HYBRIDOMA Volume 11, Number 6, 1992 Mary Ann Liebert, Inc., Publishers

Monoclonal Antibodies to Somatostatin Receptor of Rat Brain ITSUKO O. 1

NAKABAYASHI1

and HAJIME

NAKABAYASHI2

Department of Immunology, Cancer Research Institute, and 2Department of Internal Medicine (II), School of Medicine, Kanazawa University, 13-1 Takara-machi, Kanazawa, Ishikawa 920, Japan

ABSTRACT Murine Monoclonal antibodies (MAbs) to the rat brain somatostatin (SRIF) receptor produced. Sp 2/0 myeloma cells were fused with splenocytes of Balb/c mice immunized with the soluble rat brain SRIF receptor which was partially purified by gelfiltration chromatography. Screening by radioligand ([125l-Tyr11]SRIF-14) binding inhibition assay yielded three stable cell lines producing lgG1, IgM, or IgA antibody. Autoradiographic study of the polyacrylamide gel electrophoresed under nondenaturing conditions revealed that these MAbs inhibited the ligand binding to the receptor, regardless of their incubation with the receptor prior to the ligand binding. The results suggest that the MAbs produced are the antibodies to the ligand binding site of the receptor, and bind to the receptor in competition with the ligand. were

INTRODUCTION

Somatostatin (SRIF) is a cyclic tetradecapeptide widely distributed throughout the central and peripheral nervous systems, as well as in the pancreas and gastrointestinal tract (1). SRIF exerts biological actions by interacting with its membrane-bound receptors (2,3,4). The localization of the SRIF receptor in the target tissue such as the pancreatic islet where endocrine, paracrine, or neurocrine action of SRIF operates, is not yet clarified

(5).

In our previous electrophysiological study, injection of SRIF-14 at a physiological dose into the rat portal vein facilitated the sensory (afferent) activity in the hepatic branch of the vagus nerve (6). We moreover disclosed histologically the neural corpuscles located beneath the endothelium of the large branches of the portal vein. The nerve filaments in the corpuscles did bind to the exogenous SRIF (6). A similar electrophysiological phenomenon occurred in the pancreatic vagus by SRIF injection into the rat pancreatic artery. Furthermore, the SRIF-binding glomuslike neurovascular bodies were found in the peripancreatic sinusoidal vein (7). The results suggested the existence of a neural chemoreceptive system monitoring the endogenous SRIF from the splanchnic organs. To further determine the SRIF-binding nature of these neural apparatus, we designed to produce the MAbs against the SRIF receptor, preferably against the ligand biding site. 789

MATERIALS

AND

METHODS

Materials

Trizma-base, phenylmethylsulfonyl fluoride (PMSF), bacitracin, CHAPS, Freund's complete adjuvant, HAT and HT were purchased from Sigma Chemical Company (St. Louis, MO). Somatostain-14 was from Protein Research Foundation (Osaka, Japan) and [125l-Tyr11]somatostatin-14 from Amersham International pic. (Amersham, England). BioBeads™ SM-2, Bio-Gel A-5m (fine), and Protein Assay Kit I were obtained from Bio-Rad Laboratories (Richmond, CA). High molecular weight gel filtration calibration kit and electrophoresis calibration kit were from Pharmacia Inc. (Piscataway, NJ). PEG 4000 (for gas chromatography) from Merck (Darmstadt, Germany). Dulbecco's modified Eagle's medium from Nissui Pharmaceutical Co.,Ltd. (Tokyo, Japan). Fetal bovine serum was purchased from Flow Laboratories (North Ryde, Australia) and BriClone from BioResearch (Dublin, Ireland). Rabbit anti-mouse immunoglobulins A, G and M (immunoglobulin fraction) from Serotec (Oxford, England), and peroxidase-conjugated goat affinity purified anti-mouse immunoglobulins A, G and M from Cappel Research Products (Durham, NC).

Preparation of soluble SRIF Receptor Rat cortical membranes were prepared according to the method described by Epelbaum et al. (8). After homogenized and washed, brain cortexes of male Wistar rats weighing ca. 280 g were solubilized on ice in 50 mM Tris-HCI (pH 7.4) containing 5 % glycerol, 5 mM CaCI2, 5 mM MgCI2i 0.2 mM PMSF, 0.1 mg/ml of bacitracin, and 100 KIU/ml of aprotinin (Buffer A) in the presence of 1 % CHAPS and 1 M NaCI at a protein concentration of 5 mg/ml for 1 hr. The supernatant was incubated with half volume of BioBeads SM-2 at 4°C for 1 hr to remove CHAPS. After concentrating to 1/4-1/7 volume, the soluble SRIF receptor was further purified by gel-filtration chromatography using Bio-Gel A-5m equilibrated with Buffer A. Each fraction was screened for the binding activity with [125l-Tyr11]somatostatin-14, and two peaks were observed; a high molecular weight (HMW) peak eluted in the void volume, and a low molecular weight (LMW) peak eluted at ca. 100 kilodaltons (kDa). The LMW peak had higher binding activity (7.4 ± 0.8 %, n=10) than the HMW peak (2.5 ± 0.8 %) presumably consisting of the aggregated receptor. The LMW fraction was concentrated to 1-1.5 mg/ml of protein for immunization of mice. The MW was determined by calibrating the column with the MW standards (Blue Dextran 2000, thyroglobulin 669 kDa, ferritin 440 kDa, catalase 232 kDa, aldolase 158 kDa, and BSA 67 kDa). Nonspecific binding was 3.5 ± 0.2 % or 4.3 ± 0.3 % (n=10) in the absence or presence of unlabeled SRIF, respectively.

Binding Assay

chromatography screening, 100 µ of each fraction were incubated with 50 µ of 20,000 cpm) in the absence or presence of 5x10-6 M unlabeled SRIF-14 at 25°C for 1 hr. B/F separation was done by adding 500 µ of dextran-coated charcoal (0.025 % dextran and 0.25 % charcoal) (9). The specific binding was expressed as a percentage of the total radioactivity added to the assay tube after subtracting the value of nonspecific binding by the buffer (Buffer A) alone in the absence or presence of For

p25|.jyrll]SR|F-14 (ca.

excess

unlabeled SRIF.

Binding Inhibition Assay To screen hybridomas, culture supenatants were examined for inhibition of the ligand binding. Twenty five microliters of the supernatant or the culture medium as the control were mixed with an equal volume of [125l-Tyr11]SRIF-14 and 100 µ of the LMW 790

receptor protein obtained from chromatography. processed as described under "Binding Assay". Native

Polyacrylamide

Gel

Electrophoresis

and

The reaction mixture

was

further

Autoradiography

The Bio-Gel A-5m column was also examined by native polyacrylamide gel electrophoresis (PAGE) and autoradiography. Forty microliters of the starting material (solubilized brain cortex), the HMW fraction or the LMW fraction were incubated with 8 µ of radioactive SRIF (ca. 50,000 cpm) in the absence or presence of 10"5 M unlabeled SRIF at 25°C for 1 hr. The reaction mixture was applied to a 2-20 % gradient native polyacrylamide gel (10). The gel was electrophoresed at 200 V for 12 hr at 4°C, and exposed to a Kodak X-Omat AR film with an intensifying screen. On the autoradiogram (data not shown), a band of about 100 kDa was observed in the lane of the starting

material. This band was abolished in the presence of unlabeled SRIF, and was intensified in the lane of the LMW fraction. The radioactivity of the HMW fraction stayed in the top well of the gel, suggesting again the aggregation of the receptor The binding activity of the starting material, HMW and LMW fraction tested was 2.1 %, 5.3 % and 6.5 %, respectively. The result of the autoradiography was compatible with that of the Bio-Gel A5m chromatography. To examine whether SRIF binding to the receptor is inhibited by the MAb, the culture supernatant was preincubated with the LMW receptor protein at 25°C for 30 min, and further reacted with radioactive SRIF for 1 hr. The reaction mixture was subjected to native PAGE and autoradiography as described above. .

Hybridoma

Production

Balb/c mice (9 week old, female) were immunized intraperitoneally (i.p.) with the concentrated LMW fraction (400-800 µg of protein / injection) on day 0, day 28, day 31 with Freund's complete adjuvant, and the final booster injection was administered i.p. without the adjuvant on day 40. On day 44, spleen cells were fused with Sp 2/0 myeloma cells (ratio 8/1) using 50 % PEG. The selection of hybridomas was performed by growing in HAT medium (11, 12).

Hybridoma Screening Hybridoma supernatants were screened by the binding inhibition assay or the ELISA (11, 12). The ELISA was done using rabbit anti-mouse Immunoglobulins (A+G+M) and peroxidase-conjugated goat anti-mouse immunoglobulins (A+G+M) in the Falcon Assay Screening Test System (Becton Dickinson and Company, Lincoln Park, NJ) to detect the antibody secreted into the supernatant. Single-cell clonig was performed by limiting dilution in the presence of 5 % BriClone containing Interleuken 6 in stead of feeder cells. The antibody isotype was examined using Line Immunoassay Mouse Isotyping Kit (Innogenetics N.V., Antwerp, Belgium). RESULTS

AND

DISCUSSION

against the ligand binding site of the SRIF performed using the binding inhibition assay. Three positive hybridomas were obtained from the fusion between Sp 2/0 myeloma cells and splenocytes of Balb/c mice immunized with the active SRIF receptor which was partially purified by gel filtration of the solubilized rat brain cortex. The supernatants of these hybridomas inhibited the ligand biding to the soluble SRIF receptor as shown in FIGURE 1, and were isotyped as IgGI, IgM, or IgA plus IgM. Since

our

purpose

was

to obtain the MAb

receptor, screening after fusion

was

791

100 o

m *

¿oc

W

_j

o

ffi

o ü

IgA+lgM

igGi FIGURE 1.

Hybridoma Screening by Binding

Inhibition

Assay.

After fusion, three positive hybridomas producing lgG1, IgM, or IgA + IgM were obtained by screening with binding inhibition assay described under "Materials and Methods". Results were expressed as % of the control (Lane MEDIUM). Each value is mean of two assays using supernatants of the original and expanding culture.

Cloning by limiting dilution was repeated 2-3 times using the biding inhibition assay the ELISA, and three stable cell lines, secreting lgG1 ( ), IgM ( ), or IgA ( ), were obtained. In order to confirm the binding inhibition activity of the MAb, the SRIF receptor was preincubated with the supernatant, and further reacted with the radioligand. The reaction mixture was analyzed by 2-20 % gradient PAGE under nondenaturing conditions. Autoradiography of the gel showed the receptor bound with [125l-Tyr11]SRIF as a band of MW ca. 100 kDa. This band, which was abolished when the receptor and radioligand were reacted in the presence of excess unlabeled SRIF, became nearly invisible when the receptor was preincubated with the lgG1 or IgM MAb [FIGURE 2(A)]. When preincubated with the IgA MAb, the intensity of the receptor band was also decreased though to a lesser extent than with the lgG1 or IgM Ab (data not shown). When the ligand binding was tested in the presence of the lgG1, IgM or IgA MAb without preincubation, inhibition of the binding also occurred; the effect of lgG1 MAb is shown in FIGURE 2(B). The results strongly suggest that the MAbs produced are the antibodies against the binding site of the SRIF receptor, and the MAbs bind to the receptor in competition with the or

ligand.

In the present study, the molecular weight of the SRIF receptor was estimated to be about 100 kDa by gel filtration, and also by native PAGE and autoradiography. On the autoradiogram shown in FIGURE 2(A), minor bands of unidentified proteins were observed at the MW of dimeric and monomeric BSA. From crosslinking studies, the rat brain SRIF receptor has been reported to be a 60-74 kDa protein with no intramolecular disulfide bond (13,14,15). Yamada et al. recently reported the cloning and sequence of genes encoding two different SRIF receptors (SSTR1 and SSTR2) in human and mouse as one of the G protein-coupled receptors (16). SSTR1 (391 amino acids, MW 42,657) and SSTR2 (369 amino acids, MW 41,305) were strongly expressed in jejunum and stomach and in cerebrum and kidney, respectively, and were indicated as members of a larger family of the SRIF receptor. The receptor protein we partially purified might be in a G protein-coupled form (17,18,19) or a dimeric trimeric form. Either method we used is not appropriate for determination of the accurate molecular weight. In the present study, however, binding of [125l-Tyr11]SRIF-14 to the 100 kDa protein was clearly displaced with unlabeled SRIF-14 and also with the MAbs we produced. The MAbs will facilitate further -

792

()

()

m

I4H ·*

kDa 669

·

-

kDa 669

440

-

232

440 -

140

232

-

140 67 -

¡ 67

12 FIGURE 2.

Autoradiography

12

3 4

of SRIF

Binding Inhibition by

3

MAbs.

The SRIF receptor was preincubated with culture medium alone, medium containing 10"5M unlabeled SRIF, or culture supernatant (lgG1 or IgM MAb) at 25°C for 30 min. Radioligand binding was then followed for 1 hr, and analyzed by 2-20 % gradient native PAGE at 240 V for 4 hr at 4°C. Lanes : 1, medium alone; 2, unlabeled SRIF; 3, lgG1 MAb; 4, IgM MAb.

(A)

(B) The SRIF receptor was incubated with radioligand in the presence of medium alone (Lane 1), 10_5M unlabeled SRIF (Lane 2) or supernatant containing lgG1 MAb (Lane 3) at 25°C for 1 hr, and analyzed as in (A). understanding of the SRIF receptor on molecular bases. They, particularly the lgG1 MAb, should also become an effective tool to characterize the SRIF-binding nature of the neural corpuscles in the rat portal vein (6) and of the glomuslike neurovascular bodies in the rat peripancreatic sinusoidal vein (7) as a local somatostatin monitoring system. ACKNOWLEDGMENT We are grateful to Drs. Yoriaki Kurata1, Morinobu Takahashi1 and Ryoyu Takeda2 at Cancer Research Institute1 and School of Medicine2· Kanazawa University for valuable advice and support. We also thank Drs. Nobuo Sato1 and Shunnosuke NatsuumeSakai1 for advice on hybridoma preparation and for providing us with Sp 2/0 myeloma cells.

REFERENCES 1. 2. 3.

Reichlin, S. (1983) Somatostatin. N. Engl. J. Med. 309 : 1495-1501, 1556-1563. Chariot, J., C. Roze, C. Vaille, and C. Debray (1978) Effects of somatostatin on the

external secretion of the pancreas of the rat. Gastroenterology 75 : 832-837. Schonbrunn, ., and A. H. Tashjian, Jr. (1978) Characterization of functional 793

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9. 10. 11.

12.

13.

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6473-

Mclntosh, C. H. S. (1985) Gastrointestinal somatostatin : Distribution, secretion and physiological significance. Life Sci. 37 ; 2043-2058. Kawai, K., E. Ipp, L. Orci, A. Perrelet, and R. H. Unger (1982) Circulating somatostatin acts on the islets of Langerhans by way of a somatostatin-poor

compartment. Science Wash. DC 218 : 477- 478. Nakabayashi, H., A. Niijima, Y. Kurata, N. Usukura, and R. Takeda (1986)

Somatostatin-sensitive neural system in the liver. Neurosci. Lett. 67 : 78-81 Nakabayashi H., A. Niijima, Y. Kurata, Z.-Y. Jiang, N. Usukura, and R. Takeda (1987) Pancreatic vagai nerve is receptive to somatostatin in rats. Am. J. Physiol. 253 : R200-R203. Epelbaum, J., L. T. Arancibia, C. Kordon, and A. Enjalbert (1982) Characterization, regional distribution, and subcellular distribution of 125l-Tyr1 -somatostatin binding sites in rat brain. J. Neurochem. 38 : 1515-1523. Ogawa, N., T. Thompson, and H. G. Friesen (1977) Properties of soluble somatostatin-binding protein. Biochem. J. 165 : 269-277. Yarden, Y., and J. Schlessinger (1987) Epidermal growth factor induces rapid, reversible aggregation of the purified epidermal growth factor receptor.

Biochemistry 26 : 1443-1451. Fraser, C. M. and J. Lindstrom (1984) The use of monoclonal antibodies in receptor characterization and purification, in Receptor Biochemistry and Methodology vol. 3, Molecular and Chemical Characterization of Membrane Receptors (eds. Venter, J.C., and L. C. Harrison), pp.1-30, Alan R. Liss Ine, New York, NY. Harlow, E. and D. Lane (1988) Monoclonal antibodies, in Antibodies, A Laboratory Manual pp. 139-243, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Sakamoto, C, M. Nagao, T. Matozaki, H. Nishizaki, Y. Konda, and S. Baba (1988) Somatostatin receptors on rat cerebrocortical membranes. J. Biol. Chem. 263 :14441 -14445.

14.

15. 16.

He, H.-T., K. Johnson, K. Thermos, and T. Reisine (1989) Purification of a putative brain somatostatin receptor. Proc. Nati. Acad. Sci. USA 86 : 1480-1484. Kimura, N. (1989) Developmental change and molecular properties of somatostatin receptors in the rat cerebral cortex. Biochem. Biophys. Res. Commun. 160 : 72-78. Yamada, Y., S. R. Post, K. Wang, H. S. Tager, G. I. Bell, and S. Seino (1992)

Cloning

17.

18. 19.

and functional characterization of a family of human and mouse somatostatin receptors expressed in brain, gastrointestinal tract, and kidney. Proc. Nati. Acad. Sci. USA 89 : 251-255. Knuhtsen, S., J.-P. Esteve, C. Cambillau, B. Colas, C. Susini, and N. Vaysse (1990) Solubilization and characterization of active somatostatin receptors from rat pancreas. J. Biol. Chem. 265 : 1129-1133. He, H.-T., S. Rens-Domiano, J.-M. Martin, S. F. Law, S. Borislow, M. Woolkalis, D. Manning, and T. Reisine (1990) Solubilization of active somatostatin receptors from rat brain. Mol. Pharmacol. 37 : 614-621. Law, S. F., D. Manning, and T. Reisine (1991) Identification of the subunits of GTPbinding proteins coupled to somatostatin receptors. J. Biol. Chem. 266 : 1788517897.

Address

reprint requests to : Hajime Nakabayashi, M.D. Department of Internal Medicine (II)

School of Medicine, Kanazawa University 13-1 Takara-machi, Ishikawa 920, Japan. Received for publication: 6/19/92 Accepted: 6/19/92 794

Kanazawa,