The β-adrenergic receptor of turkey erythrocyte membranes: Conformational modification by β-adrenergic agonists

Vol. 86, No. 4, 1979

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

BIOCHEMICAL

Pages

February 28, 1979

1311-1318

THE B-ADRENERGIC RECEPTOR OF TURKEY ERYTHROCYTE MEMBRANES : CONFORMATIONAL MODIFICATION G. VAUQUELINx:

S. BOTTAR?,

x Biochemical

BY B-ADPENERGX

0. DURIEU",

Pathology,

Instituut

65

Paardenstraat,

B-1640

' Molecular

Immunology,

Universite Received

December

29,

C. KLUTCHKO',

VII,

and A.D.

Moleculaire

Biologie,

St.-Genesius-Rode,

Institut

Paris

AGONISTS

de Recherches Place

Jussieu

STROSBERGXo

V.U.B.,

Belgium Biologie

2, Paris,

Moleculaire, France

1978

SUMMARY. The d-adrenergic receptors of turkey erythrocyte membranes have beenidentified by the specific binding of the radiolabeled antagonist (-)-/3H[--dihydroalprenolol. Pretreatment of these membranes with either the alkylating a ent N-ethylmaleimide or with !.3-adrenergic agonists does not affect (-)- 'i 3H I - dihydroalprenolol binding to the receptor sites. However, the simultaneous presence of both types of products causes a 50 % decline in the number of binding sites. A less pronounced decline occurs when the membranes are pretreated with N-ethylmaleimide in presence of the I-)phenylephrine, and no decline in the presence of the partial agonist antagonist (-)-I HI-dihydroalprenolol. $ -adrenergic agonists thus appear to induce a conformational change of their receptor, with results in an increased susceptibility to inactivation by N-ethylmaleimide.

Adenylate the specific membrane,

cyclase

ligands

characterization

ties

requires

to B-adrenergic

several

receptors

steps

on the cell

of the signal to the enzyme and finally an The recent availability of radiolabeled AMP production.

in cyclic

f3-adrenergic

easily

by catecholamines

of the hormone

the transmission

increase

Whereas

activation

binding

such as (-j-13H1-

of the

occupation

are

adenylate physiological

usually

8-adrenergic

of these

be determined

§

G.V. is Aangesteld Onderzoek, Belgium.

&

Nonstandard abbreviations NEM, N-ethylmaleimide.

method

in tissues

Navorser

by binding

the agonist

by determining

in cell

enables

(6).

In this

of the Nationaal

and analogs or antagonist

the ability

membranes(3,5)

a direct

studies.(l,2j

by catecholamines (3,4),

indirectly

activation

responses

receptors

@receptors

by this

studied

cyclase

dihydroalprenolol

proper-

to induce

or by evaluating report,

can

we show that

the 8-adre-

Fonds voor Wetenschappelijk

: 13H/- DHA, (-)-13HI-dihydroalprenolol; 0006-291X/79/041311-08$01.00/0 1311

Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in anyform reserved.

:

Vol. 86, No. 4, 1979

nergic

receptors

by the reagent antagonrsts, two types gic

BIOCHEMICAL

of turkeyerythrocytemembranes N-ethylmaleimide

providing

for

of compounds

receptors

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

can be rapidly

in the presence a new direct

and confirming

in a different

method that

they

of agonists, to distinguish interact

with

inactivated but

not of

between

these

the S-adrener-

way.

EXPERIMENTAL PROCEDURE. MATERIALS. The following were obtained as gifts (-)-isoproterenol bitartrate (Sterling Winthrop) and phentolamine (Ciba-Geigy). (-)-Phenylephrine was purchased from Sigma Chemical Co.; pyrocatechol and N-ethylmaleimide were from Fluka. AG.(-)-13HI-dihydroalprenolol (33 Ci/ mmol) was obtained from New England Nuclear Corp.

:

membranes were Preparation of turkey erythrocyte membranes : Erythrocyte orenared according to @ve and Sutherland (7). suspended in 10 mM Tris* HCI (pH 7.4)/145 & NaCl/2 mM MgC12 containing IO&% (vol/vol) glycerol, and stored in liquid nitrogen until use. Nucleated ghosts were obtained by hemolysis of erythrocytes in 5 mM Tris-HCl(pH 7.4)/2 mM MgC12. Protein determinations were performed by the procedure of Lowry et al(8) usingbovine serum albumin as the standard. Membrane pretreatment with N-ethylmaleimide (NEM) & : Ghosts or membranes were vreincubated with NEM and washed as follows : Preincubation : 2 mg of membrane protein was preincubated with the indicated concentrations of NEM and adrenergic ligands at 30°C for 12 min. in 75 mM Tris-HCl(pH 7.4)/25 mM MgC12 (buffer A) in a final volume of I ml. Then, the reaction medium was immediately transferred into a 1.5 ml. Eppendorf micro test tube and centrifugated for 1 min in an Eppendorf centrifuge (1200 rpm) at 20°C. the preincubated membranes Washing : After removal of the supernatant, were resuspended into 1 ml. of buffer A, and centrifugated for I min. This washing step was repeated once. A

Binding of (-)-13HlDHA & to pretreated ghosts or membranes was performed as published elsewhere (9). In all figures and tables, bound (->-13H/DHA refers to-specific binding : i.e. binding of tracer displaced by 25 pM (+)-alprenolol. Data are mean at least 6 measurements, standard deviation less than 6 %. Ascending thin-layer chromatography : Equal volumes of 40 mM (-)-isoproterenol and 40 mM NEM (in buffer A) were mixed, and incubated for 12 5 1~.1 of the (-)-isoproterenol and NEM solutions and 10 ~1 of min. at 30°C. the (-)-isoproterenol plus NEM solution were spotted on the same silicagel thin-layer chromatographic plate (20 x 20 cm, pre-coated, from Merck). After elution with ethyl acetate/formic acid/water (17/2/l), plates were exposed to iodine vapors. Isoproterenol : brown spot, RF = 0.28; NEM : no colour; isoproterenol plus NEM : brown spot : RF = 0.28. RESULTS .

The radiolabeled

antagonist

(-)-13Hl-

DHA has been

shown to bind

to the Sl-adrenergic receptors of turkey erythrocytemembranes with high specificity and affinity, and with fast association and dissociation kinetics at 30°C (9). Binding occurs to a single class of non-cooperative sites with an equilibrium shown in figure 1, bound gic

agonists

( with

dissociation constant (KD) of 8 to 12 nM. As (-)-13HlDHA is completely displaced by 6-adrener-

the order

of potencies

1312

: (-)-isoproterenol

= (*)-proto-

Vol. 8.5, No. 4, 1979

BiOCHEMlCAl

DRUG

AND BIOPHYSJCAL RESEARCH COMMUNICATIONS

CONCENTRATION

(-Log

M 1

Fig.]. Competition of various drugs with (-)-13HlDHA for binding to the B-adrenergic receptor of turkey erythrocyte membranes. Membranes(2 mg/ml) are incubated with 10 nM of (-)-13HlDHA for 10 min. at 3O'C in the presence of increasing concentrations of drug.Control binding refers to binding of (-)-13H/DHA in the absence of competitor (0.11 pmol/mg protein).

kylol

> (-)-norepinephrine

receptor),

2

(-)-epinephrine,

and by the partial

phentolamine

agonist

and the non-bioactive

typical

for

(-)-phenylephrine. catechol

a Hl-adrenergic

The o-blocker weak or no displace-

produces

ment. In contrast the

fil-receptor

with

of turkey

zation

upon prolonged

Binding

of (-)-13H/-

by prior

treatment pretreatment

(-)-isoproterenol

erythrocyte

exposure

membranes

with

of the membranes

of

of a variety

to al-adrenergic

of the membranes produces

plot

receptors

DHA to the 6 -adrenergic

However, A Scatchard

the fi-adrenergic

show desensiti(Table

NEM either

a 55 % drop

(-)-13H/-

does not agonists receptors

with in

of tissues,

is not (Table

(-)-13Hl-

DHA binding

binding

affected

I and 2).

NEM in the presence

DHA saturation

1) (lo-13),

of (Table

experiments,

1).

reveals

that this drop is due to a decline in the number of receptor sites, and that the affinity of the tracer for the remaining receptors is not affected (unpublished observations). affected by improved washing membranes washing,

(i.e. instead

washing

The number of inactivated receptors of the NEM plus (-)-isoproterenol-treated

followed

of washing

only,

by incubation Table

1313

1).

with

buffer

Inactivation

is not

and a second is

apparently

Vol. 86, No. 4, 1979

Table

BIOCHEMICAL

1: (-)-

proterenol

13Hland/or

to membranes

after

treatment

with

preincubation

with:

preincubation

Second

- a)

(-)-

preincubation

13H(Hi-

NEti

of

Table binding. -

44

NEM

90

Membranes ((-)-isoproterenol twice with buffer,

with

10 nM

to membranes buffer

the

(-)-13Hl-

treated

(2 mg/ml) : 0.1 uM, preincubated

indicated DHA

with

95

is

are NEM

compounds, measured.

buffer

preincubated with the indica: 0.1 mM) for 12 minutes for a second time at the

and washed

Control

2:

Effect

of

various

buffer

preincubated

in

presence

or

- DHA binding

: 0.1 UM PM a)) , KD = 0.12

(-)-phenylephrine ( partial

: 0.1 mM agonist , KD = 0.094

I3HH(- DHA ( antagonist

:

:

1 mM

, KD = 0.26

: 1 mM ( non-bioactive

, KD.>0.5

of

NEM on

NEM

NEM

:

pretreated

with

KD-values

irreversible.

buffer

were

98

93

53

94

81

105

92

104

98

98

93

mN ) mM )

Neither

of with

causes

a pronounced

receptors

are

are

previousely

treatment

Therefore,

compounds

as

treatment

treatment,

both

in the presence and incubated membranes

only.

determined

subsequent

(-)-13Hl-DHA

IO l&M

100

Membranes (2 mg/ml) are pretreated with various drugs or absence of NEM for 12 min. at 30°C, washed twice with buffer with 10 nM (-)j3HIDHA. Control binding refers to binding to

and

buffer. binding

mM )

IO nM , KD = IO nM )

( a-antagonist

absence

no

only

phcntolamine

washing

to

( % control)to:

with:

(-)-isoproterenol ( agonist

a)

with

refers

only.

drugs

(-)-13Hl

catechol

twice

binding

only

Membranes

(-)-

45

(-)-isoproterenol

same concentration a)

+ NEM -

+ NEM

(-)-isoproterenol

Binding

103

(-)-isoproterenol

(-)-isoproterenol

)

103

NEM -

DHA bound

( % control

(-)-isoproterenol

ted compounds at 30°C, washed

(-)-iso-

NEM.

Membrane First

DHA binding

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

only

simultaneously

described

membranes

the

(9).

with

(-)-isoproterenol,

reduction inactivated

in by

present.

1314

(-)-l%lNEM and

NEM or

followed the

DHA binding (-)-isoproterenol

by reverse (Table

1). when

Vol.

86,

No.

BIOCHEMICAL

4, 1979

AND

PREINCUBATION

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

TIME(MINUTES)

Fig.2. Inactivation of &adrenergic receptors by NEM plus (-)-isoproteren01 : time dependence. Membranes (2 mgfml) are pretreated with 0.025 uM (-)-isoproterenol plus NEM (50 uM : (o---o) , 0.1 mM : (o--o) , 1 mM : (o--f))), or with 0.05 pM (-)-isoproterenol plus 50 uM NEM : (x-x) , for different periods of time at 30°C. Then membranes are washed twice with buffer and binding of 10 nM (-)-13HlDHA measured. (B) is binding to membranes preInsert : Semi-logarithmic representation. treated with NEM plus (-)-isoproterenol for the time (t). The apparent first-order rate constant of inactivation (kob) is defined by equation : In (B/100) = - kob.t.

The effect of NEM plus . . The krnetlc data, presented erythrocyte

membranes

with

50 nM NEM plus decrease

0.025

PM (-)-

a time

sites.

When the number of binding sites is plotted the decrease appears to be related 2, inset), time.

The apparent

(k,b)

approximates

0.022

twice

by doubling

0.046

min- I) or NEM (k,b

cubation

Pretreatment

the

in the number

is time- and dosethat pretreatment

duces (Fig.

- dependent

(-)-isoproterenol 2, indicate

in Fig.

= 0.044

of the membranes

of either

isoproterenol

of (-)-13Hl-

pro-

DHA binding

on a logarithmic scale linearly to the prein-

first-order rate constant -1 min . The rate of inactivation

concentration

depende,nt. of turkey

for

the inactivation increases

(-)-isoproterenol

about

(k,b

=

min-l). with

(-)-isoproterenol

(0.025

I,JM)

in the presence of a high concentration of NEM (1 mM), causes inactivation of about 50 % of the receptor sites within less than 1 min (i.e. the time in the experimenrequired for a first washing of the membranes, as described tal procedure). remains constant Higher

concentrations

After this rapid upon prolonging

the amount of receptor decline, the preincubation for at least

of (-)-isoproterenol

(0.1

131.5

sites 15 min

(Fig.

uM) and NEM (10 mM) do not

2).

Vol.

86,

No.

4, 1979

BIOCHEMICAL

affect the remaining receptors 5, 10 and 15 min preincubation, respectively

AND

in either the

54, 48, and 48 % for

ghosts.

Accordingly,

membrane

purfication

BIOPHYSICAL

membranes

(-)-13Hl-

ghosts.

activity

for

still nucleated

(-)-isoproterenol

factors

After

is

and 52, 52 and 57 % for

of NEM or

the limiting

COMMUNICATIONS

or nucleated

DHA binding

membranes,

the concentration are not

RESEARCH

or the

the extent

of inactiva-

tion. Inactivation is

in part

of the B-adrenergic

mimicked

lephrine,

but

not

in the presence

The concentrations to their pied

for

either

(-)-13Hl-

receptors

their

specific

cyte

(14,15)

ceptors

(as defined

the agonist

modification

observed

by thin-layer

that

these

Binding

cyte

membranes

inactivate

may cause

of the receptors

processes

can be summarized H

where

H is

receptor

+

towards by the

ReH.R'

the agonist;

respectively.

R, H.R'

and r the

The irreversible

step

probably due to the formation of covalent The kinetic data (Fig. 2) agree with tors.

both

relationship

between

NE&! and (-)-isoproterenol

[HR'] = [H]

).

The lack

the rate (at

of decline

and receptors.

1316

likely erythro-

in this

set

and inactivated of reactions

(Table

1)

bonds between NEM and the recepthe proposed scheme by showing a

submaximal in

is

:

of inactivation (-)-13Hl-

consecutive treatment of the membranes with.the may be explained by the reversible interaction

it

in an increased by NEM. Both

*r agonist-bound

free,

is

linear

resulting

scheme NEM

As a

of turkey

(inactivation)

following

1 ).

and

in a synergistic

receptors

change,

erythro-

of the re-

the reagent

(Table

receptor

alkylation

of two types

by NEM has not been

and Methods),

to the f31-adrenergic

of

of turkey

when both

molecule

B-adrenergic

types

in the number

simultaneously

a conformational

sensitivity

catechol

of these

receptors

sites)

(Materials

the

of agonists

occu-

desensitization

neither

decrease

(-)-isoproterenol

chromatography

compounds

fashion.

a marked

are present

of the

equal

conditions,

various

cause

(IO-12),

DHA binding

(-)-isoproterenol

chemical

agonists

the @I-adrenergic is

by (-)-13Hl-

2).

tested

or the non-bioactive

tissues

affect there

2, were

compounds

NEM inactivates

and @-adrenergic

However,

(-)-pheny-

DHA (Table

the same preincubation

agent

in several

independently

membranes.

isoproterenol

to the membranes.

the alkylating

receptors

of compounds

Under

(-)-13Hl-

the three

phentolamine

DHA binding

DISCUSSION : Although hormone

sites.

(-)agonist

used in table

so that

the a-antagonist

do not affect

ligands

the receptor,

50 % of the receptor

by NEM plus

of the partial

of the antagonist

of the B-adrenergic

KD-value

NEM plus

receptors

by NEM in the presence

and the

concentrations, DHA binding reagent between

concentration

of

where activity

after

and the agonist (Table @-adrenergic agonists

I),

Vol.

86,

No.

There changes

BIOCHEMICAL

4, 1979

are

some striking are closely 1 and ref.

duce adenylate

cyclase

ability

than

to sensitize

in presence

of the antagonist

Recently, formation

Cassel

between

involves

GTP-ase

proterenol

than

by NEM in

in its

ligands

NEM inactivates

(-)-isoproterenol(Fig.

2).

of B-adrenergic

receptors,

or the dissociation red as possible conclude

which

reagent

can form

covalent

bonds

We observed recently that of 8-adrenergic receptors

fact

argued

for

types

formation

is

tion tor

by NEM.

a different of ligands.

induced receptor,

with

sites

type

cyclase

all

the

results

of agonists

is

observation.

(->-13Hl-

interaction

be conside-

At present, by NEM.

increases antagonists(g). that

the affiThis the receptor

a change

is now under

sensitivity change

1.

3. 4. 5.

to the

of the recep-

investigation.

HABER E. and WRENN S. (1976) Physiological Revs., 56, 317-338. LEFKOWITZ R.J., LIMBIRD L.E., MUKHERJEE C. and CARON M.G., (1976) Biochim. Biophys. Acta., 457, l-39. MUKHERJEE C., CARON M.G., MULLIKIN D. and LEFKOWITZ R.J. (1976) Mol. Pharmacol., 11, 16-31. BROWN E.M., FEDAK S.A., WOODARD C.J. and AURBACH G.D., (1976) J. Biol. Chem., 251, 1239-1246. LEVITZKY A., SEVILLA N., ATLAS D. and STEER M.L. (1975) J. Mol. Biol. Chem., jy7, 35-53.

1317

of con-

to inactiva-

REFERENCES :

2.

we

This groups(lg).

not of antagonists

the conformational

of

molecule,

might

between

suggest

DHA categories

or imidazole

selectively not for

but

receptor

this

receptor

alkylated

by the increased

activation,

of (-)-iso-

represented

inactivation.

of molecular

between

erythrocytes

in the presence

on a single

sulphydryl-,amino-

The present

as indicated

explain

by agonists

but

of in-

The catecholamine-

of two equally

solubilization for agonists

by the binding

The relationship

and the adenylate

with

of the B-receptor

nity

and both

interact

of the partial

moiety

5)

transmission

in turkey

no more than half

receptor-complexes

significant

of the @-adrenergic might

of two binding

interpretations

can not

the

to NEM in presence

agonists

The existence

of binary

2 and ref. cyclase

to

by NEM.

and not

that

The inactivation

of B-adrenergic

related

(-)-isoproterenol

GTP-ase(l7).

was more sensitive

the S-adrenergic

sites(Fig.l),

S-adrenergic

and adenylate

absence.

the presence

Although

(Table

of a membrane-bound

activity

that

to inactivation

(-)-phenylephrine,

have reported

the S-receptors

closely

of the agonist

(-)-13Hl-DHA

conformational suggesting

is

receptors

in presence

and Selinger

the activation

stimulated

activity)

the B-adrenergic agonist

activation,

processes are fast and reversible of B-adrenergic ligands to in-

(intrinsic

of the partial

COMMUNICATIONS

agonist-induced

cyclase

: i) Both The ability

activation

is more pronounced

in presence

binding

related 16) ii)

RESEARCH

between

and adenylate

both phenomena (Fig. 2, Table their

BIOPHYSICAL

similarities

of the S-receptors

Inactivation

AND

Vol. 86, No. 4, 1979

6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

FURCHGOTT R.F. (1970) Fed. Proc., 29, 1352-1361 Biophys. Acta., 127, 347-354. @YE I., SUTHERLAND E.W. (1966) BiocEm. LOWRY O.H., ROSEBROUGH N.J., FARR A.L. and RANDALL R.J. (1951) J. Biol. Chem., 193, 265-275. VAUQUELIN G., GEYNET P., HANOUNE J. and STROSBERG A.D. (1977) Acad. Sci. USA., 76, 3710-3714. Proc. Natl. MUKHERJEE C. and LEFKOWITZ R.J. (1976) Proc. Natl. Acad. Sci. USA., 12, 1494-1498. KEBABIAN J.W., ZATZ M., ROMERO J.E. and AKELROD J. (1975) Proc. Natl. Acad. Sci. USA., 11, 3735-3739. Chem., SHEAR M., INSEL P-A., MELMON K.L. and COFFIN0 P. (1976) J. Biol. 251, 7572-7576. HANSKI E. and LEVITZKY A. (1978) Life Sciences, 22, 53-60. SALMAN K.N., CHAI H.S., MILLER D.D. and PATIL P.N. (1976) Eur. J. Pharmacol., 36, 41-48. (1975) Proc. Natl. Acad. Sci. USA., 11, SIMON E.J. and GROTH J. 2404-2407. BLR H.P. (1974) Mol. Pharm., '0, 597-604. Biophys. Acta., 452, 538-551. CASSEL C. and SELINGER Z. (1976) Biochim. J., 2, SMYTH D.G., BLUMENFELD 0.0. and KONIGSBERG W. (1964) Biochim. 589-595.

1318