Solubilization of the D-1 dopamine receptor from rat striatum

BIOCHEMICAL

Vol. 137, No. 3, 1986

AND BIOPHYSICALRESEARCH

COMMUNICATIONS Pages 943-949

June 30.1986

SOLUBILIZATION

OF THE D-l Anita

Membrane National

Biochemistry Institute The National

Received

April

28,

DOPAMINE RECEPTOR FROM RAT STRIATUM

Sidhu*

and Peter

H. Fishman

Section, Developmental and Metabolic Neurology Branch, of Neurological and Communicative Disorders and Stroke, Institutes of Health, Bethesda, Maryland 20892 1986

SUMMARY The D-l dopamine receptor was extracted from rat striatal membranes Pretreatment of the membranes with a with 0.7% sodium cholate and 1 M NaCl. inclusion of crude phospholipids in the solubilization D-l specific agonist, buffer, and subsequent removal of the detergent led to a maximal extraction of 48% of the receptor binding sites. The D-l antagonist, ['251]SCH 23982, bound to single class of sites with a kd of 1.8 nM and a Bmax of 1.65 pmol/mg protein. The solubilized receptors retained the ability to discriminate between active and inactive enantiomers of agonists and antagonists selective for the D-l receptor. B 1986 Academic Press, Inc.

Brain

dopamine

that

stimulate

(D-21

(1).

detergent

(2-7).

Although rat

has not

adenylate

cyclase

been

year,

D-l

receptor

*Address National

years

an appropriate

has

has become

been

and those

reported

liqand

available.

some of

the

correspondence to: Institutes of Health,

for

the

and

Due to the iodinated

biochemical

rat,

using

the

Tiz511SCH the high

ligand,

D-1. receptor

in part,

to the

un-

In

the

for

the

D-l

receptor. specific

activity

Sidhu, Building MD 20892

the

[i12511SCH and affinity,

we have used of

tissues

was extracted

P-enantiomer,

specific

dif-

of the

23390,

active

canine

activity

due,

cyclase

several

and

solubilization

for

those

adenylate

bovine

cyclase

properties

Dr. Anita Bethesda,

IAbbreviations: [lzsI]SCH 23982, methyl-5-phenyl-lH-3-benzazepine-7-01;

inhibit

may have been

ligand,

of receptors,

receptors

specific

(9)

of the

that

adenylate

This

developed

two classes

D-2 dopamine

benzazepine

binding

into

ago (8),

reported.

an iodinated

investigate

brain

has been

several

previnusly

and low nonspecific to

(D-l)

a dopamine-stimulated

of

last

of

systems

striatum

availability

239821,

have been divided

Solubilization

ferent

from

receptors

this

compound

membrane-bound 10,

Room 3004,

D-1 The

5-R enantimer of 8-iodo-2,3,4,5-tetrahydro-3PEG, polyethylene glycnl. 0006-29

943

I X/86

%I .50

Vol. 137, No. 3, 1986

receptor*. the D-l from

In order receptor,

striatal

solubilization those

BIOCHEMICALANDBIOPHYSICALRESEARCH

found

it

to further

characterize

was necessary

In this

membranes. of the D-l

the biochemistry

to develop

procedures

communication,

dopamine

receptor

COMMUNICATIONS

with

for

we report binding

and function its

solubilization

the first properties

of

successful similar

to

in membranes. EXPERIMENTAL

PROCEDURES

Materials. SK & F R- and S-38393 were from SmithKline and French Laboratories (Philadelphia, PA) and S&H R- and S-?3390 were from Schering-Plough (Bloomfield, NJ). r'251;SCH 23983 12200 Ci/mmol) was obtained from New England Nuclear (Boston, MA). Poly-L-lysine, sodium cholate, and crude phospholipids (bovine hrain extract, type VII) were purchased from Sigma Chemical Cn. (St. Louis, MO). SM-2 Siobeads were from Bio-Rad Laboratories (New York, NY). All other reagents were of the highest purity commercially available. of male Sprague Dawley rats were Membrane Frepara tion . Fresh caudate nuclei homogenized in 10-l vol (w/v) of 50 mM Tris-HCl, pH 7.4. Following a 5-min centrifugation at 1000 x g, crude striatal membranes were isolated frnm the supernatant by cpntrifugation at 18,000 x g for 20 min. The membranes were either suspended directly in Buffer S fsee below) or in Buffer TS containing 50 mM Tris-HCl, pH 7.4, 120 mM NaCl , 5 mM KCl, 7 mM CaCl? and 1 mF? MgC12. Fnr agonist pretreatment of the membranes, they were incubated in Buffer TS with 10 After 20 min, 7 vol of Buffer TS was added and the pM SK & F P-38393 at 37°C. membranes were isolated as described above. Crude striatal membranes were suspended at 1 mg Solubilization Procedures. protein/ml in Buffer S containing 50 mM Tris-HCl, pH 7.4, 5 mM KCl, ! mM EflTA, 1 mM MgC12, 2 mM CaC12, 250 mM sucrose, 0.2 mM phenylmethylsulfonylfluoride and 1 mM dithiothreitol. When present, NaCl, crude phosphnlipids and sodium cholate were added to the desired final corcentrations as indicated in the figure and table legends. The suspension was maintained on ice for 30 min with periodic agitation and was then centrifuged at 31,300 x g for 35 min. The clear, yellowish supernatant contained the soluble membrane proteins; in some experiments, the resulting pellet was washed once in Buffer TS and assayed for binding sites. This centrifugation procedure was routinely used as more prolonged centrifugation (150,000 x g for 45 min) did not sediment additional receptors. [12sIjSCH 23982 binding to membrane fractions was assayed as Binding Assays. described previously (9) by filtration onto glass fiber filters. For binding to the soluble material, it was essential to first remove the cholate sirce the detergent was found to interfere with the binding assay (data not shnwn). The soluble preparation was mixed with moist SM-2 Biobeads (1.2 g/ml of extract.) and the mixture was gently shaken for 1 h at 4°C (10). After the heads were allowed to settle, the supernatant was removed and used directly in the binding The latter contained 20 ~1 of superantant, n.5 nM rl2sIjSCH 23982, assay. unlabeled agonists or antagonists where indicated and Puffer S to 150 pL. The radioligand and drugs were diluted in Buffer S; the final concentration of NaCl in the assay was 133 mM. After incubating the samples for 60-90 min at room temperature, binding was determined by a PEG precipitation/filtration prccedure (7). Briefly, 50 ~1 of bovine y-globulin (10 mg/ml) and 200 ~1 of PEG 6000 were added; the mjxtures were chilled CP ice for 5 min and then filtered onto glass fiber filters contained in a Brandel Cell Harvester modified for receptor binding assays (Brandel, Gaithersburg, MD). After washing the filters with 4 x 2Sidhu,

A.,

Kassis,

S.,

Kebabian,

J.

W., and Fishman, 944

P. H.,

in preparation.

Vol.

137,

BIOCHEMICAL

No. 3, 1986

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

4 ml of 10% PEG in 20 mM Tris-HCl, pl-! 7.4, they were counted in a Beckman qamma Nonspecific binding, as determined in the presence of 10 nM SCH @DO counter. In a typical experiment usinq 0.5 23390, represented l&30% of total binding. nM radinligand and soluble receptnrs from agonist-treated membranes, total and nonspecific binding were 5000 and 1000 cpm, respectively. Protein was determined by the method of Lowry et aZ. (11) Other Procedures. and when cholate was present in the samples, a correction was made for the presence of the detergent. The computer fitted program LIGAND (12) was used to analyze the binding data. All values are summarized as means s S.E.M. from separate experiments where n equals the number of experiments. RESULTS AND DISCUSSION

of D-l

So?ubilimtion that

sodium

from

rat

cholate

striatal

and variable To improve

As shown

with

SM-2 Biobeads over

cessfully

0.7%

4-8X

was used

in

of

to the bindinq

cholate

alone.

incubated

with

studies,

r '2511SCH

nf

the

23987

Since

with

approach

not

the

D-l

10 PM SK & F F-38393,

was

other

nf the

Solubilization

improve

the

receptor

had been

suc-

membranes

receptor.

Striatal

a D-1 specific

agonist,

Cholate Cholate,

NaCl

Cholate,

NaCI,

Cholate,

NaCl, phospholipids

Cholate,

SM-2

on the solubilization and recovery from rat striatal membranes

Solubilized

conditions

D-l binding

Untreated 6.2 7.5 11.7

beads

12.4

from membranes

nda

nda

NaCl, phospholipids, SM-2 beads

sites

Agonist-pretreated I of total

2 1.7 +_ 1.7 i 6.1

+_ 0.6

11.9 24.5 15.9

t 2.3 -I 3.5 If 4.5

48.1

+_ 4.2

Untreated or agonist-pretreated membranes were extracted with 0.7% sodium cholate in the presence or absence of NaCl (1 M) or phsopholipids (0.6 mg/ml). Specific binding of r'251Wi 23982 to the solubilited receptots was performed either before or after removal of the detergent by SM-2 beads as described in

Methods.

The results

to the membranes Specific binding tein) were 7400 and,

are expressed

and represent to untreated and 5700 cpm,

as the percentage

the mean +_ S.E.M and agonist-pretreated respectively.

not determined.

945

of

fac-

detergent

TARLE I

The effect of various conditions of D-l dopamine receptors

low

receotors,

several

agonist-pretreated

for

sites

significantly

R-adrenerqic

from

bindinq

however,

I M NaCl and removal

the

we found

membrane-bound

combination

assa.y did

by deoxycholate a similar

preliminary

specific

from

I, addition

prior

In

degree of sotubilization,

The ranged

in Table

to attempt

were

Receptors. to extract

cholate

solubilized

we decided branes

generally

yield,

tors.

yield

was able membranes.

and the

Dopamine

of total 2-6 separate membranse

specific

binding

experiments. (8 ug of pro-

(I3), memand

Vol. 137, No. 3, 1986

these

membranes

specific

with

lipids

in

the

to

choline with

the

During

the

lipid

beads,

it

of the

studies 62% of sedimented

readily total

specific

in the

after

removal

covery both

of

of varying

specific

total

detergent:protein than

soluble

pellet

mately

30% of

represented the

total

be due to denaturation of some sites

Properties binding performed

onto

of

(Fig.

less binding

to the

SM-2 beads

E-l

binding

2).

20% of

sites

were

acety?-

by interacting (14,15).

of

based

cholate

with

be associated

this

regard,

with

the

(Miflex

initial

GVS, 0.22

membranes

the

and re-

The extractinn

1).

increased

as the

sites,

specific

binding

detected.

removal

D-l Receptor.

associated

total

during

or incomplete

rm)

was recovered

solubilization

(Fig.

activity

not

sites

946

phospho-

of

ratio

a maximum at cholate:protein

than

As can be seen,

to

on the

sites

binding

D-l

bind-

by SM-2 beads.

determined

of the solubilized

of specific

to be soluble

filter

ratios

and reached

In

original

was

of some of the

the Sohbilized

parameters

x g.

a fur-

removal

appeared

binding

The corresponding

5:l.

sites

caused

it

appeared After

a Millipore

and

was increased

greater

stabilize

mem-

phnspho-

nicotinic

helped

methods).

100,000

the

phospholipids

of the detergent

protein

for

D-l

the

of exogenous

the

through

sites

(n=6)

of

brain

cholate

f 4.?%

receptor

cholate:protein

binding

membrane

D-l

binding at

passed

and 47% of the filtrate

(see the

that

yield of

bovine

of

reported

receptor,

the

crude

The addition

been

was suggested

extraction,

however,

The effect

has

the

pretreatment

addition

the membranes.

regions

initial

extract

of

of 48.1

systems

and

Inclusion

yield

from

used,

by the

and a maximal

where

vesicles

cholate

I).

fold

to

on ultracentrifugation SM-7

2-3

RESEARCH COMMUNICATIONS

conditions

prior

solubilizing

hydrophobic

the

buffer

extracted

receptor,

Under

(Table

solubilizing

were

AND BIOPHYSICAL

was enhanced

agonist

increase

sites

lipids

extracted.

sites

the

2-fold

ing

were

binding

branes

ther

BIOCHEMICAL

In

the

the

in-

sites.

Approxi-

loss

may either

extraction,

adsorption

of cholate

by the beads.

nrdcr

characterize

saturation binding

ratios with

This

to

of

binding represented

the

curves the

bulk

were of

Vol.

137,

No. 3, 1986

BIOCHEMICAL

AND

,

,

I

1:l

BIOPHYSICAL

I

1

3:l CHOLATE:

RESEARCH

I

COMMUNICATIONS

1

I

I

I

51

7:l

9:l

PROTEIN

Effect of cholate:protein ratios on extraction of O-1 dopamine recep ors from striatal membranes. Agonist-pretreated membranes were extracted at the indicated cho?ate:protein ratios in the presence of 1 M NaCl as described in Methods. After centrifugation, the insoluble membrane pellets were washed once and suspended to the original volume. Sodium cholate in the soluble fractions was removed by SM-2 beads. Then binding of [12sI]SCH 23982 to insoluble (o ---0) and solubilized (o--o) fractions was assayed by the PEG precipitation/filtration method. Protein content in the solubilized fractions (A---A) was determined prior to removal of the detergent. Maximal values of 100% (I) for protein content and specific binding were 1 mg/ml and 5000 cpm, respectively.

-i+

CONCENTRATION

Concentration dependent receptors. Agonist-pretreated in the presence of 1 M NaCl was removed from the soluble Methods. The solubilized receptors tions of [12sIlSCH 23982 for 90 min presence (I)) of 10 UM SCH 23390. substracting nonspecific binding (0) #G$kine cholate cholate

binding

(nM)

of [lesI]SCH 23982 to solubilized membranes were extracted with 0.7% and 0.0 mg/ml of phospholipids and sodium fraction with W-2 beads as described in were incubated with increasing concentraat room temperature in the absence (0) and Specific bindinq (A---A) was obtained by from total binding (0). 947

Vol. 137, No. 3, 1986

BlOCHEMlCALANDBlOPHYSlCALRESEARCHCOMMUNlCATlONS

TABLE II Competitive inhibition of I 12511SCH 23982 binding to membrane-bound and solubilized D-l dopamke receptors by agonists and antagonists Membrane-bound

Compound SK & F R-38393 SK & F S-38393 SCH R-23390 SCH S-23390

139 28400 1.5 57.5

f. + f +

receptors 100

Solublllzed

receptors

(nM)

33 2800 0.5 25.2

192 14300 2.7 65

+ + 2 +

50 6500 1.2 35

Solubilized receptors, obtained as described in the legend to Fig. 2, and membranes were assayed for binding of [ 12511SCH 23982 in the presence of increasing concentrations of the indicated compound as described in Methods. The concentration required to inhibit binding by 50% (IC5o) was determined by computer analysis of the competition curves.

total

binding

F12511SCH Computer site

over

23982, analysis

with

protein

fn=4).

receptor

indicated

is

Kd of

the

probably

In order of

these

sites

to

specific

inhibited

of

S-entantimers than

the

the

the

forms

binding

of the

binding

D-l

of the

The soluhilized membrane-bound of both

corresponding

1.3

pmol/mg

specific

agonist R-forms

compete

with

D-l

the

radioligand

to both

948

on

the

to a single

the

site

shift

in

membrane-bound

requirements

that

receptors,

73987

for

SK & F R-38393

radioligand

of the drugs.

of

[12511SCH

Similarly,

and antagonist

pmnl/mg

studies

solubilized

receptor.

receptors;

to a single

The ?-fold

that

nK

binding.

+ 0.17

bound

protein'.

The agonist

receptors

1.65

2.5

receptor.

of

of the

bound

environmental

of the

at

total

earlier

also

over

pharmacolngy

II).

to

73987 [ 1251TSCH .!

receptor

to

(Table

inhibiting

the

the same extent. typical

drugs

30% of

r 12511SCH 23982

favourable

of the

the

than

even

nM and a 8max nf

solubilization

investigate

D-l

in

a Bmax of

less

that

compare where

concentrations;

was

+ 0.6

solubilized

upon

and membrane-bound 73390

data

was examined

equipotent

1.56

reflective

have been disrupted

ability

data

nlC and

of

radioligand

of the

D-1 receptors

affinity

of binding

These

a Kd of 0.7

the

range

nonspecific

an apparent

membrane-bound with

a wide

also

retained the

were

binding

appeared

to both

the

the

antagonist

forms

the

of the

to to be

solubilized SCH R-

receptor

the stereoselectivity

pharmacologically much less

potent

inactive inihibitars

to

vol.

137,

In that

BIOCHEMICAL

No. 3. 1986

summary, retain

we have

the

solubililation

solubilized

expected of the D-l

ultimate

purification

directed

at elucidatinp

of

AND

BIOPHYSICAL

binding

sites

characteristics receptor the

of

should

receptor,

the mechanisms

the

RESEARCH

from D-l

be of great as well

of receptor

as

rat

COMMUNICATIONS

striatal

dopamine value

regulation

The

receptor.

in studies

reconstitution

membranes

aimed

at

experiments and function.

REFERENCES 1. Kebahian, J. W., and Calne, D. (1979) Nature 277, 93-96. 2. Gorissen, H., and Laduron, P. (1979) filature 279, 72-74. 3. Gorissen, t!., Ilien, B., Aerts, G., and Laduron, P. fI.980) FERS I,ett. 121, 133-138. 4. Lerner, M. H., Rosengarten, H., and Friedhof, A. J. (1981) rife sci. 29, 2367-7374. 5. Kilpatrick, B. F., and Caron, M. G. (1983) J. Bi02. Chem. 258, 13538-13534. 6. Hall, J. M., Frankham, P. A., and Strange, P. G. (1983) J. Neurockem. 41, 1526-1532. 7. LPW, J. Y., and Goldstein, M. (1984) cT. Neurockem. 42, 1798-1305. 8. Hoffman, F. M. (1979) J. BioZ. them. 254, 255-258. 9. Sidhu, A., and Kehabian, ~1. W. (1985) Eur. J. Pkarmaeol. 113, 437-440. 10. Gal, A., Braun, S., Feder, D., and Levitzki, A. (1983) &r. J. Rio&em. 134, 391-396. 11. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, P. 2. (1951)

J. Biol. 12. 13. 14. 15.

Munson, Nevidi, Epstein, Anholt, 4387.

Chem. 193, 765-275. P., and Rodhard, 0. E., and Schramm, M. M., and Racker, E. R., Lindstrom, J.,

(1980) AnaZ. Biockem. 107, ?20-739. (1984) J. BioZ. Ckem. 259, 5R@3-5808. (1978) J. Biol. Ckem. 253, 66@D-6669. and Montal, M. (1981) J. BioZ. Ckem. 256, 4377-

949