Agonist-induced internalization of the β adrenergic receptor in a smooth muscle cell line

Vol. 124, No. 3, 1984

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

November 14, 1984

Pages 863-870

AGONIST-INDUCED

INTERNALIZATION

OF THE 6ADRENERGIC RECEPTOR

IN A SMOOTH MUSCLE CELL LINE E. Sher CNR Center Received

September

and F. Clementi

of Cytopharmacology, Department University of Milano, Milano, 21,

of Pharmacology,

Italy

1984

SUMMARY. The mechanism of agonist-induced desensitization of the 6 adrenergic receptor coupled adenylate cyclase has been studied in a smooth muscle cell line, BC3H-1, tiich expresses both (X and 6 adrenergic receptors and nicotinic receptors. 6 receptors have been investigated in intact cells using as radioligand 3HCGP-12177, an hydrophilic canpound tiich labels only surface The treatment of 8C3H-1 cells with the receptors. agonist Isoproterenol, at 3P but not at 4", induced a dose dependent internalization of the6 adrenergic receptor. Agonist-induced internalization was very rapid, in the order of few minutes. B adrenergic receptor internalization was very specific:the a adrenergic agonist Phenylefrine had almost no effect onfreceptor levels, tiile Isoproterenol treatment had no effect on the number of a adrenergic or nicotinic receptors expressed at the cell surface of these cells. 8 adrenergic receptor internalization is probably the major mechanism responsible for catecholamine desensitization in smooth muscle cells. 0 1984AcademicPress. I~~.

In

different

tissues

desensitization cyclase.

of This

a treatment

8 adrenergic

phenanenon

with

receptors

occurs

both

(ISO)

Isoproterenol (Bars)

in vivo

coupled

to

and in vitro

induces

the

(for

a

adenylate

review

1

see

and 2). Although process,

much work

the molecular

has been done basis

in the recent

underlying

this

years

phenanenon

for

are

clarifying

not yet

this

canpletely

understood. There

are

evidences

desensitization receptor tion

In occurs receptor

agonist

lymphoma other

and glioma types,

without internalization from

cell

mechanisms types.

other

have been cells

provided

for

be

responsible

diffuse

for

mechanism

receptor

frog

for is

internaliza-

erithrocytes,

astro-

(3,4,5).

however, of the canponents

can

A widely

Evidences

by endocytosis. treatment

cell

different

different

internalization

after

cytoma,

in

that

as avian Bar, of the

erithrocytes,

but

through

ccmplex,

desensitization

an "uncoupling" acccmpanied

of the by

a

CAMP

0006-291X/84 $1.50 863

Copyright 0 1984 by Academic Press, Inc. AI1 rights of reproduction in any form reserved.

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 124, No. 3, 1984 dependent

phosphorylation

studying

this

process

regulated

in vivo

human pathology

of

receptors and

ati

in smooth

receptor

factors,

and therapeutics

(7).

this

purpose

smooth

muscle

nicotinic

cells

We were

and are

particularly

involved

which

has several

both

o and

line,

expresses

receptors.

their

interested

since

and

characteristics

(6).

cells,

the BC3H-1 cell

cholinergic

pharmacological

itself

muscle

by different

We used for teristics

of the

Bars

Furthermore,

of these

receptors

are

finely both

in

charac-

B adrenergic

the have

in

biochemical

been

described

(8). To these

study

the

cells,

we took

first

described

12177, variance

with

all

hydrophilic

on intact

in this

intact

BC3H-1

sadrenergic tivity

do

of

paper cells;

agonist Isoproterenol

of agonist-induced

advantage

of the

by Staehelin

traditional

and

selectively, report

characteristics

use of the and

ligands the

plasma

cells,

only

the

1) the 2)

effect

receptors

of a

on the on

MATERIALS

binding This

This

number

adrenergic

of surface

3H-CGP ligand,

studies, allows

at the cell

short-term

in

rajioligand

at highly

measure

surface

treatment ars;

is to

of 3H-CGP 12177 binding

effect

Isoproterenol

Bar

internalization

(9).

membrane.

characteristics the

new Bar

coworkers

used for

not cross

Bar

(5).

to Bars with 3) the

We on the

selec-

receptors.

AND METHODS

- The BC3H-1 cell line was obtained by the American Type Culture Cell culture Collection and grown as described (10). In particular, the cells were platd on gelatin coated 35 mm Petri dishes, at a concentration of 2, 3.5x103/cm2 arki used for experiments after 13-15 days, den adrenergic and cholinergic receptors were maximally expressed (not shown). For all the experiments reported in this paper, the cells were grown in a 5% C02, 95% air incubator, at 37 C, in Dulbecco's modified Eagle's medium, supplemented with 10% fetal calf serum, 100 I.U./ml Pennicilin and 100 ug/ml Streptomicin. Receptor binding assays in intact cells -a ars were identified by 3H-CGP 12177 binding performed directly in the Petri dishes. Different groups of dishes from the same lot were incubated with increasing concentrations of 3H-CGP 12177 (0.1-10 nM), in 50 mM Tris, 10 mM MgCl 2, 1 mg/ml Ascot-bate, pH 7.5, in a 7 total volume of 2 ml. Non specific binding was determined for each group in sister cultures in the presence of 10m4M Propranolol, and was always less than 20%. After 90 minutes at 37@,or 16 hours at 4", of incubation with the radioligand (time after which the binding reaches equilibrium; not shown), the cells ware scraped with a rubber policeman and then rapidly collected and filtered through GF/C Whatman filters, using a Millipore multifilter apparatus. The cells were scraped in the presence of the radioligand to minimize the dissociation of 3H-CGP 12177. The filters were washed with 15 ml of buffer and 864

BIOCHEMICAL

Vol. 124, No. 3, 1984

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

their radioactivity determined in a Beckman LS 7500 Bcounter. Wrenergic a receptors and nicotinic receptors were labeled with 3H-Prazosin (1 nM) and 1z51-a8ungarotoxin (20 nM). Bindings with these radioligands were performed as described (11). Non specific binding of 'H-Prazosin and k251- a8ungarotoxin were determined with 50 pM Phentolamine and 100 PM d-Tubocurarine, respectivelY. Drug treatments and measurement of receptor internalization - To study the effect of agonist exposure on the number of surface Bars. 8C3H-1 cells ware incubated for 30 minutes at 37" with different concentrations of Isoproterenol or Phenylefrine (from 10sg to 10M3M), dissolved in the culture medium, without fetal calf serum. The medium was supplemented with 10e4 Phentolamine to avoid indirect effect of the agonists through the a adrenergic receptors. Phentolamine, at this concentrations, has no effect on 3H-CGP 12177 binding (not shown). After drug treatment, the cells were chilled at 4" by transferring the dishes in melting ice, and washed five times with 2 ml of cold buffer. The number of cell surface Bars was then determined by incubating the cells at 4" overnight with a saturating concentration of 3H-CGP 12177 (2.5 nM). On the other hand cells treated in the same way with Isoproterenol were used to determine the specificity of Bar internalization with respect to aadrenergic and nicotinic receptors using their specific ligands, as described. Drugs and supplies - Plastic culture dishes were obtainti fran Corning (France). . DME, Pennicilin, Streptomicin and Fetal Calf Serum were fran Flow Gelatin, 1-Isoproterenol d, 1-Propranolol, Phenylefrine, lab. (U.K.). Phentolamine ard d-tubocurarine were fran Sigma Chem. Co. (St. Louis, USA).3HCGP 12177 (41 Ci/mrrml) was franl~~rsham (U.K.) and 3H-Prazosin (17.4 Ci/mol) frcm New England Nuclear (USA). I-a Bungarotoxin was prepared as described (lo), at a specific activity of s20 Ci/mol. RESULTS BC3H-1 for

cells

the Badrenergic

had an high receptor

affinity, radioligand

The Kd of 3H-CGP 12177 binding dently

if

the

binding

was performed

displaceable

ard

3H-CGP 12177

(fig.1).

to 8C3H-1 Bars,

saturating

binding

was %O.~X~O-~M,

indepen-

at 4" or at 37°C (fig.1).

3H-CGP12177

1nMl

curves of 3H-CGP12177 birding to intact BC3H-1 cell Figure 1 - Saturation Binding was performed as described in Methods both at 37O (0) or monolayers. 4O (0). Non specific binding was less than 2a near the Kd both at 37O and 4'. There were no statistically significant differences between the two groups in which were calculated from the Scgtchard representation Bmax and Kd values, shown in the inset (Bmax 10 fomles/dish; kl: ~0.6~10 M). Each point IS the mean of three dishes.

865

8lOCHEMlCAL

Vol. 124, No. 3, 1984

AND BIOPHYSICAL RESEARCH COMMUNlCATlONS

zoI

I

I

1

10-b

KY4

10.2

1

lo"0

lo-8

Agonist

1

(M

Figure 2 - Dose depenrlence of Isoproterenol (0) and Phenylefrine (0) induced internalization of the sadrenergic receptor. Cells wet-g incubated with the drugs for 30 minutes at 37' C and Bars were measured as H-CGP12177 binding The birding in control cells was 11+0.12 (fmoles/petri dishes + S.E.). sites. Each point represents the mean of three dTshes ard the experiment sliown is representative of other three giving similar results.

The Max each

cell,

ture

using

was of

10 fmoles/Petri

a number different

tiich

Bars,

minutes

at 37" ard

after

bindings,

performed

sites

moved. to avoid

loss

The effect

to reach

adrenergic

from the

cell

surface

at 10m5.

equilibrium

after

equilibrium

the

was evident

of

For this

with

of

treatment

agonist of

the

all

60 the

Isoproterenol

"H-CGP of

3H-CGP 12177 if

reason

3H-

overnight.

at a concentration

No loss

litera-

of association

reached

shown).

in the

on

12177 Isoproterenol

binding

incubation

binding of

sites

with

the

was

agonist

at 4" (fig.3).

The binding drug

allowed

to %8000 Bars

reported

course

binding

at 4" (not

by a 10e6M Isoproterenol

was performed

any

8 hours

corresponds those

The time that

of BC3H-1 cells

10d8 M and was maximal induced

with

indicated

a dose dependent (fig.2).

in agreement (8).

at 4" were

treatment

induced

&ich

radioligands

CGP 12177 to BC3H-1

A

is

dish,

was performed

possibly The washing recycling

after

extensive

interfering

with

procedures,

and the

of the

receptors.

washings

3H-CGP 12177 binding subsequent

binding

of the dishes, was ware

canpletely performed

so that reat 4"

BIOCHEMICAL

Vol. 124, No. 3, 1984

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

20 t I

I

I

I

I

1

10

20 30 40 50 60 Time (minutes) Figure 3 - Time course of Isoproterenol-induced internalizationof the? adrenerglc receptor. Cells wre incubatai for the indicated times with 19-6 Isoproterenol. after rhich the cells were chilled ard the bindinc with HCGPi2177 was performed at 4O as described in Methods. Isoproterenol treatment induced at 37O (0) a very rapid loss of surface Bars, while at 4O (0), even a treatment for 1 hour had no effect.

The a adrenergic

agonist

without

effect

induced

a little

loss

course

of lo-aM

Isoproterenol

fig.3.

The effect

and maximal

proteins, involved Tab.1 incubation induced, the

binding

adrenergic

are

the

in this

of

induced

of agonist

Bar

was interesting

agonist-induced

endocytosis.

as expected, of 3H-Prazosin and nicotinic

cells

with

10e6M

a loss

from

the

receptors),

at lo-3M).

significant

it The time

Bars

after

internalization

of experiments

and 1251-

was

concentrations

of the

to see Aether

and nicotinic

BC3H-1

(-23%

lo-5M

is

shown in

2.5

minutes

exposure.

that

results

below

high

internalization

a adrenergic

the

sites

was already

evidences it

reports

at very

of 3H-CGP 12177 binding

30 minutes

(5,12), as

Only

of Isoproterenol

there

endocytosis

at concentrations

onB ar internalization.

after

Since

Phenylefrine,

ment. 867

integral

receptors,

were

which

demonstrate

surface

aBungarotoxin were

other

Isoproterenol

cell

proceeds

unaffected

for of

through membrane

unspecifically

that

30 minutes

50% of the Bars,

(typical

radioligands

by Isoproterenol

the

at

37" while for a

treat-

Vol. 124, No. 3, 1984 Table

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1 - SPECIFICITY OF ISOPROTERENOL-INDUCEDLOSS OF RECEPTOR FROM THE SURFACE OF BC3H-1 CELLS 3H-CGP12177 - %

3H-Prazosin

- %

ADRENERGIC

1251- Bgtx - %

Untreated

334 + 15.4

100

412 + 6.5

100

3379 + 102

100

Isoproterenol

174 + 10.2

52

439 + 7.5

106

3866 + 287

114

Specificity of Isoproterenol-induced internalization of the+? adrenergic receptor. Cells were incubated for 30 minutes at 37" with 10 M Isoproterenol, chilled, aq the bindings perfor@ as described in Methods. Recepsgrs were labeled with H-CGP12177 forBar, H-Prazosin foraairenergic ard I- Bungarotoxin for nicotinic cholineqic receptors. The values are expressed as cpm binding sites/dish + S.E.. A typical experiment performed in triplicate is shown.

DISCUSSION The BC3H-1 tions

presence smooth

and the characteristics

muscle

of the cells,

cell

Bars

hydrophilic

in this

across

receptors Until

now

cells

internal receptors,

fractions

zation

induced

line

also

experiments

the

ligand,

we

dependent

the

possible

(9).

cell

receptors

using

in

membrane

the

prepara-

as radioligands.

to label

intact

and quantify

mOnolayers,

method

for

the

cell

by using

The use of radioligards

which

the do not

quantifying

results

were

and Simons radioligand

the Bar,

similar

loss

which

inconclusive.

surface

kinetic

of which

allowed Only

on a rapid

the

Bars

label

in also

recycling

of

experiments

on subcel-

of receptor

internali-

(3,4). (5),

first

described,

by Isoproterenol.

that from the

The dose dependence to that

radioligands

3H-CGP 12177,

induced

demonstrated

internalization

hydrophobic

temperatures

Staehelin

vitro. is

at

treatment

and specific in

(8),

in

using

by agonist

also

or1251-CYP is

gave some evidences

of

described

on agonist-induced

and

hydrophilic

endocytosis

already

is a simple

performed

thus

Finally,

proterenol

cell

B adrenergic

cells.

were

ard

lular

cells

it

3H-CGP 12177

receptors,

using

that

plasmamembrane

in intact

intact

paper

radioligand

diffuse

were

and 3H-DHA (13)

We show in this surface

line

of

Isoproterenol cell

of adenylate

868

fast

a

of the Bars

internalization

cyclase

intact and

cells, reversible

Using 3H-CGP 12177 as induced

surface

of Bar

a very

in

activation

very

a

fast,

dose

in smooth

muscle

induced (13)

by

Iso-

\mich,

in

BIOCHEMICAL

Vol. 124, No. 3, 1984 turn,

is

fast

time

course

known to be parallel course

to that

of receptor

of desensitization

the

Bar represents

these

smooth

smooth anti-Bars

some

cells

It

driven

diseases

use cells

of

as asthma

Bagonists

f3 agonists

clarification

induce

of the mechanisms particular

suggested

cells,

Also

that

of

desensitization

in

of Bars

of catecholamines feature

limiting

factor

The demonstration

internalization

of Bars

is

process

of desensitization

involved

and

in the

opens

new

ways

for

rational

rapid in

in asthma.

a first

by

underthe

that

on

or

Certainly

the

time

internalization

most significant

is

very

the

the decrease

or hypertension. Bars

the

with

that

secretion

be one of the

of

likely

of agonist-induced

by increased

could

(14).

is canparable

seems thus

has been

desensitization

therapeutic

these

It

(15)

human

agonist-induced

muscle

internalization

(14).

cells.

antibodies

lying

of desensitization

the main mechanism

muscle

muscle

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the

in

smooth

step

in the in

therapeutic

strategies. Another

important

of8

ar internalization;

at

so high

by

at tiich of the

Isoproterenol, Recently

evidences

of

Bar;

shown,

with

respect

of the

against

this

results

suggest

that

in erdocytotic

a very vesicles

but

fi agonists also

because

point

may fail it

does

is the

specificity

Isoproterenol

(or

Phenylefrine

activity) only

the

(16),

4ar the

in astrocytoma

internalization muscarinic is,

that

receptor

is

affect

a. or

specific

after

agonist these

because receptors 869

out of

it

reported

Ligand-induced phenanenon.

also

selective,

not

Isoproterenol

specific

B adrenergic

sorting

of view,

not affect

highly

induce

were

cells,

receptor.

same BC3H-1 cells,

to

internalized

after

a very

not only

Pars Kere receptors

thus,

occurs

was able

and cholinergic

receptors

do not

paper

like

hard,

et al.

to

nicotinic

receptors

other

of

on the

From a therapeutic with

Waldo

specificity

indeed,

has some beta

c( adrenergic

also

in this

agonist

on the

of membrane

endocytosis

tion

it

a

internalization have

the

while

affected.

treatment,

we demonstrated

only

doses

internalization

point

We

antibody-induced since

receptors

antibodies (11 ).

Bars and their

These

accumula-

treatment.

results

suggest

reduces

the

with

opposite

that

number

of

function

a treatment B receptors (as the a

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 124, No. 3, 1984 adrenergic control

and

the

cholinergic),

of smooth muscle

producing

contraction,

an imbalance

underlying

in

the

some human diseases

autonanic (7).

ACKNOWLEDGEMENT Supported tive

in part

by a CNR grant,

applied

project

"Preventive

and Rehabilita-

Medicine". REFERENCES

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D.M., Costa, E. (1979) Proc. Natl. Acad. Sci. USA 76, 3024-3028. J.K., Cotton, C.V., Waldo, G.L., Lutton, J.K., Perkins, J.P. (1980) Science 210, 441-443. Staehelin, M., Simons, P. (1982) EMBO J. 1, 187-190. Stadel, J.M., Nambi, P., Shorr, R.G.L., Sawyer, O.F., Caron, M.G. Lefkowitz, R.J. (1983) Proc. Natl. Acad. Sci. USA 80, 3173-3177. Lefkowitz, R.J., Caron, M.G., Stiles, G.L. (1984) New Eng. J. Med. 310,

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