Substitution of an extracellular cysteine in the β2-adrenergic receptor enhances agonist-promoted phosphorylation and receptor desensitization

Vol.

165,

No.

November

BIOCHEMICAL

1, 1989

30,

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Pages

1989

SUBSTITUTION

OF AN EXTRACELLULAR

P2-ADRENERGIC

257-263

CYSTEINE IN THE

RECEPTOR ENHANCES AGONIST-PROMOTED

PHOSPHORYLATION AND RECEPTOR DESENSITIZATION S.B.

Liggettl,

M. Bouvier, R.J.

B.F.

Lefkowitz

Departments

of Medicine,

Biochemistry,

September

25,

Cell

M.G.

Biology

Duke University

and HHMI, Durham, Received

O'Dowd,

Caron,

and A. DeBlasi and

Medical

North

Center

Carolina

1989

SUMMARY: We constructed and expressed in a permanent cell line a p2-adrenergic receptor with a valine substitution for cysteine 184 of the second putative extracellular loop. The mutant receptor was partially uncoupled from adenylyl cyclase with impaired ability to form the high affinity agonistreceptor-G protein complex, yet displayed more rapid and extensive agonistinduced desensitization. The enhanced desensitization was accompanied by increased agonist promoted, but not CAMP promoted, receptor phosphorylation in intact cells. Thus, not only is impaired desensitization associated with decreased phosphorylation, as we have shown with several mutant p2-adrenergic receptors recently, but enhanced desensitization is accompanied by increased agonist promoted receptor phosphorylation. In the case of this cysteine mutant, this may be due to the greater accessibility of the uncoupled receptor for phosphorylation by the P-adrenergic receptor kinase. 0 1989 Academicmess, Inc.

The

phenomenon

response

despite

/3-adrenergic

continuous

receptor

mechanisms

that

phosphorylation receptor

the

In the in

to

PEA or

desensitization course

j32AR, the

be

important

for

example,

which intact

cells

the

a mutant

1 To whom correspondence

second

should

(1,2,3).

Of the

the

several process,

and the B-adrenergic

during that

in

short cells

term bearing

sites

results

have

agonist mutated depressed

with

the

loss

of

have

been

shown

by

(3).

importance p2AR

extracellular

a biological

desensitization A (PEA)

correlated

Similar

developed

putative

is

of

demonstrated

BARK phosphorylation

of evaluating we

readily

the

kinase

(1,2). in

a dampening

system in

by protein shown,

CAMP accumulation

human

present

recently

roles

is been

cyclase

phosphorylation,

agonist-promoted

the

has

play

appears

putative

agonist-promoted measuring

to receptor

(PARK)

We have

lacking

activation,

appear

kinase

which

(PAR)/adenylyl

of the

exposure. p2AR

of desensitization,

of extracellular lacking loop.

cysteine In

stably

cysteines 184,

in

normally

transfected

be addressed. 0006-291X/89 257

$1.50

Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 165, No. 1, 1989

cells,

this

than

did

mutant

the

wild

of

agonist-promoted

of

the

Thus,

mutant not

tization, accompanies

only but

BIOCHEMICAL

receptor type

desensitized

receptor

upon

phosphorylation ,82AR was is

for enhanced

accompanied

a loss the

first

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

more short

term

showed

that

time

we have

and to

exposure the

by an enhanced

of phosphorylation

agonist-induced

rapidly

shown

that

extent

to agonist.

enhanced receptor

accompanied

a greater

Studies desensitization

phosphorylation.

by a loss

increased

of desensi-

phosphorylation

desensitization.

METHODS Mutagenesis. transfection and cell culture Wild type p2AR cDNA was subcloned into the plasmid PTZ at the Eco RI/Hind III restriction sites, and oligonucleotide directed mutagenesis of the /J2AR cDNA carried out by methods previously utilized (1,2). The ,9AR mutant described here has a valine substituted for cysteine 184 in the second extracellular loop and is designated 184CYS(VAL). The mutation was verified by dideoxy sequencing. Mutant cDNA was then cloned into the plasmid pBC12MI and co-transfected with pSVNeo into Chinese hamster fibroblast (CHW) cells by co-precipitating the DNA with calcium phosphate. Clonal cells were selected in media containing geneticin. CHW cells transfected with the normal human described, were used as control cells (4). Cells B2AR gene, as previously were grown in monolayers in Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum, 100 U/ml penicillin and 100 pg/ml Streptomycin in a 95% air, 5% CO2 atmosphere at 37OC. Agonist-promoted desensitization 184CYS(VAL) and wild type expressing were cells exposed to 2 PM isoproterenol in DMEM supplemented with 0.5 mM ascorbic acid for 60, 30, 15, 6, 3 and 0 min. at 37OC, then washed three times by rinsing the plates with Cells were then scraped in hyptonic buffer (5 mM Tris, 2 mM EDTA) cold PBS. and homogenized for 5 set with a polytron. A 400 xg centrifugation pelleted and membranes of the supernatant were pelleted by nuclei and clumped cells, centrifugation at 38,000 xg, washed once by a similar centrifugation, and resuspended in 75 mM Tris, 12.5 mM MgC12, 2 mM EDTA buffer. Adenylyl cyclase were determined by the method of Salomon as modified (4). activities Activities were determined in the presence of 10 PM isoproterenol, 100 PM Forskolin or buffer alone. Agonist-promoted nhosnhorvlation Cells were detached by incubation with collagenase (1.0 mg/ml) and soybean trypsin inhibitor (.05 mg/ml), washed three times and resuspended in Cells were then phosphate free DMEM at a density of 3 X lo6 cells/ml. then equilibrated for two hours at 37OC with carrier free 32Pi (7 mCi/.sample), samples exposed to either 2 PM isoproterenol or 1 mM dibutyryl CAMP or buffer After a 5 min centrifugation at 400 xg, the reaction was stopped for 8 min. by addition of cold PBS, the cells washed an additional two times and then disrupted by sonication in 20 mM Tris, 5 mM EDTA, 10 mM Na2P04 buffer. This and all subsequent buffers included the protease inhibitors benzamidine 10 The 5 pg/ml and soybean trypsin inhibitor 5 pg/ml. hWm1 I leupeptin particulate suspension was then washed and solubilized in 2% digitonin. Solubilized receptor was purified by Sepharose/alprenolol chromatography as by 10% SDS-polyacrylamide gel previously described (1,2), and examined electrophoresis and autoradiography. Radiolieand binding cells were detached and homogenized as above for For membrane studies, adenylyl cyclase activities and radioligand binding studies carried out with Intact cell binding was carried out [1251]-pindolol, as described (2,4). using cells detached by gentle scraping as previously described (2,3). (300 PM) in the absence or Briefly, cells were incubated with [ 1251]-pindolol presence of 0.5 JLM CGP12177 (for quantitation of cell surface receptors) or 1 258

BIOCHEMICAL

Vol. 165, No. 1, 1989

PM propranolol sequestered) Solubilized absence or Sephadex G50

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(for quantitation of all receptors, both cell surface and Reactions were terminated as above. for three hours at 13OC. receptor binding was performed using [1251]-cyanopindolol in the with bound ligand separated by presence of 10 PM alprenolol, column chromatography (1,2).

RESULTS Receptor + 0.2 the

densities

pmol/mg)

or

184CYS(VAL)

dissociation

stimulated

adenylyl

tion

bearing

were

not

since

the

lower

cyclic

of

184 CYS(VAL)

cells

those

To explore

this

functional

studies

were

performed

(Figure

1).

The

model

affinity

(KH)

these

data

and

wild

the

KH for

revealing

that

low

of

in

mutant

the

the

of

of receptor

the

(30 + 2%) and ls4CYS(VAL)

the

same.

mutant,

as compared stimula-

Consistent

basal lower

(17.2

pmol/l07

GTP were

high

competition of

best

similar

100 PM GTP

fit

by a one

low

affinity

providing

high

constants.

higher

than

+ 3.4

cells).

to two sites

for

with

intracellular

or presence

fold

higher

T 5 pM,

17 -+ 3 no for ~*~cYsproximal to the catalytic

binding

affinity in

69

isoproterenol

isoproterenol

p2AR had fit

was -2

lower

as were

isoproterenol

dissociation

was -9 fold

pM vs

(2.2

and

absence

were

184CYS(VAL)

a substantially

for

type

similar,

184CYS(VAL)

T 10.1

type

data

10

significantly

(50.1

presence

(KL)

t

further,

and wild

affinity

the

were type

in

of GTP, the KL for

was were

uncoupling

membranes

1*4CYS(VAL)

The proportions type

of the wild

derived

absence

conditions,

In addition, P2AR, receptor.

in

and showed In the

for

activity

than

binding.

uncoupling

wild

and maximal in

type

activities

cyclase

cells)

lower

The EC5O's

functional

adenylyl

(47

wild

bearing

B2AR were

1, basal

were

_+ 5 no for

stimulated

AMP contents

site

receptors.

apparent

basal

pmol/l07

type

forskolin

pmol/mg)

in Table

activities

(24

cells

[1251]pindolol

cyclase

different

This

@AL) ) . unit,

for

from

T 0.2

as shown

wild

dervied

(1.9

constants However,

respectively). to cells

of membranes

that agonist

affinity

than of

Under wild

the of

form

type.

wild the

were

type mutant

similar

(42 + 7%).

TABLE I Wild Basal

Isoproterenol (10 pM) Forskolin (100 PM)

26.2 114.4 266.1

TYD~

~*~YS(VAL)

+ 4.4 T 12.1 -i 35.0

9.4 40.7 262.5

+ 3.1 i 2.6 T- 85.1

Adenylyl c clase activities of membranes derived from CHW cells expressing wild type or 18J+CYS(VAL) mutant p2AR. Activities are expressed as pmol/min/mg and are means of 5 independent experiments. Both basal (p <0.02) and isoproterenol stimulated (p
BIOCHEMICAL

Vol. 165, No. 1, 1989

100

m v L 0 w a VI

. 0 KL= ‘9 ‘LL WILD TYPE

(3 Z $

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

‘84CYS (VAL)

409+56

KL=449+95

50

K,, = 8.5

23

0

? 2.3

0

-9-8 -7

-6

-5

-4

-9

-7

-8

log[ISOPROTERENOL]

nol,

a more

which be

cells rapid

occurred seen,

adenylyl exposure

in

this

expressing and

with wild

cyclase

extensive

cells

bearing receptor

activity

the

control

adenylyl

receptor

type

cyclase

over

after

1 hour

from

to

2 PM isoprotere-

as

compared

(Figure

isoproterenol

cells,

decreased level

exposed developed

bearing

gradually the

wild

-4

Wild type and mutant p2AR for the times indicated, isoproterenol stimulated as described in Methods.

B2AR were

desensitization

type

to 73 -+ 8% of isoproterenol stimulated

mutant

-5

M

Figure 1. Agonist-induced desensitization of j32AR. bearing cells were incubated with 2 PM isoproterenol Maximal washed extensively and membranes prepared. adenylyl cyclase activities were then determined Shown are the means of five experiments.

When

-6

the

course

exposure.

membranes

to

that

As can

2). stimulated of

agonist In contrast,

of 184CYS(VAL)

cells

go-

80-

WILD

TYPE

706050-, 0

I IO IS0

I

I

20

30

EXPOSURE

‘-CYS I

40 TIME

(VAL) I 50

I

6C

(Mid

for [125I]pindolol binding to B2AR in Figure 2. Isoproterenol competition Membranes were membranes from wild type and lL14CYS(VAL) bearing cells. incubated in the absence (0) or presence (0) of 100 PM GTP, and the results KL, KR = low and high affinity analyzed by iterative least squares techniques. Results are means of three experiments. dissociation constants in nM.

260

BIOCHEMICAL

Vol. 165, No. 1, 1989

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

90 80 70 60

Wild Type ‘*?YS (VAL)

50

I 60

“ TIME

(Min)

w

Agonist-induced loss of cell surface p2AR. Cells bearing wild type CYS(VAL) &AR were incubated with 2 PM iso roterenol for the indicated carried out as times, washed extensively, and binding with [ 1% Ilpindolol described in Methods. Shown are the means of three experiments.

reached

a nadir

after

-55% of control

levels

of desensitization cell

surface

shown

for To

receptors, both

this

enhanced

were

wild

exposed

with

than

wild basal that

in

phosphorylation of

the

wild

cells

which then

had

been

the &AR

loaded

purified

(1,2,4),

32Pi

the of

to

present lower 184~~~-

(i/AL)

(x10-3)

9467 43lsoproterenol

Figure bearing

4.

isoproterenol,

wild

Autoradiographs type or purified

-

+

of phosphorylated lS'kYS(VAL) and subjected

+

p2AR.

fl2AR were incubated to 10% SDS-PAGE.

261

Cells with

loaded or

with without

on

as a

exposure

was markedly

phosphorylation

‘84CYS

with and run

In

receptor

points

migrates upon

increased.

lg4CYS(VAL)

time

of

accompanied

receptor

agonist-induced

loss

3).

phosphorylation

phosphorylated

TYPE

the

at

pattern

promoted at

is

WILD

agonist

remained

altered

(Figure

phosphorylation the

This

was identical

reported

The

and

bearing

cells the

the

As previously of

type.

in

receptor

8 minutes, 4,

exposure,

up to 1 hour.

receptor in

for Figure

receptor

agonist

parameter

profile,

Mr = 70,000. type

for

alterations

to isoproterenol

band

exposure this

desensitization

broad

prior

and mutant

whether

As shown

of

due to a difference

since type

SDS-PAGE.

study,

after

was not

assess

agonist

6 minutes

[32Pi] 2 PM

Vol. 165, No. 1, 1989

(VAL),

BIOCHEMICAL

expressed

was greater

as net

than

that

receptor.

In three

2.1 z 0.07

fold

differentiate

phosphorylation of

the

such

higher

the

this

184 CYS(VAL)

dibutyryl

AMP was

phosphorylation dibutyryl not

of

cyclic

type

examined.

the mutant

mutant

the

p2AR by isoproterenol, than

type

type.

in

marked

the

was To

or PKA mediated

,9AR phosphorylation to

basal)

wild

to wild

@ARK mediated,

In contrast

AMP was no different

the

phosphorylation

as compared

to enhanced

minus

of

agonist-induced

184 CYS(VAL) and wild

stimulated

phosphorylation

the

was due

phosphorylation, cyclic

(isoproterenol

agonist-induced

experiments,

in

whether

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

response

to

enhancement

of

increment

induced

that

found

with

the wild

processes

play

key

regulatory

type

by (data

shown).

DISCUSSION Phosphorylation/dephosphorylation a number key

of

receptor

findings

mechanism

systems

implicate

cyclase

the

during time

promoted

identical,

as are

phosphate/receptor

by

phosphorylated

The

mutant

show

system

receptor

as

a

the

In

rates

such

several

the of

turkey adenylyl

of resensitization

(6).

Purified

receptors

an impaired

in

regulatory

desensitization

stoichiometry

PKA and PARK and when receptors

cyclase

desensitization.

promoted

to control

G,,

the

of agonist are

be phosphorylated

pAR/adenylyl of

agonist

courses

and phosphorylation

and return with

In the

phosphorylation

occurring

erythrocyte,

(5).

roles

are

ability

BAR can

reconstituted

to

couple

to

G,

(7,S). receptor

described

enhanced

desensitization

and

setting.

The enhanced

desensitization

of

the

which

receptor. have

quence

shown

1*4CYS(VAL)

maintaining

In

the

that

in

uncoupled the

and

1*4CYS(VAL) from

form

(ternary

complex)

is

impaired

receptor

converted

to the

the high

in

in

this

affinity 262

form

in

is

similar

all

in

experiments the to

Moreknown

critical

expressed

While

phosfor

(10,ll).

role. cells

complex percentage

that

is

suggest

agonist-receptor-G,

mutant.

the

extracellular important

its

Competition

affinity

these

with and

conserved

suggesting

(9),

substitution

the

properties

universally

cyclase. high

that

studies

bonds,

/32AR permanently

adenylyl

to

al.

desensitization

located

further

et

as a conse-

whereby

disulfide

cell

uncoupling

suggesting

mechanisms

between

whole

delayed

Current

binding

is

far,

a

marked Cheung

is

enhanced of

ligand

184 thus

ability

of

coupling,

as others

formation

link

in the

studies

receptor. the

causes

position

study,

the

as well in

cloned

the

receptor-G, elucidate

structure

receptors present

partially

cysteine, implicated

cysteine

to

of

first

despite

desensitization

depress fully

the

phosphorylation

contrast

cysteine

tertiary

the

adrenergic

been

provides

occurred

common domains do not

This has

domain,

in

extracellular

phorylation.

increased

agonist-induced that

share mutant

of a single

is

that

of mutations

two phenomena

over,

This

here

of

the

of wild

Vol.

type

165,

the

I

This

No.

affinity

unique

stabilize

form

of uncoupling

more

ternary

it

in

ed

for

analogous

rhodopsin

is

transducin

a like

complex not

in the

b2AR would

tion.

Rather,

enhance regions

imply

of the

assistance supported

by

an

by

a Centennial

enhanced

receptor

responsible

for

for

manuscript Research

in

where

than the

the

are tissue

Award Medical

rhodopsinmutant

and phosphorylasuggesting

the

uncoupled

alterations

uncoupling,

Scholar

is report-

184CYS(VAL)

structural

preparation. from

to that

of

for

to G, and

phosphorylation

system,

properties

to

phosphorylation

alterations

Irons

isoprotere-

binding

similar

with

receptor.

inability

its

desensitization

these

Grace

Fellowship

increase

due to other

specific

thank

from

is

type

of

kinase

studies

despite

RJR-Nabisco

the

because

This

to enhanced

case,

The authors

supported

for

CAMP mediated

rhodopsin

current

lead

and Mary Holben

wild

rhodopsin

uncoupling

this

molecule

Acknowledgments:

for

These

desensitization

the

This mutant.

COMMUNICATIONS

than

released

coupled

receptor

necessarily in

RESEARCH

that

dibutyryl

substrate

(12).

as

less

rapidly

in this

G-protein

that

is

since

manner

a better

should

fold

phosphorylation.

to be BARK mediated,

enhanced

-9

BIOPHYSICAL

We hypothesize

for

not

the

is

complex,

available

AND

may be responsible

desensitization. the

appears

of this

form

no1 induced thus

BIOCHEMICAL

1, 1989

the

that

may the

distinct. culture

Stephen and

receptor

technical

B. Liggett

is

Michel

Bouvier

is

Research

Council

of

Canada. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

Bouvier, M., Hausdorff, W.P., DeBlasi, A., O'Dowd, B.F., Kobilka, B.K., Caron, M.G., and Lefkowitz, R.J. (1988) Nature 333:370-372. Hausdorff, W.P., Bouvier, M., O'Dowd, B.F., Irons, G.P., Caron, M.G., and Lefkowitz, R.J. (1989) J. Biol. Chem. 264:12657-12665. Liggett, S.B., Bouvier, M., Hausdorff, W.P., O'Dowd, B.F., Caron, M.G., and Lefkowitz, R.J. (in press) Mol. Pharmacol. Bouvier, M., Hnatowich, M., Collins, S., Kobilka, B.K., DeBlasi, A., Lefkowitz, R.J., and Caron, M.G. (1988) Mol. Pharmacol. 33:133-139. Sibley, D.R., Benovic, J.L., Caron, M.G., and Lefkowitz, R.J. (1988) Endocr. Rev. 9:38-56. Sibley, D.R., Peters, J.R., Nambi, P., Caron, M.G., and Lefkowitz, R.J. (1984) J. Biol. Chem. 259-9742. Benovic, J.L., Pike, L.J., Cerione, R.A., Stansizewski, C., Yoshimasa, T. Codina, J., Caron, M.G., and Lefkowitz, R.J. (1985) J. Biol. Chem. 260:7094-7101. Benovic, J.L., Strasser, R.H., Caron, M.G., and Lefkowitz, R.J. (1986) Proc. Natl. Acad. Sci. USA 83:2797-2801. Cheung, A.H., Sigal, I.S., Dixon, R.A.F., and Strader, C.D. (1989) Mol. Pharmacol. 34:132-138. Dixon, R.A.F., Sigal, I.S., Candelore, M.R., Register, R.B., Scattergood, W., Rands, E., and Strader, C.D. (1987) EMBO J. 6:3269-3275. C. (1989) J. Biol. Chem. 264:9266-9270. Fraser, Kelleher, D.J. and Johnson, G.L. (1988) Mol. Pharmacol. 34:452-460.

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