Recognition by anti-idiotypic anti-thyrotropin-releasing hormone (TRH) antibody of rat TRH receptors

Neuropeptides(1991) 18, 121-127 0 Longman Group UK Ltd 1991

Recognition by Anti-ldiotypic Anti-ThyrotropinReleasing Hormone (TRH) Antibody of Rat TRH Receptors T. SATOH, M. MORI, M. MURAKAMI, S. KOBAYASHI

T. IRIUCHIJIMA,

M. YAMADA,

I. KOBAYASHI”

and

First Department of Internal Medicine, and *Central Laboratory Medicine, Gunma University, School of Medicine, Maebashi 371, Japan. (Correspondence to MM)

Abstract-We asked whether anti-idiotypic antibodies raised against anti-TRH antibody could bind to TRH receptors in the rat anterior pituitary and brain. Six rabbits were immunized with IgG from a rabbit anti-TRH antiserum. One anti-idiotypic antibody caused strong, dosedependent inhibition in anti-TRH antibody-binding to [‘2511-TRH. This inhibition was not observed after treatment with goat anti-rabbit IgG antibody. The anti-idiotypic anti-TRH antibody significantly immunoprecipitated digitonin-solubilized pituitary TRH receptors. When eluates of digitonin-solubilized membranes which were adsorbed by either an antiidiotypic anti-TRH IgG-, normal rabbit IgG-linked affinity column or control column were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and visualized by silver stain, only the former eluate showed two bands under nonreducing conditions; one corresponded to a molecular weight marker of 200K, the other to 100K. Western blotting analysis with an anti-idiotypic anti-TRH antiserum showed a single band of molecular weight 56K under reducing conditions. The present study indicates that one can make anti-idiotypic antibodies that specifically recognize TRH receptors.

Introduction Brain neuropeptides exert their functions by interacting with specific binding sites on target cells. Thyrotropin-releasing hormone (TRH) is a hypothalamic tripeptide which stimulates thyrotropin and prolactin release from the anterior Date received 10 July 1990 Date accepted 6 September 1990

pituitary and also functions as a neurotransmitter in the brain. A number of investigators have found receptors for TRH in the anterior pituitary and brain (l-3). The TRH receptors existing in those two different tissues seem to be indistinguishable in nature (l-3). Recently, detailed structures of several brain neuropeptide receptors have been determined. Although antibodies specific for TRH receptors could be used for receptor cloning, it would be very difficult to obtain adequate 121

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quantities of purified TRH receptors to raise antibodies (4). Oren et al. (5) found that Xenopus oocytes injected with mRNA 4kb in length from pituitary tumor cells expressed TRH receptor, and an abstract by Straub et al. (6) has indicated that this receptor has been cloned. In recent years, the application of an anti-idiotypit antibody as a specific receptor probe has been used with unique benefits over the more conventional ligand-based methdology. Anti-idiotypic antibodies capable of binding receptors, for example, may be produced in the absence of purified receptors. Employment of this strategy has enabled investigators to produce sundry antibodies for receptors, including acetylcholine receptor, insulin receptor, 8-adrenergic receptor, thyrotropin receptor, substance P receptor, glucocorticoid receptor, and adenosine receptor (7-9). Our previous observations (10) have indicated that preincubation of TRH with its specific antibody resulted in the loss of TRH-stimulating activity in vivo. These observations suggested that the anti-TRH antibody may possess the same active site as pituitary receptors which recognize TRH. In the present study we attempted to generate an antiidiotypic antibody using an anti-TRH antiserum. Materials and Methods TRH was supplied by Tanabe Pharmaceutical Co., Osaka, Japan. t3H-MeHis]TRH was purchased from New England Nuclear, Boston, Mass. (specific activity = 57.8 Ci/mmol). Protein A-linked Sepharose 4B and diethyl-aminoethyl (DEAE) gels were purchased from Sigma Chemical Co., St Louis, MO and Bio-Rad Laboratories, Richmond, CA, respectively. Digitonin was supplied by Wako Pure Chemical Industries Ltd., Osaka, Japan. Protein markers (Amersham International plc., Buckinghamshire, England) used were myosin (200K), phosphorylase b (92.513), bovine serum albumin (BSA, 69K), ovalbumin (46K), carbonic anhydrase (30K), trypsin inhibitor (21.5K) and lysozyme (14.3K). Immunization

with ZgG from anti- TRH antiserum

An anti-TRH antiserum was generated in rabbits against synthetic TRH conjugated with BSA using bis-diazotized benzidine. This antiserum was

observed to detect at least 2pg of TRH at 1:lOOOOO dilution in radioimmunoassay system, and had the very restricted specificity for TRH as described previously (10). The anti-TRH antiserum was dialyzed against 0.02M KH2P04 (pH 8.0) at 4°C overnight and applied to DEAE gel. The eluted fraction was further dialyzed against 0.01 M NaHzPO4 and 0.15M NaCl buffer (pH 8.2) and subjected to protein A affinity chromatography. After washing with the dialysis buffer, IgG was eluted using O.lM citrate buffer (pH 3.0). This fraction was neutralized and adjusted to 2mg/ml with saline. The anti-TRH IgG was emulsified with an equal volume of complete1 Freund’s adjuvant, and injected subcutaneously into six rabbits. Two booster injections were done. Concentrations of IgG were measured by the method of Lowry et al. (11) using bovine gamma globulin as a standard. The activity of the anti-idiotypic antibody was measured by inhibition of anti-TRH antiserumbinding to [1251]TRH. One hundred p.1 rabbit serum, lOOl.~l anti-TRH antiserum at 1:50000 dilution, and lOO$ phosphate buffered saline (PBS, O.OlM P04, 0.15M NaCl, pH 7.5) containing 0.25% BSA(PBS-BSA) were incubated with 10O)~l [1251]TRH in PBS-BSA at 4°C for 2 days. After adding 200 l.~lanti-rabbit IgG goat antibody in PBS containing 0.05M EDTA and 3.5% polyethylene glycol (molecular weight = 6000), the samples were incubated for an additional 24h at 4°C. The incubation mixture was centrifuged at 1500 x g for 20 min, and the precipitate counted. To evaluate effects of anti-rabbit IgG goat antibody on anti-idiotypic antibody activity, one ml anti-idiotypic antibody was incubated with an equal volume of anti-rabbit IgG goat antiserum at 4°C for 24h, and centrifuged at 1500 x g for 20 min. The supernatant was used for measurement of anti-idiotypic antibody activity. Solubilization of TRH receptors

Adult male Wistar rats were decapitated, and either 20 anterior pituitaries or 10 brains excluding cerebellum were homogenized in 10 vol of TrisHCl buffer (40mM Trishydroxymethylaminomethane-HCl, pH 7.4) and centrifuged at 100 X g for 10 min. The supernatants were centrifuged at 56000 x g for 60 min. The preciptates were

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reconstituted in 3 ml of Tris-HCl buffer containing varying amounts of digitonin, incubated for 30 min in an ice box, and centrifuged at 100000 x g for 60 min. The supernatants were used in the TRH binding assay. Two hundred ~1 of solubilized membranes were incubated with 10 p,l (1.73 pmol) [3H-MeHis]TRH at 0°C in the presence or absence of lOl.~l of 2 nmol TRH. A rapid gel filtration technique was used to separate bound from free TRH, as described previously (12). The incubation was terminated by layering 100~1 of the reaction mixture over a Sephadex G-50 minicolumn (gel was packed in a 1 ml plastic tuberculin syringe and pre-equilibrated with Tris-HCl buffer). The loaded minicolumn was centrifuged at 100 x g for 2 min. The eluates were collected in scintillation vials containing Aquasol 2 (New England Nuclear) and [3H] counted. Specific binding was calculated by substracting nonspecific binding (with unlabeled TRH) from total binding (without unlabeled TRH). Proteins of solubilized membranes were measured using bicinchoninic acid (Pierce Chemical Co., Rockford, IL) (13). Immunoprecipitation idiotypic antibody

of TRH receptors

by anti-

One hundred and fifty ~1 of solubilized pituitary membranes and 150 l~,lTris-HCl buffer were incubated with 50~1 of anti-idiotypic antibody at 4°C for 24h. After adding 200~.~1anti-rabbit IgG goat antiserum, the tubes were placed at 4°C for 3h. followed by centrifugation at 1.500 x g for 20 min. The supernatants were used in the TRH binding assay described above. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of TRH receptors

Either 15mg of anti-idiotypic anti-TRH IgG or normal rabbit IgG was linked to 2ml Glycosylhard gel (Hohnen Corporation Co., Tokyo, Japan) which was equilibrated with Tris-HCl buffer containing 0.1% digitonin. The gel without IgG was used as a control column. Solubilized membranes obtained from 200 rat pituitaries were applied on the columns and washed with 1Oml Tris-HCI buffer with digitonin, and the fractions eluted by 5ml of 0.1 M sodium acetate buffer (pH 2.0) were concentrated to 100 times using Amicon’s macrosolute concentrator (Division of W.R.

Grace & Co., Danvers, MA). The concentrated samples were analyzed by SDS-PAGE as described by Laemmli (14) using a 6% running gel. Molecular weight protein markers were included as standards. Electrophoresis was carried out for lh at constant 20mA. Proteins in the gel were visualized by silver staining (Wako Pure Chemical Industries Ltd.) using the method of Morrissey (15). Western blotting analysis of TRH receptors

The eluates of solubilized brain membranes which were adsorbed by the anti-idiotypic anti-TRH IgG-linked affinity column were concentrated and analyzed by SDS-PAGE as described above. The separated proteins were transferred from gel to nitrocellulose (Hybond-C, Amersham International plc.) using electrophoresis transfer apparatus (FC-0030-51, Funakoshi Yakuhin Co., Tokyo, Japan) for 70 min at BOrnA, as described elsewhere (16). The nitrocellulose strips were blocked with Tris-buffered saline (20mM Tris-HCl, 137mM NaCl, pH 7.6) containing 0.1% Tween 20 and 5% dried milk, and incubated for 1 h at room temperature with a 1:70 dilution of anti-idiotypic anti-TRH antiserum or normal rabbit serum. After washing the strips in Tris-buffered saline, they were incubated with biotinylated anti-rabbit IgG donkey antibody (Amersham International pit.) for lh at room temperature. After washing with Tris-buffered saline, the streptavidin-biotinylated alkaline phosphatase complex was added and the enzyme was finally visualized with nitro-blue tetrazolium and bromo-chloro-indolyl phosphate. Results

As shown in Figure 1, serum from one rabbit (no. 1) immunized with anti-TRH IgG apparently inhibited the binding of anti-TRH antiserum to [“‘I]TRH. This inhibition was not observed after treatment with anti-rabbit IgG goat antiserum and was dependent on the concentrations of anti-idiotypic IgG. Figure 2 shows the effect of varying digitonin concentration on solubilization of TRH receptors and incubation time of TRH binding. The solubilization of TRH receptors increased with the increasing concentrations of digitonin, and TRH binding increased with the period of incubation.

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observed if solubilized membranes were incubated with 25 p.M TRH at 4°C for 2 h before being applied to the affinity column (data not shown here). Figure 5 shows Western blotting analysis of TRH receptors on SDS-PAGE under reducing conditions. The anti-idiotypic antibody detected a single band with an apparent molecular weight of 56K, in contrast to normal rabbit serum which revealed no bands.

Discussion

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I

50 d,l”t,ad

IgG

Co”centrat\on

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Fig. 1 Generation of anti-idiotypic antibody. (a)-Activities of anti-idiotypic anti-TRH antibody. Rabbits were immunized with IgG of anti-TRH antisenun, and activities of anti-idiotypic antibodies were measured by estimating the inhibitory effects of those rabbit sera on anti-TRH antiserum-binding to [l*sI] TRH, as described in Materials and Methods. (b)-Effects of treatment with anti-rabbit IgG goat IgG (AGG) on anti-TRH antiserum-binding to [1251]TRH. (c)-Effects of concentrations of anti-idiotypic IgG on anti-TRH antiserum-binding to [“‘I]TRH. Each point represents a mean of duplicate determination.

Based on these observations, 1% digitonin was used to solubilize TRH receptors which were incubated with [3H-MeHis]TRH at 0°C for 3 h in subsequent experiments. Figure 3 depicts immunoprecipitation by antiidiotypic antibody of TRH receptors. The addition of anti-idiotypic antibody immunoprecipitated 80% TRH binding activity from the supernatant (the control group, 100.0 + 20.9 vs. the anti-idiotypit antibody-added group, 20.1 + 4.4%) P < 0.01). Figure 4 shows analysis of TRH receptors on SDS-PAGE under nonreducing conditions. Eluates which were adsorbed by the control and normal rabbit IgG-linked columns showed no apparent band, whereas the anti-idiotypic IgGlinked column yielded two bands, one of which corresponded to a molecular weight marker of 2OOK, the other to 1OOK. These bands were not

The present study demonstrated that serum from one of six rabbits immunized with anti-TRH IgG caused a significant inhibition of binding of antiTRH antiserum to [l*‘I]TRH. This inhibitory activity was attributed to the IgG fraction and depended on the concentration of IgG used. The anti-idiotypic antibody immunoprecipitated solubilized TRH receptors. Since Jerne proposed that the variable regions of antibodies may act as immunogens , the idiotypic approach has been used to make anti-receptor antibodies (7). In fact, several anti-receptor antibodies have been generated without purifying receptors using antiidiotypic antibodies (7-9). Moreover, anti-idiotypit antibodies have been observed to interact with the ligand-binding site of the receptor, whereas

(b)

(a) 501

rM

/-

.

Fig. 2 Solubilization of TRH receptors. (a)-Effects of digitonin concentrations on solubilization of TRH receptors. Pituitary membranes were sohtbihzed with digitonin and TRH binding was measured as described in Materials and Methods. (b)-Incubation time and TRH binding. Solubilized pituitary membranes were incubated with [3H-MeHis]TRH at 0°C and the bound form was separated from the free form using a rapid gel filtration method as described in Materials and Methods. Each point represents a mean of duplicate determination.

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BY ANTI-IDIOTYPIC ANTI-THYROTROPIN-RELEASING

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Ogawa et al. (18) reported that the molecular weight of Triton X-solubilized receptors was approximated at 300K. A different molecular weight - 224K - was reported by Johnson et al. (19). Phillips and Hinkle (4) found that the t3HMeHis]TRH-bound receptor complex was eluted at the position near catalase (240K) on a gel filtration. Recently, a pituitary protein with molecular weight of 64K, to which t3H]TRH was crosslinked by UV illumination, was proposed to be the TRH receptor (20). In the present study, the eluate which was adsorbed by the anti-idiotypit antibody-affinity chromatography showed two different molecular weight proteins with 200K and

I N=6

control

HORMONE (TRH) ANTIBODY OF RAT TRH RECEPTORS

Anti-idiotypic antibody

Fig. 3 Immunoprecipitation of digitonin-solubilized TRH receptors by anti-idiotypic antibody. Solubilized TRH receptors were incubated with anti-idiotypic antibody or normal rabbit serum (as control) followed by addition of anti-rabbit IgG goat antiserum. After centrifugation, the supernatants were used for measurement of TRH binding as described in Materials and Methods. The data are shown as % of the control binding with mean + S.E.M. The statistical significance was analyzed by Student’s t test.

most anti-receptor antibodies obtained by immunization with purified receptor react with portions of the molecule other than the binding site (7). Although the anti-idiotypic antibody used in the present study was not tested with respect to its effects on TRH-biological activities, the present results indicate that the anti-idiotypic route generated a specific antibody that recognized TRH receptors. Many attempts have been made to characterize TRH receptors, but little information is as yet available about the detailed structure of the recentor. Purification of the receptor is an important step in understanding the molecular mechanism of TRH receptors. Hinkle and Lewis (17) solubilized [3H]TRH-bound receptor with Triton X-100 and found that the TRH-receptor complex was 150K. I

123 -2OOk

-92.5k -69k -46k -30k -21.5k Fia. 4 SDS-PAGE analysis of TRH receptors. Solubilized pituitary membranes were adsorbed by either anti-idiotypic anti-TECH IgG- or normal rabbit (NRS) IgG-linked affinity column or control column (without IgG), and the eluates ran on SDS-PAGE under nonreducing conditions and was visualized by sdver stain as described in Materials and Methods. The arrows show the positions of molecular weight markers.

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-92.5k -69k

-46k

Fig. 5 Western blotting analysis of TRH receptors. Solubilized brain membranes were adsorbed by anti-idiotypic antiTRH IgG-linked affinity column. The eluates ran on SDSPAGE under reducing conditions and were transferred to nitrocellulose membranes, and Western blot was done using anti-idiotypic antibody and normal rabbit serum (NRS) as described in Materials and Methods. The arrows show the positions of molecular weight markers.

1OOK on SDS-PAGE under nonreducing conditions. In contrast, on SDS-PAGE under reducing conditions, a single band was found, which corresponded to a 56K protein. These molecular weights estimated for digitonin-solubilized TRH receptors differ from previously reported values described above. It remains to be seen whether the differences arise from variations in the extent of solubilizing detergents from the different separation methods, i.e., gel filtration vs. SDS-PAGE, from the disparate methods used to detect solubilized TRH receptors, photoaffinity crosslinking vs. anti-idiotypic antibody, and/or from the dissimilar tissues examined, tumor tissues vs. normal

tissues. The proteins with the larger molecular weights described in the present study were not observed on SDS-PAGE under reducing conditions. These results indicate that the protein with the smaller mo!ecular weight may represent a sub-unit of an even larger protein, indicating that TRH receptor may be composed of sub-units linked via disulfide bonds. We could not rule out, however, the possibility that the larger molecules may be aggregates of the smaller protein. The existence of receptor complexes with various molecular weights has been shown for some physiologically active receptors. Srikant and Pate1 (21) found pancreatic somatostatin receptors with three different molecular weights of 2OOK, 80K and 70K. With respect to the adenosine receptor, Western blotting revealed a 62K band under non-reducing conditions, and a major band of 36K under reducing conditions (9). Nissenson et al. (22) observed that the large molecular weight form (180K) of solubilized renal parathyroid hormone (PTH)-receptors may be a complex combining PTH-binding moiety (60-70K) and the stimulatory nucleotide-binding guanine component (G protein, 90-lOOK). Because TRH receptors are also conjoined to G protein (23), it is possible that the protein with the larger molecular weight as shown in the present study may result from the binding complex of the small molecular protein with G protein. Although the results are not unambiguously interpretable, we have concluded that the 56K band may represent the TRH receptor. This 56K protein was comparable to the molecular weights of hypothalamic hormone receptors such as pituitary luteinizing hormonereleasing hormone receptor (60K) (24) and pituitary somatostatin receptor (88K) (25). Acknowledgements Part of the study was presented at the 64th Meeting of American Thyroid Association, San Francisco, 1989 (Abstract no. 106). The present work was supported in part by a grant-in-aid (to M. Mori) for scientific research from the Ministry of Education, Science and Culture, Japan.

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