Vol.

172,

October

No. 30,

BIOCHEMICAL

2, 1990

AND

B(OPH’fSICAL

RESEARCH

COMMUNICATIONS Pages

1990

709-714

SOLUBILIZATION OF RECEPTOR FOR PITUITARY ADENYLATE CYCLASE ACTIVATING POLYPEPTIDE FROM BOVINE BRAIN Yasushi

Masuda,

Tsukuba

Tetsuya Ohtaki,lChieko Kitada, and Masahiko Fujino hrimura" Takeda Ibaraki

Research Laboratories, Wadai 7, Tsukuba,

Masao Tsuda,

Chemical Industries, 300-42, Japan

hkira

Ltd.

lrU.S.-Japan

Biomedical Research Laboratories, Tulane University Hebert Center, Belle Chasse, LA 70037, and Departments of Medicine, Anatomy and Physiology, Tulane University School of Medicine,New Orleans, LA 70112

Received

September

19,

1990

Summary: A receptor for pituitary adenylate cyclase activating polypeptide (PhChP) was solubilized from bovine brain membranes io]-l-propanesulfonate. with 3-[(3-cholamidopropyl)dimethylam T% I]PACAP27 revealed that Saturable binding experiments using [ the solubilized extract contained a single class of binding sites with a Kd of 200 pM and a Bmax of 0.9 pmol/mg protein. The Kd did not change significantly after the solubilization. Competitive binding experiments confirmed that the solubilized receptor Thus, the solubilization of retained a specificity for PACAP. the receptor in an active form was successfully achieved by the '19"OA,.;rdimlc P1L55, Inc. present procedure. PACAP Arimura

was recently

and his

intracellular

terminus

cyclic

They

have

degree To

AMP

of

whom

noticeable homology

correspoc,!ecce

levels

(PACAP27)

to have

the

from

using

a shorter

27 residues

and was found

1

colleagues

Subsequently,

(1).

isolated

same

sequence

an index in

rat

peptide

biological homology portion be

hypothalamus

of

the

anterior

pituitary

from

with with

VIP

of cells

to

the

potency

by

elevation

corresponding

was isolated

in N-terminus should

ovine

the

N-

hypothalamus

as PACAP

(2).

and a certain

GRF, peptide

histi-

addressed.

Abbreviations and chemical names of detergents: CHAPS, 3-[(3cholamidopropyl)dimethylammon~-copanesulfonate: Zwittergent 3-12, N-dodecyl-N,N-dimethyl-3-ammonio-l-propanesulfonate: Thesit, dodecylpoly(ethyleneglycolether)9; Triton X-100, Octylphenolpoly(ethleneglycolether)lO: VIP, vasoactive intestinal peptide; GRF, Growth hormone releasing factor; PMSF, phenylmethylsulfonyl fluoride: BSA, bovine serum albumin.

Vol.

172,

dine

No.

2, 1990

isoleucine

amide,

physiological

central

that

but

they

have

In

the

solubilization

affinity

of using and

CHAPS.

specificity

and reports

other

related

study,

The for

we

PACAP

were

both

solubilized

acinar

from

native

and such

describe

in cells

properties

PACAP

receptor the

recently

similar

peptides will

the

investigation,

pancreatic

receptor

PACAP as

Although

found

share

for

active

under

in

COMMUNICATIONS

(l-3).

still

affinity

present

RESEARCH

receptors

these

with

an

is

(4-6)

a high

interact

secretin.

PACAP

system in

BIOPHYSICAL

and glucagon

membrane

receptors

scarcely

membranes

of

affinity nervous

PACAP

(7). in

high

AND

secretin

function

specific the

BIOCHEMICAL

as

PACAP VIP

or

successful bovine

brain

retained

a high

membrane

recep-

tor. MATERIALS

AND METHODS

Materials: Na[ 1251] was purchased from Amersham Japan Co.. Digitonin and sodium deoxycholate were obtained from Wako Pure Chemical Ind. (Osaka, Japan). Octylglucoside, CHAPS, Triton X100, Thesit, and Zwittergent 3-12 were from Boehringer Mannheim. All the peptides in this study besides PACAP were from the Peptide Institute (Osaka, Japan). Peptide synthesis and radioiodination: PACAP was synthesized by the solid phase method and radioiodinated as described before (6). Preparation of bovine brain membranes: Fresh bovine brains were homogenized in 4 volumes of Buffer A consisting of 20 mM TrisHCl, 0.25 M sucrose, 2 mM EDTA, 0.5 mM PMSF, 20 ug/ml leupeptin and 1 ug/ml pepstatin (pH 7.4) with a Waring blender followed by Polytron homogenization. The homogenate was centrifuged at 690 x g for 10 min. The supernatant was further ultracentrifuged at The 100,000 x g for 1 h and the membrane pellet was obtained. pellet was suspended in Buffer A deprived of sucrose, ultracentrifuged again and resuspended at 13 mg protein/ml in the same buffer. The membrane suspension was stored at -70 OC until use. Protein concentration was determined by the bicinchoninic acid method (8) using BSA as a standard. The membrane suspensions (7 mg proSolubilization of receptor: tein/ml) were mixed with various concentrations of a detergent The mixtures were ultracentrifuged and stirred for 1 h at 4 OC. at 174,000 x g for 30 min and the resultant supernatant fractions were obtained. fractions were incubated with 50 Ei..gtf~qpj assay : The solubilized I]PACAP27 for 1 h at 25 OC in a 0.1 ml total volume of Buffer B consisting of 20 mM Tris-HCl, 5 mM MgC12, 0.1% BSA, leupeptin and 1 pg/ml pepsta0.05% CHAPS, 0.5 mM PMSF, 20 pg/ml The reaction mixtures were diluted with 2 ml of tin (pH 7.4). ice cold Buffer B and immediately filtered through glass fiber filters (Whatman GF/F) which were pre-wetted with Buffer B conThe glass fiber filters were taining 0.3% polyethylenimine (9). subjected to gamma-counting. Non-specific binding was determined 710

Vol.

172,

No.

BIOCHEMICAL

2, 1990

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

of 1 uM unlabeled PACAP and specific binding in the presence was obtained by subtraction of the non-specific binding from the total binding. For saturable binding experiments, the solubi(4 ug/lOO ul total volume) or the membrane fraclized fractions tions (3 pg protein/ 100 1.11 total volume) were incubated with increasing concentrations of the labeled PACAP under the same The ligands bound to the membrane fractions conditions as above. were separated by centrifugation following the previous method For competitive binding experiments the solubilized frac(6). with 50 pM of tions (20 ug/lOO ul total volume) were incubated the labeled PACAP with increasing concentrations of PACAP27, VIP, GRF or secretin. RESULTS AND DISCUSSION We tried

to

solubilize

membrane fraction our

using

experimental

it

gave

als.

Although

effective

in

fective

in

the

to

other

the

solubilizing

were

investigate experiments

CHAPS at

fixed

used

in this

for

out

concentration

of

The amount

of

detergents),

materi-

deoxycholate they

were

were

inef-

Digitonin membrane

for

using

various

the

membrane

the

In

and

receptor

condition.

concentration

CHAPS

1).

active

activity.

commonly

brain

solubilization,

biologically

receptor

were carried

protein/ml)(Fig.l).

the

and sodium

ineffective

optimal

0.58

for

of

bovine (Table

membrane proteins,

the are

detergents

detergents

3-12

solubilizing

from

(7 mg protein/ml,

Zwittergent

solubilizations,

of

different

amount

which

zation,

PACAP receptor

highest

octylglucoside,

TO

six

conditions

CHAPS was superior since

the

the

solubili-

concentrations

solubilized

protein

(7

proteins

Table 1 Solubilization

Digitonin Octylglucoside Triton X-100 Thesit CHAPS Zwittergent

of

3-12

Sodium deoxycholate pecific

IlPACAP27

PACAP receptor

with

various

detergents

Supernatant protein (w/ml 1

[1251]PACAP27 specific binding (8)

0.46 0.59 2.2 1.8 1.8 3.8 5.7

3 N.D. 24 9 50 N.D. 2

binding

added.

was expressed as the ratio N.D.

means

not

detectable.

to the

mg

in-

Vol.

172,

No.

2, 1990

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

50 .

- 50

40

-

- 40

30

-

- 30

20 -

- 20

10 -

-10

O0

05

10

concentration

CHAPS

0

15

( % w/v )

Fig.l.CHAPS dependence of the solubilization of PACAP receptor. mbrane protein solubilized (0) and the ~~",,~'fT~~~"Rd"in~fo~h",l~~ IlPACAP27 to each 50 pg of the solubilized protein (0) were plotted versus CHAPS concentration. creased

depending

1.0% CHAPS. in

this

Nearly

of

bilized

activity.

trations

the

the

0.5% CHAPS at The binding

This

plot sites

of

constant were

indicates

significant

as to

of

in the

the

the

the

of

concenthe

decreasing

efficiency

did

(data

experiments

solu-

concen-

2 mg protein/ml)

was saturable

not

were

preparations

the the

to

class

the

not

shown).

performed

of

receptor

and 0.9 712

high

and of

procedure activity.

pmol/mg

affinity

The dissocithe

be 100 pM and 200 pM,

solubilizing

solubilized (Fig.ZA,B).

(Fig.ZC,D).

membranes and in the be 2.5

to

fractions

membrane

membrane receptor

determined

to

(8 to

showed a single

both

damage to

were estimated

increase

0.5% CHAPS under

[ 1251]PACAP27

that

receptors

further

increased

Higher

0.5%.

at

be extracted

activity

up to

solubilization

analysis for

could

receptor

in

a plateau

7 mg protein/ml.

as well

Scatchard

and reached protein

solubilizing

subsequent

fractions

result

membrane protein

alter

Therefore,

receptor

not

The use of

significantly

ation

total

The solubilized

CHAPS did

of

binding

the

to CHAPS concentration

tration

of

38% of

experiment.

proportionally

with

on CHAPS concentration

solubilized

respectively. did

not

The maximal solubilized protein,

cause number

preparations respectively.

Vol.

172,

No.

BIOCHEMICAL

2, 1990

0

1

AND

2

1251-PACAP

b ound

BIOPHYSICAL

3

0

(omol/mg)

02

RESEARCH

04

06

“%PACAP

08

bound

COMMUNICATIONS

10

12

:Dmol/mg)

Fi$$2Saturable

binding of [1251]PACAP27. The ;F;i:;la",b;;d;;z I]PACAP27 to the membrane receptor ( [ 95 IlPACAP27 solubilized receptor (B) were plotted versus added. Scatchard plots (C) and (D) were obtained from the data shown in respectively.

(A) and (B), Considering about

the

10 to

soluble

solubilizing

15% of

form.

the

Although

efficiency total

of

binding

this

yield

the

sites

membrane protein,

were

seems rather

released low,

it

as a is

s +-ET

z

0

’

11

10

Peottde *,,““,“~“;fg

binding secretin

was (n)

9

a

concentration

've binding to the I]PACAP27 expressed plotted versus unlabeled and GRF (m).

713

7

6

5

( - log M 1

solubilized as percent PACAP

receptor. The of the maximal ( l ), VIP (O),

com-

Vol.

172,

No.

2, 1990

parable

to

CHAPS

that

(10,

The

binding

half

maximal

PM,

respectively.

PACAP is

competitive

of

unlabeled

similar

the

BIOPHYSICAL

case

of

the

results

does

not which

PACAP the

RESEARCH

other

COMMUNICATIONS

receptors

using

1000

times

that

might

experiments

(Fig.3).

that

of were

the

i.e.

The

to

2.5

solubilized than

for

VIP

was

give

the

nM and

2.5

receptor

that

for

membrane

secretin

and

for

VIP.

This

receptor

(6).

GRF did

not

[1251]PACAP27.

indicate induce

the

greater

peptides,

of

receptor

PACAP

of

obtained

related

solubilized

and

labeled

affinity

binding

the

binding

The

to

VIP

These

of

about

Furthermore,

molecule

the

of

by

binding

was

displace

in

specificity

concentration

dure

obtained

AND

11).

investigated

result

BIOCHEMICAL

that

the

conformational affect

the

present

solubilizing

changes affinity

and

of the

the

procereceptor

specificity

of

receptor. REFERENCES

(1)

Miyata, A., Arimura, A., Dahl, R.R., Minamino, N., Uehara, A Jiang, L., Culler, M.D., and Coy, D.H. (1989) Biochem. Biophys. Res. Commun. 164, 567-574. (2) Miyata, A., Jiang, L., Dahl, R.D., Kitada, C., Kubo, K., Fujino, M., Minamino, N., and Arimura, A. (1990) Biochem. Biophys. Res. Commun. 170, 643-648. (3) Kimura, C., Ohkubo, S., Ohgi, K., Hosoya, M., Itoh, Y. Onda, H * I Miyata, A., Jiang, L. Dahl, R.R., Stibbs, H.H., Arimura, and Fujino, M. (1990) Biochem. Biopys. Res. Commun. 166, A 8il89. (4) Tatsuno, I., Gottschall, P.E., Koeves, K., and Arimura, A. (1990) Biochem. Biophys. Res. Commun. 168, 1027-1033. (5) Gottchall, P-E., Tatsuno, I., Miyata, A., and Arimura, A. (1990) Endocrinology, in press. (6) Ohtaki. T., Watanabe, T., Ishibashi, Y., Kitada, C., Tsuda, M Gottschall', P.E., Arimura, A., and Fujino, M. (1990) B&hem. Biophys. Res. Commun. 171, 838-844. (7) Buscail, L., Gourlet, P., Cauvin, A., De Neef, P. Gossen, D ., Arimura, A., Miyata, A., Coy, D.H., Robberecht, P., and Christophe, J. (1990) FEBS Lett. 262, 77-81. (8) Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartner, F.H, Provenzano, M.D., Fujimoto, E-K., Goeke, N.M., Olson, B-J., and Klenk, D.C. (1985) Anal. Biochem. 150, 7685. (9) Bruns, R.F., Lawson-Wendling, K., and Pugsley, T. (1983) Anal. Biochem. 132, 74-81. (10) Helmeste, D.M. and Li, C.H. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 67-71. (11) Knuhtsen, S., Esteve, J.P., Cambillau, C., Colas, B., SusiN. (1990) J. Biol. Chem. 265, 1129-1133. ni, C., and Vaysse, 714