of

the

C

terminus

containing

possible

phosphorylation

sites

did

not

have

any

effect

on

radioligand

binding

adenylyl

cyclase

activity

or

desensitization

kinetics

compared

to

the

wild-type

However

when

two

possible

phosphorylation

sites

-LRB-

Thr

and

Ser

-RRB-

just

upstream

the

deleted

amino

acids

were

mutated

to

Ala

short-term

-LRB-

min

-RRB-

agonist-induced

desensitization

was

attenuated

while

the

long-term

-LRB-

h

-RRB-

desensitization

was

not

affected

Single

mutations

revealed

that

mutation

of

Thr

alone

was

sufficient

to

reduce

receptor

phosphorylation

and

agonist-induced

short-term

desensitization

This

study

also

shows

that

short-term

and

long-term

desensitization

have

distinct

structural

requirements

and

do

not

occur

via

the

same

mechanism

-LSB-

-RSB-

GRKs

and

arrestins

Which

GRK

isoforms

are

involved

in

the

phosphorylation

of

the

is

not

entirely

clear

yet

However

a

putative

role

for

and/or

has

been

suggested

-LSB-

-RSB-

The

role

of

in

agonist-induced

phosphorylation

and

subsequent

desensitization

of

and

was

thoroughly

investigated

by

Kelly

and

co-workers

Wild-type

was

stably

expressed

in

cells

which

endogenously

express

and

The

acute

stimulation

of

adenylyl

cyclase

by

activation

of

was

markedly

reduced

in

cells

overexpressing

wild-type

This

was

probably

caused

by

pre-desensitization

of

the

by

extracellular

adenosine

This

effect

could

be

reversed

by

pretreating

the

cells

h

with

unit/ml

ADA

-LSB-

-RSB-

The

same

research

group

investigated

the

effect

of

a

dominant-negative

mutant

on

the

desensitization

of

the

-LSB-

-RSB-

Stable

transfection

of

a

mutant

in

cells

reduced

desensitization

of

the

by

following

a

treatment

with

the

adenosine

agonist

NECA

Tumour

necrosis

factor

-LRB-

TNF

-RRB-

α

treatment

of

human

monocytoid

cells

expressing

the

prevented

desensitization

of

this

receptor

occurring

under

control

conditions

upon

pretreatment

with

or

NECA

-LSB-

-RSB-

It

was

discovered

that

the

TNF-α

treatment

prevented

translocation

to

and

decreased

association

with

the

plasma

membranes

of

these

cells

as

a

consequence

of

the

activation

of

a

sphingomyelinase-dependent

pathway

In

another

study

Mundell

and

Kelly

investigated

the

effect

of

inhibitors

of

receptor

internalization

on

desensitization

and

resensitization

of

ARs

in

cells

-LSB-

-RSB-

Before

agonist

exposure

cells

were

pretreated

with

hypertonic

sucrose

or

concanavalin

A

-LRB-

ConA

-RRB-

both

inhibitors

of

internalization

This

pretreatment

did

not

affect

the

agonist-induced

desensitization

of

the

However

the

resensitization

of

the

upon

agonist

removal

was

abolished

in

the

presence

of

ConA

or

sucrose

Arrestins

and

have

been

implicated

in

the

downstream

desensitization

process

stimulation

of

a

tagged

transiently

transfected

in

cells

induced

the

translocation

of

GFP-tagged

arrestins

and

towards

the

plasma

membrane

A

dominant-negative

arrestin

mutant

inhibited

agonist-induced

internalization

-LSB-

-RSB-

Other

protein

partners

The

long

C

terminus

of

the

has

been

coined

a

coincidence

detector

as

it

recognizes

quite

a

number

of

other

proteins

such

as

α-actinin

and

ARNO

the

Arf

nucleotide

site

opener

-LRB-

for

a

review

see

Gsandtner

and

Freissmuth

-LSB-

-RSB-

-RRB-

α-Actinin

may

play

a

role

in

receptor

internalization

not

unlikely

for

a

protein

involved

in

cytoskeletal

arrangements

-LSB-

-RSB-

however

ARNO

does

not

-LSB-

-RSB-

Receptor-receptor

interactions

The

colocalizes

with

dopamine

receptors

in

the

basal

ganglia

and

their

interaction

has

been

documented

on

several

occasions

-LSB-

-RSB-

For

instance

the

protein-protein

interaction

between

was

confirmed

in

cells

The

most

likely

mode

of

interaction

is

that

helix

and/or

helix

and

the

N-terminal

portion

of

in

the

approach

helix

and

the

C

terminus

of

the

-LSB-

-RSB-

Within

the

scope

of

this

review

fits

the

observation

that

activation

of

the

receptor

actually

sensitizes

increases

in

cAMP

production

in

CHO

cells

expressing

both

receptors

-LSB-

-RSB-

as

well

as

in

CAD

and

neuroblastoma

cells

in

which

the

receptor

was

expressed

-LSB-

-RSB-

In

neuroblastoma

cells

co-stimulation

of

and

accelerated

desensitization

probably

by

causing

or

enhancing

internalization

-LSB-

-RSB-

Recently

it

was

found

that

in

these

neuroblastoma

cells

the

also

formed

heteromers

with

the

endogenously

expressed

The

is

negatively

coupled

to

adenylyl

cyclase

and

requires

previous

or

simultaneous

activation

of

to

signal

in

these

cells

-LSB-

-RSB-

receptor

Cellular

and

physiological

studies

The

adenosine

receptor

-LRB-

-RRB-

is

endogenously

expressed

on

most

artificial

cell

lines

such

as

COS

and

cells

The

exception

is

formed

by

CHO

cells

that

lack

this

adenosine

receptor

subtype

The

like

the

other

three

adenosine

receptor

subtypes

is

subject

to

agonist-induced

desensitization

This

was

measured

on

the

level

of

cAMP

production

by

Peters

et

al.

-LSB-

-RSB-

Pretreatment

of

cells

endogenously

expressing

receptors

with

the

non-selective

agonist

NECA

-LRB-

μM

-RRB-

for

up

to

h

led

to

a

significant

reduction

in

cAMP

production

upon

acute

agonist

challenge

already

after

h

of

pretreatment

Maximal

reduction

of

cAMP

production

was

reached

after

h

of

pretreatment

CHO

cells

stably

expressing

a

FLAG

epitope-tagged

receptor

showed

the

same

result

albeit

that

maximal

reduction

of

cAMP

response

was

already

achieved

after

h

of

pretreatment

Also

mouse

cells

and

human

cells

endogenously

expressing

the

showed

a

decreased

cAMP

response

after

h

and

h

of

pretreatment

with

μM

NECA

respectively

-LSB-

-RSB-

In

cells

expressing

both

and

the

rate

of

desensitization

for

both

receptor

subtypes

appeared

similar

with

a

half-life

of

min

Pretreatment

with

NECA

of

these

cells

-LRB-

mM

min

-RRB-

reduced

stimulation

of

adenylyl

cyclase

by

by

approximately

-LSB-

-RSB-

Rat

pheochromocytoma

cells

also

endogenously

express

both

and

Prolonged

exposure

-LRB-

h

-RRB-

to

nM

NECA

or

μM

significantly

inhibited

the

cAMP

response

of

the

cells

to

subsequent

stimulation

with

the

agonist

Although

a

higher

NECA

concentration

is

needed

to

stimulate

the

compared

to

Chern

et

al.

found

that

the

receptor

cAMP

response

was

also

desensitized

upon

desensitization

of

the

receptor

This

observation

can

be

explained

by

the

fact

that

the

protein

level

and

the

adenylyl

cyclase

activity

were

diminished

upon

long-term

desensitization

of

the

by

-LSB-

-RSB-

The

domain

in

which

the

receptor

resides

is

also

important

for

desensitization

events

according

to

Sitaraman

et

al.

NECA

-LRB-

μM

-RRB-

added

to

either

the

apical

or

basolateral

side

of

intestinal

cells

resulted

in

desensitization

of

the

on

the

corresponding

side

within

h.

However

whereas

applying

NECA

to

the

apical

side

of

the

membrane

had

no

effect

on

the

basolateral

basolateral

NECA

induced

a

complete

desensitization

of

the

apical

receptor

Since

receptor

trafficking

may

play

a

role

in

this

cross-desensitization

process

this

effect

may

contribute

to

the

desensitization

and

subsequent

downregulation

of

the

-LSB-

-RSB-

Trincavelli

et

al.

observed

a

very

rapid

desensitization

-LRB-

min

-RRB-

of

in

a

human

astrocytoma

cell

line

Both

G

protein

coupling

efficiency

and

cAMP

production

were

diminished

after

pretreatment

of

the

cells

with

NECA

in

the

presence

of

an

antagonist

-LSB-

-RSB-

Mundell

et

al.

reported

that

acute

exposure

of

human

airway

smooth

muscle

-LRB-

ASM

-RRB-

cells

to

adenosine

receptor

agonists

resulted

in

a

rapid

accumulation

of

cAMP

most

probably

via

Treatment

with

adenosine

deaminase

-LRB-

ADA

-RRB-

suggested

that

ASM

cells

produce

adenosine

which

feeds

back

on

the

cells

thereby

regulating

basal

cAMP

levels

and

inducing

a

small

degree

of

desensitization

Chronic

treatment

with

adenosine

agonists

had

a

dual

effect

both

desensitization

and

adenylyl

cyclase

sensitization

-LRB-

an

increased

responsiveness

of

adenylyl

cyclase

upon

stimulation

-RRB-

were

observed

-LSB-

-RSB-

Haynes

et

al.

-LSB-

-RSB-

studied

desensitization

in

both

pulmonary

artery

smooth

muscle

cells

and

a

more

integrated

preparation

the

isolated

perfused

lung

Pretreatment

of

the

smooth

muscle

cells

with

NECA

for

min

abrogated

the

increase

in

cAMP

response

otherwise

observed

for

both

NECA

and

isoproterenol

suggesting

heterologous

desensitization

Indeed

experiments

with

cholera

toxin

showed

that

the

desensitization

took

place

at

the

level

of

the

Gs

protein-adenylyl

cyclase

complex

In

the

lung

preparation

NECA

when

dosed

acutely

caused

a

rapid

vasodilation

which

was

not

observed

when

the

tissue

was

treated

again

with

the

same

compound

min

after

the

first

dose

-LSB-

-RSB-

Taken

together

these

results

suggest

that

desensitization

of

the

receptor

is

a

robust

phenomenon

rather

independent

from

the

cell

type

or

tissue

in

which

it

is

expressed

Molecular

mechanisms

Effects

of

receptor

mutations

on

desensitization

and/or

internalization

Matharu

et

al.

studied

the

regions

of

the

which

are

responsible

for

the

desensitization

and

internalization

of

the

-LSB-

-RSB-

For

this

purpose

they

introduced

point

mutations

or

deletions

in

the

C

terminus

of

the

Deleting

the

final

two

amino

acids

-LRB-

-RRB-

did

not

affect

the

internalization

properties

of

the

The

and

mutants

however

were

resistant

to

agonist-induced

desensitization

and

internalization

GFP-tagged

arrestin

did

not

translocate

from

the

cytosol

to

the

plasma

membrane

upon

agonist

stimulation

of

these

two

truncated

receptors

From

a

single

point

mutation

in

the

C

terminus

it

became

clear

that

this

serine

residue

is

responsible

for

the

rapid

agonist-induced

desensitization

and

internalization

Surprisingly

a

further

deletion

mutant

was

able

to

undergo

rapid

agonist-induced

desensitization

and

internalization

however

arrestin

was

not

attracted

to

the

plasma

membrane

upon

agonist

stimulation

It

appeared

that

internalization

of

this

truncated

receptor

occurred

via

an

arrestin

and

clathrin-independent

pathway

probably

via

caveolin-mediated

internalization

since

the

internalization

of

was

dynamin

dependent

The

destination

of

this

truncated

receptor

was

also

different

a

compartment

close

to

the

plasma

membrane

instead

of

early

endosomes

Whether

the

receptor

will

undergo

rapid

agonist-induced

desensitization

and

internalization

or

influence

the

rate

extent

and

mechanism

of

internalization

depends

on

specific

sites

in

the

C

terminus

of

the

and

can

be

influenced

by

their

mutation

or

deletion

GRKs

and

arrestins

The

endogenously

expressed

in

cells

is

desensitized

through

phosphorylation

by

This

effect

was

selective

for

and

since

the

other

endogenously

expressed

receptors

-LRB-

secretin

R

and

IP-prostanoid

R

-RRB-

were

not

desensitized

in

this

way

-LSB-

-RSB-

In

addition

stable

transfection

of

a

dominant

negative

-LRB-

-RRB-

mutant

in

the

cells

reduced

the

NECA-induced

-LRB-

min

μM

-RRB-

desensitization

of

the

by

-LSB-

-RSB-

Peters

et

al.

studied

the

role

of

phosphorylation

by

PKA

and

PKC

rather

than

in

agonist-induced

desensitization

in

cells

transiently

expressing

the

FLAG

They

concluded

that

these

second

messenger-dependent

kinases

are

not

involved

in

phosphorylation

-LSB-

-RSB-

A

similar

conclusion

for

phosphorylation

in

cells

was

reached

by

Mundell

and

Kelly

-LSB-

-RSB-

Trincavelli

et

al.

found

that

treatment

of

astrocytoma

cells

with

TNF-α

increased

the

functional

response

without

changing

receptor

protein

or

mRNA

levels

TNF-α

treatment

appeared

to

markedly

reduce

agonist-dependent

receptor

phosphorylation

and

also

attenuated

agonist-mediated

desensitization

These

effects

could

be

inhibited

by

the

addition

of

the

antagonist

MRS

-LSB-

-RSB-

These

findings

resemble

data

obtained

with

the

-LRB-

vide

supra

-RRB-

Mundell

et

al.

found

that

within

the

arrestin

family

both

arrestin

and

arrestin

are

involved

in

the

internalization

process

of

the

receptor

Within

min

of

agonist

exposure

both

GFP-tagged

arrestin

isoforms

were

translocated

to

the

plasma

membrane

Longer

agonist

exposure

-LRB-

min

-RRB-

however

revealed

that

only

arrestin

colocalizes

with

the

receptor

in

the

early

endosomes

One

explanation

for

this

may

be

a

higher

affinity

of

the

for

arrestin

compared

to

arrestin

Interestingly

arrestins

are

not

only

involved

in

the

internalization

of

the

receptor

but

also

in

its

recycling

It

appeared

that

the

expression

of

stable

arrestin

anti-sense

constructs

reducing

the

levels

of

endogenous

arrestins

not

only

resulted

in

less

internalization

of

the

receptors

but

also

impaired

the

recycling

process

significantly

In

contrast

to

the

internalization

process

arrestin

induced

a

significantly

faster

rate

of

recycling

compared

to

arrestin

-LSB-

-RSB-

Penn

et

al.

also

studied

the

behaviour

of

GFP-tagged

arrestins

now

in

human

airway

smooth

muscle

cells

Arrestin

and

arrestin

were

able

to

reduce

the

cAMP

production

upon

stimulation

of

endogenous

by

NECA

and

stimulation

of

by

isoproterenol

In

addition

a

punctate

clustering

was

observed

in

the

membrane

upon

exposure

to

either

NECA

-LRB-

μM

-RRB-

or

isoproterenol

-LRB-

μM

-RRB-

indicating

that

arrestins

play

a

role

in

receptor

trafficking

On

the

contrary

signalling

and

trafficking

of

the

prostaglandin

another

endogenously

expressed

receptor

was

not

affected

by

arrestin

or

arrestin

-LSB-

-RSB-

receptor

Adenosine

receptors

-LRB-

-RRB-

evoke

considerable

interest

as

novel

drug

targets

to

address

cerebral/cardiac

ischaemia

cancer

inflammation

asthma

and

chronic

obstructive

pulmonary

disease

So

far

potent

and

selective

antagonists

for

the

have

been

identified

the

disadvantage

however

is

that

those

antagonists

show

extremely

low

binding

affinity

for

the

rodent

typically

times

lower

than

in

humans

Since

rodent

models

are

essential

for

the

pharmacological

evaluation

of

new

therapeutic

agents

this

forms

a

serious

drawback

In

addition

the

adenosine

receptor

-LRB-

-RRB-

sequence

holds

a

nuclear

localization

signal

in

its

C-terminal

tail

-LSB-

-RSB-

This

typical

stretch

of

four

amino

acids

-LRB-

KKFK

-RRB-

in

helix

may

direct

the

receptor

to

the

cell

nucleus

not

necessarily

as

a

consequence

of

desensitization

or

internalization

This

should

be

kept

in

mind

when

appreciating

the

studies

described

below

Cellular

and

physiological

studies

Ramkumar

et

al.

studied

the

characteristics

of

rat

endogenously

expressed

on

mast

cells

In

one

of

their

experiments

the

authors

pretreated

the

cells

with

NECA

and

found

a

partial

desensitization

of

the

initial

response

to

NECA

-LSB-

-RSB-

Trincavelli

et

al.

studied

the

relationship

between

agonist-induced

desensitization

internalization

and

resensitization

of

in

transfected

CHO

cells

Agonist-induced

endocytosis

of

was

investigated

by

immunogold

electron

microscopy

of

plasma

membranes

and

intracellular

vesicles

and

shown

to

occur

with

a

half-life

of

min

Subsequent

removal

of

the

agonist

led

to

recycling

of

of

the

receptor

population

to

the

cell

surface

with

a

half-life

of

min

Short-term

exposure

to

agonist

caused

rapid

desensitization

as

assessed

in

cAMP

assays

Removal

of

the

agonist

led

to

resensitization

of

the

cAMP

signal

to

of

the

original

signal

within

min

Internalization

did

not

affect

signal

transduction

as

was

demonstrated

after

blockade

of

internalization

and

recycling

Internalization

occurred

via

clathrin-coated

pits

These

results

show

that

undergoes

agonist-induced

endocytosis

which

is

not

responsible

for

desensitization

Moreover

in

the

case

of

receptor

sequestration

rather

than

desensitization

seems

to

be

the

first

step

in

a

cycle

of

internalization

dephosphorylation

and

recycling

to

the

plasma

membrane

-LSB-

-RSB-

A

similar

cell

line

was

used

by

Palmer

et

al.

In

stably

transfected

CHO

cells

prolonged

treatment

with

NECA

-LRB-

μM

h

-RRB-

induced

uncoupling

of

recombinant

from

Gi

proteins

and

a

functional

desensitization

Upon

this

desensitization

an

approximately

twofold

increase

in

adenylyl

cyclase

activity

was

found

in

the

presence

or

absence

of

forskolin

This

sensitization

of

adenylyl

cyclase

activity

was

not

caused

by

an

altered

level

of

inhibitory

or

stimulatory

G

protein

expression

The

occurrence

of

the

sensitization

was

also

not

due

to

new

protein

synthesis

but

probably

to

an

increased

coupling

efficiency

between

Gs

and

adenylyl

cyclase

Compared

to

control

cells

long-term

exposure

of

stably

transfected

CHO

cells

to

NECA

caused

an

increase

in

phosphorylation

of

the

cAMP-responsive

element

binding

protein

upon

addition

of

suboptimal

concentrations

of

forskolin

The

sensitization

of

adenylyl

cyclase

activity

upon

long-term

treatment

might

provide

a

molecular

basis

for

the

observation

that

for

several

adenosine

receptor-mediated

events

acute

agonist

exposure

produces

opposite

effects

to

those

after

chronic

agonist

treatment

-LSB-

-RSB-

Ferguson

et

al.

studied

the

fate

of

the

rat

expressed

in

CHO

cells

The

receptors

internalized

rapidly

after

treatment

with

NECA

or

R-PIA

over

a

time

frame

-LRB-

t

min

-RRB-

that

followed

receptor

phosphorylation

-LRB-

t

min

-RRB-

-LSB-

-RSB-

The

desensitization

internalization

and

downregulation

of

was

also

investigated

in

human

astrocytoma

cells

natively

expressing

Short-term

-LRB-

min

-RRB-

exposure

of

the

cells

to

nM

Cl-IBMECA

an

agonist

caused

rapid

receptor

desensitization

subsequently

followed

by

receptor

internalization

within

min

With

the

help

of

immunogold

electron

microscopy

the

localization

of

the

was

revealed

After

min

of

exposure

the

was

found

in

smooth-surfaced

pits

and

in

uncoated

vesicles

in

the

cytoplasm

After

min

of

exposure

the

was

found

in

vesicular

endosomes

Upon

removal

of

the

agonist

resensitization

of

the

occurred

within

min

through

receptor

recycling

to

the

cell

surface

Long-term

agonist

exposure

-LRB-

h

-RRB-

resulted

in

a

marked

downregulation

of

to

of

control

after

h

In

conclusion

multiple

temporally

distinct

and

sequential

processes

are

involved

in

the

regulation

of

upon

short

and

long-term

exposure

to

agonists

-LSB-

-RSB-

were

found

to

be

highly

expressed

on

murine

melanoma

cells

-LSB-

-RSB-

The

authors

examined

the

association

between

trafficking

and

receptor

functionality

and

tumour

growth

inhibition

upon

activation

with

the

agonist

IB-MECA

Exposure

to

nM

IB-MECA

-LRB-

min

-RRB-

led

to

rapid

internalization

of

to

the

cytosol

upon

which

receptors

were

directed

to

the

endosomes

for

recycling

or

to

lysosomes

for

degradation

The

addition

of

nM

MRS

-LRB-

min

-RRB-

an

antagonist

was

able

to

counteract

the

internalization

process

as

well

as

the

modulation

of

the

Wnt

pathway

leading

to

proliferation

thereby

emphasizing

the

involvement

of

in

this

process

When

the

melanoma

cells

were

injected

into

nude

mice

tumours

rapidly

developed

Tumour

growth

was

significantly

inhibited

after

administration

of

IB-MECA

to

the

animals

paralleled

by

a

decrease

in

expression

in

tumour

lesions

In

hypoxic

human

and

glioblastoma

cell

lines

it

was

found

that

adenosine

upregulates

the

expression

of

hypoxia-inducible

-LRB-

-RRB-

and

vascular

endothelial

growth

factor

-LRB-

VEGF

-RRB-

via

the

receptor

is

a

key

regulator

in

the

development

of

tumours

The

addition

of

the

MRE

inhibited

the

adenosine-induced

and

VEGF

accumulation

in

these

hypoxic

cells

thereby

showing

a

putative

role

in

inhibiting

tumour

growth

-LSB-

-RSB-

To

circumvent

the

species

differences

mentioned

in

the

introduction

to

the

section

Yamano

et

al.

generated

humanized

-LRB-

-RRB-

mice

in

which

the

was

replaced

by

its

human

counterpart

The

expression

level

of

in

the

humanized

-LRB-

-RRB-

mice

was

equal

to

the

expression

levels

of

in

wild-type

mice

agonists

were

able

to

elevate

the

intracellular

concentration

in

bone

marrow-derived

mast

cells

from

the

humanized

-LRB-

-RRB-

mice

However

the

rate

of

internalization

was

markedly

reduced

compared

with

that

of

in

these

mast

cells

-LSB-

-RSB-

Molecular

mechanisms

Receptor

phosphorylation

GRKs

and

arrestins

Palmer

and

Stiles

as

well

as

Ferguson

et

al.

investigated

which

amino

acid

residues

in

the

C

terminus

are

responsible

and

crucial

for

the

rapid

desensitization

of

the

-LSB-

-RSB-

A

triple

mutant

-LRB-

and

to

Ala

-RRB-

exhibited

dramatically

reduced

phosphorylation

desensitization

and

internalization

of

the

rat

Individual

mutation

of

each

Thr

residue

showed

that

and

were

the

most

important

sites

for

phosphorylation

In

addition

phosphorylation

of

was

necessary

to

observe

phosphorylation

of

but

not

vice

versa

Moreover

changing

for

a

negatively

charged

residue

-LRB-

Glu

-RRB-

was

not

sufficient

to

retain

phosphorylation

at

Mutating

two

predicted

palmitoylation

sites

to

Ala

resulted

in

agonist-independent

basal

phosphorylation

of

the

rat

Such

findings

strongly

suggest

that

palmitoylation

of

these

Cys

residues

is

an

important

factor

in

controlling

accessibility

of

the

C

terminus

of

the

in

the

process

of

recruiting

GRKs

In

fact

the

palmitoylation

sites

are

highly

conserved

between

different

species

e.g.

rat

mouse

human

dog

sheep

Taking

these

results

together

it

appears

that

GRK-mediated

phosphorylation

of

the

C

terminus

follows

a

sequential

mechanism

with

the

receptor

palmitoyl

moieties

in

an

important

regulatory

role

and

phosphorylation

of

being

particularly

crucial

as

an

essential

first

step

In

an

earlier

study

Palmer

et

al.

replaced

the

carboxyl

terminus

of

the

by

that

of

the

-LSB-

-RSB-

This

chimaeric

construct

was

able

to

undergo

agonist-stimulated

phosphorylation

and

functional

desensitization

similar

to

It

was

also

found

that

purified

and

were

all

able

to

enhance

agonist-dependent

phosphorylation

of

as

well

as

the

chimaera

Trincavelli

et

al.

studied

the

involvement

of

extracellularly

regulated

kinases

-LRB-

and

-RRB-

members

of

the

mitogen-activated

protein

kinase

-LRB-

MAPK

-RRB-

family

in

phosphorylation

It

was

found

that

within

min

of

exposure

to

μM

NECA

and

were

already

phosphorylated

in

CHO

cells

stably

expressing

An

inhibitor

of

MAPK

activation

-LRB-

-RRB-

also

caused

inhibition

of

phosphorylation

desensitization

and

internalization

probably

by

preventing

the

membrane

translocation

of

These

results

indicate

that

the

MAPK

cascade

is

involved

in

regulation

by

a

feedback

mechanism

which

controls

activity

and

probably

involves

direct

receptor

phosphorylation

-LSB-

-RSB-

The

rat

basophilic

leukaemia

cell

cell

line

-LRB-

cells

-RRB-

endogenously

expresses

equal

levels

of

arrestin

and

arrestin

Both

arrestin

isoforms

also

have

a

high

and

comparable

affinity

for

clathrin

thereby

promoting

agonist-induced

internalization

cells

also

endogenously

express

a

high

level

of

which

however

neither

recruited

arrestin

nor

arrestin

upon

stimulation

with

NECA

Also

no

changes

in

distribution

were

observed

One

explanation

is

that

follows

an

endocytic

mechanism

upon

agonist

stimulation

that

does

not

involve

arrestin-mediated

clathrin-coated

pit

internalization

Another

possible

explanation

is

that

arrestin

recruitment

was

below

the

limit

of

detection

in

the

cells

-LSB-

-RSB-

Ferguson

et

al.

observed

that

upon

phosphorylation

of

by

GRK

arrestin

is

redistributed

into

punctate

vesicles

both

at

the

plasma

membrane

and

within

the

cytoplasm

Nevertheless

these

authors

also

noticed

that

there

was

no

colocalization

between

and

arrestin

-LSB-

-RSB-

Conclusions

The

adenosine

receptor

subtypes

are

differentially

regulated

when

exposed

to

agonist

treatment

are

not

-LRB-

readily

-RRB-

phosphorylated

and

slowly

internalize

with

a

typical

half-life

of

several

hours

This

feature

may

cause

less

than

average

tachyphylaxis

upon

chronic

agonist

administration

for

instance

in

type

II

diabetes

and

show

much

faster

downregulation

with

similar

kinetics

usually

h

for

short-term

desensitization

Agonists

for

the

receptor

currently

profiled

in

wound

healing

may

thus

suffer

from

declining

efficacy

upon

chronic

administration

in

contrast

to

those

for

the

receptor

The

undergoes

even

faster

downregulation

often

a

matter

of

minutes

The

latter

receptor

also

holds

a

nuclear

localization

signal

in

its

carboxy

terminal

tail

possibly

obscuring

true

agonist-induced

downregulation

The

fast

desensitization

of

the

after

agonist

exposure

may

be

therapeutically

equivalent

to

antagonist

occupancy

of

the

receptor

adenosine receptors
caveolae
desensitization
lipid rafts
internalization
palmitoylation
phosphorylation

β-arrestins
g protein-coupled receptor kinase