Vol. 172, No. 2, 1990 BIOCHEMICAL AND B(OPH’fSICAL RESEARCH COMMUNICATIONS

October 30, 1990 Pages 709-714

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

Yasushi Masuda, Tetsuya Ohtaki,lChieko Kitada, Masao Tsuda, hkira hrimura" and Masahiko Fujino

Tsukuba Research Laboratories, Takeda Chemical Industries, Ltd. Wadai 7, Tsukuba, Ibaraki 300-42, Japan

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 with 3-[(3-cholamidopropyl)dimethylam

T% io]-l-propanesulfonate.

Saturable binding experiments using [ I]PACAP27 revealed that 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. Competi- tive binding experiments confirmed that the solubilized receptor retained a specificity for PACAP. Thus, the solubilization of the receptor in an active form was successfully achieved by the present procedure. '19"O A,.;rdimlc P1L55, Inc.

PACAP was recently isolated from ovine hypothalamus by

Arimura and his colleagues using an index of the elevation of

intracellular cyclic AMP levels in rat anterior pituitary cells

(1). Subsequently, a shorter peptide corresponding to the N-

terminus 27 residues (PACAP27) was isolated from the hypothalamus

and was found to have the same biological potency as PACAP (2).

They have noticeable sequence homology with VIP and a certain

degree of homology in N-terminus portion with GRF, peptide histi-

1 To whom correspoc,!ecce should be addressed. Abbreviations and chemical names of detergents: CHAPS, 3-[(3- cholamidopropyl)dimethylammon~-copanesulfonate: Zwittergent 3-12, N-dodecyl-N,N-dimethyl-3-ammonio-l-propanesulfonate: Thes- it, dodecylpoly(ethyleneglycolether)9; Triton X-100, Octylphenol- poly(ethleneglycolether)lO: VIP, vasoactive intestinal peptide; GRF, Growth hormone releasing factor; PMSF, phenylmethylsulfonyl fluoride: BSA, bovine serum albumin.

Vol. 172, No. 2, 1990 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

dine isoleucine amide, secretin and glucagon (l-3). Although the

physiological function of PACAP is still under investigation,

specific high affinity membrane receptors were found recently in

the central nervous system (4-6) and in pancreatic acinar cells

(7). PACAP receptors in these reports share similar properties

in that they have a high affinity for both PACAP and PACAP

but scarcely interact with other related peptides such as VIP or

secretin. In the present study, we will describe successful

solubilization of an active PACAP receptor from bovine brain

membranes using CHAPS. The solubilized receptor retained a high

affinity and specificity for PACAP as the native 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 X- 100, 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 Tris- HCl, 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 100,000 x g for 1 h and the membrane pellet was obtained. The pellet was suspended in Buffer A deprived of sucrose, ultracen- trifuged 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. Solubilization of receptor: The membrane suspensions (7 mg pro- tein/ml) were mixed with various concentrations of a detergent and stirred for 1 h at 4 OC. The mixtures were ultracentrifuged at 174,000 x g for 30 min and the resultant supernatant fractions were obtained. Ei..gtf~qpj assay : The solubilized fractions were incubated with 50

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, 0.05% CHAPS, 0.5 mM PMSF, 20 pg/ml leupeptin and 1 pg/ml pepsta- tin (pH 7.4). The reaction mixtures were diluted with 2 ml of ice cold Buffer B and immediately filtered through glass fiber filters (Whatman GF/F) which were pre-wetted with Buffer B con- taining 0.3% polyethylenimine (9). The glass fiber filters were subjected to gamma-counting. Non-specific binding was determined

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Vol. 172, No. 2, 1990

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

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

RESULTS AND DISCUSSION

We tried to solubilize the PACAP receptor from bovine brain

membrane fraction using six different detergents (Table 1). In

our experimental conditions (7 mg protein/ml, 0.58 detergents),

CHAPS was superior to other detergents for the solubilization,

since it gave the highest amount of biologically active materi-

als. Although Zwittergent 3-12 and sodium deoxycholate were

effective in solubilizing the membrane proteins, they were inef-

fective in solubilizing the receptor activity. Digitonin and

octylglucoside, which are commonly used for membrane receptor

solubilizations, were ineffective in this condition.

TO investigate optimal CHAPS concentration for the solubili-

zation, experiments were carried out using various concentrations

of CHAPS at fixed concentration of the membrane protein (7 mg

protein/ml)(Fig.l). The amount of the solubilized proteins in-

Table 1

Solubilization of PACAP receptor with various detergents

Supernatant [1251]PACAP27 protein specific binding (w/ml 1 (8)

Digitonin 0.46 3 Octylglucoside 0.59 N.D. Triton X-100 2.2 24 Thesit 1.8 9 CHAPS 1.8 50 Zwittergent 3-12 3.8 N.D. Sodium deoxycholate 5.7 2

pecific binding was expressed as the ratio to the IlPACAP27 added. N.D. means not detectable.

Vol. 172, No. 2, 1990 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

50 . - 50

40 - - 40

30 - - 30

20 - - 20

10 - -10

O- 0 05 10 15

0

CHAPS concentration ( % 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 solubi-

lized protein (0) were plotted versus CHAPS concentration.

creased depending on CHAPS concentration and reached a plateau at

1.0% CHAPS. Nearly 38% of the total protein could be extracted

in this experiment. The solubilized receptor activity increased

proportionally to CHAPS concentration up to 0.5%. Higher concen-

tration of CHAPS did not result in further increase of the solu-

bilized activity. The use of 0.5% CHAPS under decreasing concen-

trations of the membrane protein (8 to 2 mg protein/ml) did not

significantly alter the solubilizing efficiency (data not shown).

Therefore, subsequent solubilization experiments were performed

with 0.5% CHAPS at 7 mg protein/ml.

The binding of [ 1251]PACAP27 was saturable to the solubilized

fractions as well as to the membrane fractions (Fig.ZA,B).

Scatchard plot analysis showed a single class of high affinity

binding sites for both the preparations (Fig.ZC,D). The dissoci-

ation constant of the membrane receptor and of the solubilized

receptor were determined to be 100 pM and 200 pM, respectively.

This indicates that the solubilizing procedure did not cause

significant damage to the receptor activity. The maximal number

of receptors in the membranes and in the solubilized preparations

were estimated to be 2.5 and 0.9 pmol/mg protein, respectively.

712

Vol. 172, No. 2, 1990 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

0 1 2 3 0

1251-PACAP b 02 04 06 08 10 12

ound (omol/mg) “%PACAP bound :Dmol/mg)

Fi$$2Saturable binding of [1251]PACAP27. The ;F;i:;la",b;;d;;z I]PACAP27 to the membrane receptor (

solubilized receptor (B) were plotted versus [ 95 IlPACAP27 added. Scatchard plots (C) and (D) were obtained from the data shown in (A) and (B), respectively.

Considering the solubilizing efficiency of the membrane protein,

about 10 to 15% of the total binding sites were released as a

soluble form. Although this yield seems rather low, it is com-

s ET +-

- z 0 ’

11 10 9 a 7 6 5

Peottde concentration ( - log M 1

*,,““,“~“;fg 've binding to the solubilized receptor. The I]PACAP27 expressed as percent of the maximal

binding was plotted versus unlabeled PACAP ( l ), VIP (O), secretin (n) and GRF (m).

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Vol. 172, No. 2, 1990 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

parable to that obtained in the case of other receptors using

CHAPS (10, 11).

The binding specificity of the solubilized receptor was

investigated by competitive binding experiments (Fig.3). The

concentration of unlabeled PACAP and that of VIP to give the

half maximal binding of the labeled PACAP were 2.5 nM and 2.5

PM, respectively. The affinity of the solubilized receptor for

PACAP was about 1000 times greater than that for VIP. This

result is similar to that obtained for the membrane receptor (6).

Furthermore, VIP related peptides, i.e. secretin and GRF did not

displace the binding of [1251]PACAP27.

These results indicate that the present solubilizing proce-

dure does not induce conformational changes of the receptor

molecule which might affect the affinity and the specificity of

the 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, A and Fujino, 8il89.

M. (1990) Biochem. Biopys. Res. Commun. 166,

(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, 76- 85.

(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., Susi- ni, C., and Vaysse, N. (1990) J. Biol. Chem. 265, 1129-1133.

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