THE JOURNAL OP BIOLOGICAL CHEMISTRY 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 265, No. 34, Issue of December 5, pp. 21302-21306,169O Printed in U.S.A.

Characterization of the Glucagon Receptor and Its Functional Domains Using Monoclonal Antibodies*

(Received for publication, May 24, 1990)

Victoria Iwanij$ and Alexandra C. Vincent From the Department of Genetics and Cell Biology, University of Minnesota, St. Paul, Minnesota 55108

Four monoclonal antibodies, designated 4H11,6E 10, 2C6, and 3E9 were prepared against partially purified rat hepatic glucagon receptor. These antibodies were characterized by their ability to recognize the glucagon receptor in target tissues using immunoblot and im- munoprecipitation procedures. The antibodies recog- nized a 62-kDa receptor band in rat liver, kidney, and adipose tissue but not in lung, adrenals, and erythro- cytes, indicating a high degree of specificity. These antibodies recognize different antigenic determinants; the 6ElO and 2C5 bind protein epitopes, while 4Hll and 3E9 bind carbohydrate epitopes. Furthermore, proteolytic mapping of the glucagon receptor estab- lished that monoclonal antibodies 6ElO and 2C6 rec- ognize different domains of the receptor molecule. These antibodies were used to study the immunochem- ical similarities among the receptors from different species and to assess the topological location of the ligand-binding site. By combining the techniques of affinity cross-linking, proteolytic mapping, and anti- body binding, we have identified the location of the glucagon-binding site near to the COOH-terminal do- main of the receptor.

The peptide hormone glucagon has been long recognized as an important regulator of the hepatic fuel metabolism (1). In addition to the effects upon liver, glucagon exerts important physiological effects upon other tissues such as adipose (2, 3), kidney (4), brain (5), and gastrointestinal epithelia (6). In most of the target tissues, the diverse biological responses to glucagon are initiated by its binding to a membrane receptor followed by stimulation of adenylyl cyclase activity via intra- cellular, membrane-associated G proteins (1). Although the hepatic glucagon receptor has been extensively studied (l), to date it has not been purified, and antibodies against the receptor are unavailable. Some progress in the structural characterization of the glucagon receptor has been made by the application of affinity labeling approaches (7-10). The glucagon receptor is a 62-kDa glycoprotein that contains at least four iv-linked oligosaccharide chains and intramolecular disulfide bonds (9, 10).

Studies of other receptor systems such as the nicotinic acetylcholine receptor (ll), the insulin receptor (12, 13), and the /3-adrenergic receptor (14, 15) have been greatly assisted by the availability of specific anti-receptor antibodies. These

* This work was supported in part by Grant AM-34732 (to V. I.) from the National Institutes of Health and grants from the American Diabetes Association (Minnesota Chapter) and from the University of Minnesota Graduate School Fund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom correspondence should be addressed.

antibodies have been utilized for the study of the regulation, biosynthesis, and identification of the receptors functional domains. Using a partially purified hepatic glucagon receptor as an immunogen, we have undertaken the preparation of anti-glucagon receptor monoclonal antibodies (mAbs).’ We report here, for the first time, the isolation and characteriza- tion of an anti-glucagon receptor antibodies. This study dem- onstrates that the antibodies recognize different antigenic determinants located within distinct domains of the glucagon receptor. These antibodies were used to investigate the im- munochemical similarities among the receptors from different species and to analyze the topology of the glucagon receptor.

EXPERIMENTAL PROCEDURES

Materials-Glucagon, protease V8, carboxypeptidase Y, bacitracin, PMSF, and detergents (Triton X-100, Nonidet P-40) were purchased from Sigma, leupeptin and pepstatin from Vega Biochemicals, Tra- sylol, from FBA Pharmaceuticals, and Iodine-125 from Amersham Corp. All other chemicals used in this work were of analytical grade.

Purification of Hepatic Glucagon Receptor-Membranes from the rat liver were prepared according to Neville (16) in the presence of the following protease inhibitors: leupeptin (5 pg/ml), pepstatin (7 pg/ml), Trasylol(lO0 units/ml), and EDTA (2 mM). In order to follow up receptor purification, we have carried out affinity cross-linking of ‘251-glucagon to the plasma membrane receptor using modification of our direct UV irradiation protocol (10). Membranes were suspended at 0.5 mp/ml in 20 ml of 50 mM Tris-HCl, pH 7.5, binding buffer contain& a mixture of protease inhibitors ‘(TO) and incubated with ‘251-glucagon (5 X lo’-10’ cpm) for 1 h at 4 “C. Membranes were washed three times bv centrifunation (20 min at 20,000 x .g, Sorval) to remove unbound 1;gand, and-resuspended in the initial Volume of buffer. Five-ml portions of membrane suspension were placed on lOO- mm tissue culture dish and subiected to a UV irradiation for 30 min.

The first step in the receptor isolation was preparative sodium dodecvl sulfate-Dolvacrvlamide gel electroohoresis (SDS-PAGE) of

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rat liver plasma membranes using affinity labeled receptor as a marker. Preparative gels (11% acrylamide) of 19 cm X 40 cm X 3 mm were prepared according to Laemmli (17). Purified liver plasma membranes (3-5 mg/gel) were treated with solubilization buffer (17), and applied across the width of the gel. The gels were stained with Coomassie Blue G as described bv Scheele et al. (18). The region of the gel between 66 and 60 kDa molecular mass markers containing the receptor, was cut out, gel strips were combined, and the protein was electroeluted. The protein from electroeluted samples was con- centrated by chloroform/methanol precipitation according to Wessel et al. (19). The second step of the receptor isolation procedure was wheat germ agglutinin (WGA) affinity chromatography. The concen- trated protein pellet was sonicated in 50 mM phosphate buffer, pH 7.5 (0.2-1.0 ml), and solubilized by adding 10% SDS for a final concentration of 0.5%. then a small volume of concentrated Nonidet P-40 was added to obtain a IO-fold excess over the SDS. The sample was diluted lo-fold with WGA column buffer (50 mM Tris-HCI. PH 7.5, with 0.2% Nonidet P-40 containing protease inhibitors leupepiin at 5 pg/ml, Trasylol at 100 units/ml, and pepstatin at 5 pg/ml), and all insoluble material was removed by centrifugation (100,000 X g for

’ The abbreviations used are: mAbs, monoclonal antibodies; PMSF, phenymethylsulfonyl fluoride; SDS-PAGE, sodium dodecyl sulfate- polyacrylamide gel electrophoresis; WGA, wheat germ agglutinin.

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60 min). Prior to the application of the sample to the WGA affinity column. it was aeain diluted with the column buffer to achieve a 20- fold dilution of t?le initial detergent concentration. The sample was applied to the WGA column (2 ml of settled volume, Vector Labora- tories) by gravity and the flow-through was recycled four times. The column was washed with 20 ml of WGA column buffer. Adsorbed glycoproteins were eluted with the same buffer containing 0.2 M N- acetylglucosamine. Five ml of the buffer was sufficient to collect most of the adsorbed glycoprotein. Eluted proteins were concentrated by precipitation as described above and analyzed by SDS-PAGE, iso- electric focusing, and autoradiography. This preparation yielded be- tween 100 and 200 fig of partially purified receptor from 30 to 60 mg of plasma membranes.

Preparation of Morwcl~n~l Antibodies-The glucagon receptor preparation was concentrated by chloroform/methanol precipitation (19), washed three times with ethanol, and suspended by sonication in a small aliquot of sterile 0.9% saline for intravenous injections or was mixed with an equal volume of Freund’s adjuvant for intraperi- toneal injections. After the initial inoculation, the animals were boosted with two to five additional injections at 4-week intervals. Splenic lymphocytes from BALB/c mice immunized with the gluca- gon receptor preparation were fused with NSl myeloma cells essen- tially as described by Hybridoma Techniques manual (20). Media from the resulting hybridomas were screened for anti-receptor anti- bodies using a dot blot procedure. A nitrocellulose sheet was fitted into the manifold (Bio-Rad), 1-2 fig of the antigen were spotted into each well, and the nitrocellulose was processed as described by Jahn et al. (21). Hybridoma medium was transferred into the wells and incubated with the immobilized antigen. Antibody binding was visu- alized by the use of a secondary anti-mouse antibody conjugated to alkaline phosphatase (Promega Biotech). A duplicate set of nitrocel- lulose sheets with rat serum as the antigen was screened with hybrid- oma medium to eliminate any nonspecific reaction. Positive hy- bridomas were further screened using immunoblots of an affinity labeled, partially purified glucagon receptor preparation. Antigen was applied across the width of a minigel and following electrophoresis, the proteins were transferred onto nitrocellulose. The nitrocellulose was-blocked as described below and then placed onto a multilane manifold (Immunetics. Cambridee) for the antibodv testine. This screen identified antibodies that-recognized proteins of the correct M, and selected the antibodies that stained the radioactively labeled band. Positive clones isolated through these screens were subcloned twice by limited dilution. The antibodies were typed using a kit from Boehringer Mannheim Biochemicals. Ascites fluid was prepared in pristine-treated mice according to the Cold Spring Harbor protocol 120), aliquoted, and stored in a--87 “C until needed.

Zmmurwblot Analvsis-SDS-PAGE was nerformed as described bv Laemmli (17) with- 11% polyacrylamide -separation gel cast in g minigel apparatus (Hoeffer Scientific). Proteins were electrophoreti- tally transferred as described by Towbin et al. (22), with the exception that SDS was omitted from the transfer buffer. The quality of transfers was checked by staining the nitrocellulose with 0.2% Pon- ceau in 0.3% trichloroacetic acid. Blots were pretreated with a solution containing 2% gelatin, 0.2% bovine serum albumin in 50 mM Tris- HCl at pH 7.5 with 150 mM NaCl and 0.2% Triton X-100. The nitrocellulose was incubated with hybridoma medium or diluted as- cites preparation for l-2 h at room temperature. Following four 20- min washes in the buffer described above but without the protein, the blots were incubated with anti-mouse antibody (1:5,000 dilution) conjugated to alkaline phosphatase. After another series of washes, the bound antibodies were visualized as recommended by the supplier.

Zmmunoprecipitation-Affinity cross-linked liver plasma mem- branes were solubilized with 0.5% octyl glucoside in Tris-HCl buffer, pH 7.4, as described by Bartles et al. (23). Insoluble residue was removed by centrifugation at 100,000 X g for 45 min. The detergent extract was then diluted P-fold with the same buffer and incubated with ascites fluid (lo-20 ~1) overnight at 4 “C. Following the incuba- tion step, an aliquot (50-100 ~1) of Sepharose-bound protein G (Genex Corp.) was added, and additional incubation was carried out for 3 h at 4 “C with constant agitation. The Sepharose beads were washed four times with the buffer containing octyl glucoside, then twice with the buffer without the detergent. Finally, they were treated with Laemmli sample preparation buffer (10). The total volume of the sample was applied to the polyacrylamide gel. Alternatively, affinity labeled membranes were subjected to preparative SDS-PAGE and “‘I-glucagon-receptor complex partially purified as previously de- scribed. Receptor was solubilized in 0.5% SDS, followed by the addition of lo-fold excess of Nonidet P-40. Samples were diluted 1:lO

with incubation buffer and incubated with aliquots of ascites fluid followed by the anti-mouse IgG-Sepharose (Coppel Laboratories), in identical manner as described above. Precipitated samples were sol- ubilized in SDS buffer and analyzed by SDS-PAGE and autoradiog- raphy.

Preparation of Plasma Membranes from Various Tissues-The procedure of Neville (16) was used for the isolation of the plasma membranes from chicken, mouse, and dog liver with the exception that the dog liver was homogenized with Polytron as described by Sheetz and Tager (24). Plasma membranes from the rat adrenals were prepared according to the procedure of Paglin and Jamieson (25), lung membranes were isolated according to Leroux et al. (26), and erythrocyte ghosts were prepared as described by Dodge et al. (27). Plasma membranes from adipose tissue were isolated according to Williams et al. (28), and membranes from kidneys were prepared as described by Segre et al. (29). All of these preparations were carried out in the presence of the protease inhibitors at the same concentra- tions described for the liver plasma membrane isolation. The isolated membranes were frozen and stored in aliquots in liquid nitrogen. Binding of ‘251-glucagon to the glucagon receptor in isolated plasma membranes and UV-directed cross-linking was carried out as de- scribed by Iwanij and Hur (10).

Treatment of Plasma Membranes with N-Glycanase and Pro- teases-Treatment of membranes with N-glycanase (Genzyme Corp.) was carried out in the following manner: 20-40 pg of membranes were suspended in the 0.1 M phosphate buffer, pH 8.6, containing 0.2% SDS, 1% Nonidet P-40, 50 mM dithiothreitol, and 2 mM PMSF and incubated with 0.5-1.0 units of N-glycanase for 14-24 h at 37 “C. The reaction was terminated by addition of SDS-solubilization buffer and samples were analyzed by SDS-PAGE and immunoblotting as de- scribed above.

Prior to the V-8 treatment, liver plasma membranes (100 rg) were washed by centrifugation (Eppendorf) with 50 mM phosphate buffer, pH 8.0, in order to remove protease inhibitors, then suspended by brief sonication in 50 ~1 of the same buffer. Staphylococcus aureus V- 8 protease was used at 10 pg/sample, and reaction was allowed to proceed for 15-30 min at room temperature. Digestion was stopped by addition of 2 mM PMSF followed by SDS-solubilization buffer and samples were subjected to SDS-PAGE.

For the treatment with carboxypeptidase Y, liver plasma mem- branes (So-100 pg) were washed in 50 mM phosphate buffer, pH 6.5, to remove protease inhibitors, then suspended in the same buffer. Enzyme was added at 5-10 pg and digestion was carried out for 30- 60 min at room temperature. Digestion was stopped by addition of 2 mM PMSF followed by SDS-solubilization buffer, and samples were analyzed by SDS-PAGE and immunoblotting.

Miscellaneous Procedures-The isoelectric focusing procedure was performed in the pH range 3.5-10 (l-2% Ampholine, LKB) in 2 x 120-mm tubes according to Ames and Nikaido (30). Isoelectric focus- ing gels were stained as described by Spencer and King (31). For autoradiography, gels were dried and exposed to Kodak XRP-5 film with a Du Pont Quanta III screen for 2-6 days. The protein concen- tration was determined by the fluorescamine procedure of Udenfriend et al. (32). Glucagon was iodinated as described by Lin et al. (33), and separation of iodinated glucagon from unincorporated iodine was carried out as described by Iwanij and Hur (10).

RESULTS

Purification of th.e Glucagon Receptor from Liver Plasma Membranes-The purification of glucagon receptor has not yet been reported. Since the glucagon receptor represents a minor component of the plasma membrane, significant en- richment was needed to improve the chances of preparing anti-receptor antibodies. To this end, we have developed a rapid and simple procedure for the isolation of denatured glucagon receptor, combining preparative SDS-polyacryl- amide gel electrophoresis with WGA affinity chromatography. First, the receptor was isolated by preparative SDS-PAGE of liver plasma membranes using affinity labeled receptor as a marker. The regions of the gels containing the receptor (be- tween 66 and 60 kDa molecular mass markers) were cut out, the gel strips were combined, and the protein was electroe- luted. Since this region of the gel does not contain any major protein bands (Fig. 1, inset 1), a substantial purification was

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FIG. 1. Chromatography of ‘261-labeled glucagon receptor complex on WGA-Sepharose. The receptor complex was isolated from preparative SDS gels, concentrated by precipitation, and solu- bilized as described under “Exoerimental Procedures.” The samnle was applied to the column and washed until there was no change-in the radioactivity found in the eluate. The bound material was eluted with 0.2 M N-acetylglucosamine (see arroru). Eluted material was analyzed by SDS-PAGE and autoradiography (inset). Lane 1, protein pattern of liver plasma membrane (starting material) visualized by Coomassie Blue staining. Small arrowheads indicate region of the gel excised for the receptor electroelution. Lane 2, protein pattern of the material eluted from the WGA column. Lane 3, autoradiography of lane 2. Arrouhad points to the 1’51-labeled glucagon receptor band.

achieved in this step. The protein in the electroeluted samples was concentrated by chloroform/methanol precipitation (19). The protein was purified further by WGA affinity chromatog- raphy (Fig. 1). The column was loaded and developed with buffers as described under “Experimental Procedures.” Rou- tinely, 90% of applied radioactivity was recovered from the WGA column, with 55-60% of counts eluted with the N- acetylglucosamine-containing buffer. Material that did not adsorb to the column and was eluted in the initial wash fractions contained affinity labeled receptor, but we have observed that the M, of the radioactive band was slightly lower than that of the adsorbed receptor. The molecules in the flow-through most probably represent a desialiated form of the receptor as was observed for the P-adrenergic receptor (34). The SDS-PAGE analysis of partially purified material (Fig. 1, inset 2) revealed the presence of a distinct Coomassie Blue-stained band of molecular mass 62 kDa accompanied by two to three bands of slightly lower molecular weight. Auto- radiographic analysis showed that the major protein band corresponds to the affinity radiolabeled band (Fig. 1, inset 3). Based on the protein determination, about 0.1 mg of partially purified receptor was routinely obtained from 40 to 50 mg of liver plasma membrane. Therefore, based on the yield, a conservative estimation of purification is 400-500-fold, al- though SDS-PAGE results suggest higher level of purification. In order to further characterize the receptor preparation, we have carried out isoelectric focusing. As shown in Fig. 2, six distinct Coomassie Blue-stained bands could be observed with isoelectric points ranging from pH 5.9 to 6.9. Five of these protein bands were radioactively labeled indicating that they contain affinity cross-linked glucagon receptor. Since two independent protein separation techniques showed comigra- tion of the Coomassie Blue positive bands with radiolabeled bands, we conclude that our purification procedure generates

FIG. 2. Isoelectric focusing of the affinity labeled glucagon recentor isolated bv nrenarative SDS-PAGE and WGA affin- ity chromatography. The protein solubilization and focusing pro- cedure was carried out as described under “Experimental Procedures.” Panel A, Coomassie Blue protein pattern. Panel B, autoradiogram of proteins shown in panel A. The (-) sign indicates the acidic region of the gel and the (+) sign indicates the basic region of the gel.

a highly enriched glucagon receptor preparation. Preparation and Characterization of Monoclonal Antibodies

against the Glucagon Receptor-Partially purified glucagon receptor was used as an immunogen for the preparation of mAbs. Three independent fusion procedures were carried out, and the resulting hybridomas were screened first by the dot- blot procedure of Jahn et al. (21), using the purified receptor preparation as the antigen. Positive hybridomas were screened again using immunoblots of partially purified, affin- ity labeled receptor. Through this screen, we have identified 64 positive clones from an initial 2800 clones. Of these, we have selected four antibodies that gave the strongest signal on immunoblots for further characterization. The properties of these antibodies are summarized in Table I.

Hybridoma clones were tested first for the ability to recog- nize the glucagon receptor in rat liver homogenate, purified plasma membranes, and in the soluble and particular fractions obtained from the initial liver homogenate. As shown in Fig. 3, antibody 6ElO stained a single band of 62 kDa in the homogenate, particular fraction, and plasma membrane prep- aration lanes, but did not stain any species in the soluble fraction. Identical results were obtained with three other mAbs. Immunostaining of the frozen liver section with mAbs aslo indicates plasma membrane location of the antigen pre- dominantly within sinusoidal and lateral domains of hepato- cytes.2 We tested the specificity of the antibodies by probing immunoblots of purified plasma membrane preparations from tissues that do not respond to glucagon and thus should not contain glucagon receptors. Plasma membranes isolated from rat adrenals, lung, and erythrocytes showed no staining (Fig. 4), whereas liver membranes always showed a single positive band.

We tested the ability of the mAbs to compete with glucagon for the binding site. The receptor binding assay in the pres- ence and absence of antibodies showed no inhibition of hor- mone binding, indicating that the antibodies recognition epi- topes do not overlap with the receptor’s hormone binding site. We then examined the antibodies for their ability to immu- noprecipitate the glucagon receptor. For these experiments, we used liver plasma membranes that were affinity labeled with ‘251-glucagon. Detergent extracts of the plasma mem- branes were prepared as described under “Experimental Pro- cedures,” and were consecutively incubated with ascites fluid and Sepharose-bound protein G or Sepharose-bound anti- mouse IgG. All four antibodies were capable of immunopre- cipitation of the glucagon receptor from crude liver plasma membrane extracts, however, antibodies 3E9 and 4Hll were significantly more efficient in the precipitation procedure. Denaturation of proteins by SDS treatment improved effi- ciency of the immunoprecipitation with 2C5 and 6E10, prob- ably due to better accessibility of antigenic sites. Fig. 5 shows results of the immunoprecipitation experiments with mAbs 4Hll and 2C5, identical results were obtained with mAbs 3E9 and 6E10, respectively.

’ J. Ritland and V. Iwanji, unpublished observations.

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TABLE I

Summary of properties of the monoclonal antibodies recognizing the glucagon receptor mAbs characterization was carried out as described under “Experimental Procedures.” Four (+), indicates the

strongest positive signal for each category tested.

Monoclonal antibody Isotype Epitope Immunoblot Immunoprecipitation Immunofluorescence

2C5 I&.&, K Protein +++ + ++ 3E9 Id&, K Carbohydrate +++ ++ +++ 4Hll I&,, K Carbohydrate +++ ++ +++ 6ElO I&1, K Protein +++ + +++

1 234 5

FIG. 3. Immunoblot analysis of the 6ElO antigen distribu- tion within different fractions of rat liver homogenate. Rat liver homogenate was fractionated as described under “Experimental Procedures,” aliquots from these fractions were separated by SDS- PAGE, transferred to nitrocellulose, and probed with mAbs 6ElO. Lane 1, homogenate; lane 2, supernatant; lane 3, particulate fraction; lane 4, plasma membrane; lane 5, M, markers.

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FIG. 4. Immunoblot of plasma membrane preparations from rat liver and rat peripheral tissues. Panel A, immunoblot probed with mAbs E610;panel B, immunoblot probed with mAbs 4Hll. Lane 1, liver; lane 2, lung; lane 3, adrenal gland; lane 4, erythrocyte ghosts. Arrowhead points to the glucagon receptor band.

FIG. 5. Immunoprecipitation of affinity labeled glucagon receptor with mAbs 4Hll and 2C5. Glucagon receptor was affinity labeled with ‘T-glucagon and membranes were processed and incubated with primary and secondary reagents as described under “Experimental Procedures.” Panel A: lane I, control ascites (NSl cells); lane 2, ascites 4Hll. Panel B: lane I, control ascites (NSl cells); lane 2, ascites 2C5. Arrowhead indicates position of the gluca- gon receptor.

Characterization of the Antibody Epitopes-We examined the properties of the recognition epitopes of the monoclonal antibodies. Affinity cross-linked membranes were digested with N-glycanase, an enzyme that cleaves both complex and high mannose type oligosaccharides (35). SDS-PAGE and autoradiography indicated that the native form of the gluca- gon receptor was converted to the intermediate forms of lower M, as we have previously described (10). An aliquot of this preparation was used for an immunoblot procedure. In the case of mAb 4Hl1, the N-glycanase treatment resulted in complete loss of staining (Fig. 6B). An identical result was observed for mAb 3E9. In contrast, mAb 6ElO did recognize the deglycosylated glucagon receptor. As shown in Fig. 6A, staining with 6ElO revealed the presence of four intermediate deglycosylated forms that we have previously observed for the affinity labeled glucagon receptor (10). Identical staining pat- tern was also obtained with antibody 2C5. Thus, we generated two mAbs that recognize protein epitopes and two antibodies that react with the oligosaccharide portion of the molecule.

Further experiments were carried out with plasma mem- branes solubilized in the presence or absence of -SH reducing agents. Samples with and without dithiothreitol were sepa- rated by SDS-PAGE, transferred onto nitrocellulose, and probed with antibodies. Both reduced and unreduced forms of the receptor were recognized by the antibodies (data not shown). Moreover, the antibody positive band showed de- creased electrophoretic mobility in the presence of dithiothre- itol, as we have previously observed for the affinity labeled glucagon receptor (10).

Analysis of the Species and Tissue Specificity of the Anti- bodies-we have analyzed the species specificity of mAbs 2C5 and 6EIO. Liver plasma membranes prepared from rat, mouse, chicken, and dog were shown to possess glucagon receptors by binding and cross-linking to 1251-glucagon (Fig. 7). These

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FIG. 6. Immunoblot analysis of deglycosylated glucagon receptor. Liver plasma membranes were treated with N-glycanase, samples were separated on the same SDS gel, transferred onto the same sheet of nitrocellulose, and analyzed by immunoblot as described under “Experimental Procedures.” Panel A, immunoblot probed with mAb 6ElO; panel B, immunoblot probed with mAb 4Hll. Lane I, N- glycanase-treated membranes; lane 2, untreated control membranes. Arrowheads indicate position of the glucagon receptor.

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WC. 7. UV-induced cross-linking of 1251-glucagon to liver plasma membranes isolated from rat, mouse, dog, and chicken. Panel A: lane I, rat plasma membranes; lane 2, mouse plasma membranes. Panel B: lane 1, rat plasma membranes; lane 2, dog plasma membranes. Panel C: lane I, rat plasma membranes; lane 2, chick plasma membranes. Arrowheads indicate position of the glucagon receptor. Glucagon receptor band in the dog plasma mem- brane preparation appears as a doublet in agreement with data of Sheetz and Tager (24).

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FIG. 9. Immunoblot analysis of the glucagon receptor from adipocyte and kidney plasma membranes probed with mAbs 6ElO. Membranes were prepared according to the procedures out- lined under “Experimental Procedures.” Panel A: lane L, liver plasma membranes; lane A, adipocyte plasma membrane. Panel B: lane L, liver plasma membrane; lane K, kidney plasma membrane.

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FIG. 8. Immunoblot analysis of plasma membranes isolated from rat, mouse, dog, and chick livers. Membrane preparation and immunoblotting was carried out as described under “Experimen- tal Procedures.” Panel A, immunoblot probed with mAb 6ElO; panel B, immunoblot probed with mAb 2C5. Lane 1, rat plasma membranes; lane 2, mouse plasma membranes: lane 3, dog plasma membranes; lane 4, chick plasma membranes.

membrane preparations were subjected to SDS-PAGE, fol- lowed by electrophoretic transfer to nitrocellulose for immu- noblotting. As shown in Fig. SA, antibody 6ElO recognized the mouse glucagon receptor in addition to the rat receptor, while antibody 2C5 stained only the rat liver plasma mem- branes (Fig. 8B). This experiment demonstrated that the mAbs show significant species specificity.

We analyzed the ability of these antibodies to recognize the glucagon receptor in target tissues other than liver, in adipo- cytes (2, 3), and in kidney (4). Antibody 6ElO detected a protein species of identical M, as seen for the liver receptor in membranes prepared from rat adipose and kidney tissues (Fig. 9). However, mAb 6ElO failed to recognize the canine kidney receptor and the Madin-Darby canine kidney cell line receptor (data not shown). Antibody 2C5 recognized the adi- pocyte receptor but failed to recognize the kidney receptor. The inability of antibody 2C5 to recognize the kidney receptor may be due to low levels of expression of the receptor. When the sensitivity of the antibody staining on immunoblots of decreasing antigen concentration was examined, the signal by mAb 2C5 was lost at a 1:5 dilution while it remained detectable by 6ElO at a 1:lO dilution.

Antibody and Ligand-binding Domains of the Glucagon Receptor-The differences in species specificity observed be- tween mAbs 6ElO- and 2C5-staining patterns suggested pres- ence of different epitopes. We investigated this possibility

FIG. 10. Proteolytic mapping of the hepatic glucagon recep- tor with 5’. aureus V-8 protease. Affinity labeled membranes were digested with V-8 according to the protocol outlined under “Experi- mental Procedures.” Membranes were separated by SDS-PAGE and analyzed by autoradiography and by immunoblotting with mAbs. Panel A, autoradiography of the affinity labeled glucagon receptor before (lane I), and after (lane 2) the protease treatment. Panel B, immunoblot probed with mAb 6ElO; lane 1, control; lane 2, protease V-8 digest. Panel C, immunoblot probed with mAb 2C5; lane I, control; lane 2, protease V-8 digest. Filled arrowheads indicate posi- tion of the intact glucagon receptor; open arrowheads indicate position of V-8-generated fragments of the glucagon receptor.

using proteolytic cleavage of the glucagon receptor with S. aureus V-8 protease. As shown in Fig. lOA, V-8 digestion of the glucagon receptor generated three distinct radiolabeled proteolytic fragments, one of about 57 kDa and a doublet of 25-30 kDa. When the immunoblots of the V-8 treated mem- branes were probed with the antibodies, mAb 6ElO stained only the lower M, doublet (Fig. lOB), while mAb 2C5 selec- tively stained the higher M, bands, one that corn&rated with upper radioactive band and the second band not observed in the affinity labeling experiment (Fig. 1OC). Therefore, this proteolytic fragment of about 47 kDa contains the 2C5 epitope site but does not contain glucagon-binding site. Since the low M, doublet was radioactive as well as mAb 6ElO immuno- reactive, these fragments contain both the glucagon-binding site as well as the mAb 6ElO antigenic site. All of the observed V-8 fragments were found in the pellet following centrifuga- tion of the proteolyzed membranes, indicating that these fragments remain firmly associated with lipid bilayer (results not shown). We next studied epitope localization using car- boxypeptidase Y, an enzyme that cleaves the COOH-terminal domains of proteins. As shown in Fig. 11, carboxypeptidase treatment abolished staining by mAb 6ElO while staining by mAb 2C5 was unaffected, placing 6ElO epitope on the COOH terminus. Significantly, these experiments established that the 25kDa V-8 proteolytic fragment contains both the glu- cagon-binding site, as revealed by autoradiography, and the

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second class binds to protein epitopes. The mAbs 4Hll and 3E9 recognize an oligosaccharide but stain a single polypep- tide in the blots of purified plasma membranes and of total liver homogenate. If the epitopes were exclusively oligosac- charide, more than a single positive band would be expected. Most probably, the epitope in question is a combination of the oligosaccharide moiety and the peptide sequence that surrounds the glycosylation site. At present, we cannot deter- mine if the epitopes for mAbs 4Hll and 3E9 are identical.

3 03 FIG. 11. Immunoblot analysis of the hepatic glucagon recep-

tor following treatment with carboxypeptidase Y. Membranes were digested with carboxypeptidase Y, separated by SDS-PAGE and probed with mAbs as described under “Experimental Procedures.” Panel A: immunoblot probed with mAb 6ElO; lane I, control; lane 2, carboxypeptidase digest. Panel B: immunoblot probed with mAb 2C5; lane 1, control; lane 2, carboxypeptidase digest. Arrowheads indicate position of the glucagon receptor.

COOH-terminal domain, as revealed by antibody 6ElO rec- ognition.

DISCUSSION

Antibodies provide a powerful tool for the investigation of the structure-function relationship of receptor molecules (ll- 15). To aid our study of the glucagon receptor, we have set out to prepare anti-receptor mAbs. Since the glucagon recep- tor has not been purified to homogeneity, we have developed a simple and rapid procedure for the isolation of glucagon receptor suitable for use as an immunogen. Several reports indicate that isolated muscarinic (36) and P-adrenergic recep- tors (15) proved to be poor immunogens for mAbs prepara- tions, perhaps because similarity among rodent receptors prevents the mounting of a strong response by recipient mice. We, however, were able to obtain a positive response for each of three separate fusion experiments that were carried out. This high level of success may be due to the immunogenicity of SDS-denatured glucagon receptor. In agreement with our observations, excellent immunogenicity of the epidermal growth factor receptor isolated by SDS-PAGE has been re- ported (37).

The four mAbs we have generated recognize the glucagon receptor. This claim is supported by following observations. 1) The antibodies recognized the receptor on immunoblots of liver plasma membranes, whereas no positive bands were observed when adrenal, lung, and erythrocyte membranes were probed. These tissues do not possess glucagon receptors but express VIP receptors, @-adrenergic receptors (lung), cor- ticotropin and angiotensin receptors (adrenals), /3-adrenergic receptors and several glycoproteins (red blood cell). Thus, these tissues serve as good controls for the specificity of antibody binding. 2) The antibodies recognized the receptor on the immunoblots of adipocyte and kidney plasma mem- branes. 3) The antibodies immunoprecipitated the detergent solubilized receptor. 4) mAbs 6ElO and 2C5 recognize four intermediate receptor species generated by controlled degly- cosylation with N-glycanase, the same pattern as observed for affinity labeled receptor. 5) The antibodies bind to the same set of proteolytic fragments that were obtained for the affinity labeled receptor. 6) The immunolocalization of the antigen at the sinusoidal and lateral surfaces of the hepato- cytes is consistent with the location of the glucagon receptor.

The mAbs described here fall into two classes. One class (4Hll and 3’E9) recognizes carbohydrate epitopes while the

With regard to the second class of antibodies, we have clear indications that mAbs 6ElO and 2C5 recognize different sites within the receptor molecule. When the glucagon receptor was cleaved into several distinct fragments with S. aureus V- 8 protease, each of the antibodies recognized a different set of fragments.

Our studies revealed that the antibodies do not inhibit the binding of glucagon to its receptor. This finding is not sur- prising since we used an SDS-denatured receptor preparation as the immunogen. Furthermore, we have identified a carbo- hydrate as a recognition site for two of the antibodies. The treatment with V-8 protease places the mAbs 2C5 epitope on the portion of the receptor distal to the binding site, while in the case of 6E10, the carboxypeptidase Y digestion results place the epitope at the COOH-terminal domain. Thus, both epitopes for mAbs 6ElO and 2C5 are distant from the hor- mone-binding site.

Glucagon receptors are present in tissues other than liver such as adipose (2,3) and kidney (4), although at much lower densities. Although the concentration of receptors is 20-50- fold lower than that of liver, mAb 6ElO was able to recognize the receptor in these tissues. This antibody will be of special importance for the detection of small quantities of the recep- tor. Antibody 2C5 stained immunoblots of adipose and renal tissues poorly, perhaps due to lower binding affinity. The experiments presented here indicate that the antibodies show a high degree of species specificity. We consider mAb 6ElO to be rodent specific since both rat and mouse receptors are recognized. Antibody 2C5 recognized only the rat receptor. Both antibodies failed to recognize the chicken or canine forms of the receptor although the presence of these receptors could be shown easily by ‘251-glucagon binding and cross- linking experiments. This result demonstrates species specific differences among glucagon receptors. Since our antibodies recognize at least three separate antigenic determinants de- tected differences may include variations in N-linked glyco- sylation as well as variations in the primary structure of the COOH-terminal domain of the receptor.

The use of mAbs has brought new insights into the structure of the glucagon receptor. A combination of affinity labeling, antibody staining, and proteolytic mapping allowed us to place the glucagon binding site on the 25-kDa fragment that con- tains the intact COOH-terminal domain. Furthermore, if we take under account that V-8 protease also produced a 47-kDa fragment that lost the glucagon-binding site, the size of the fragment containing the ligand may be as a low as 15 kDa. In agreement with our results a 15-kDa fragment of the glucagon receptor was observed by Iyengar and Herberg (9) after exten- sive digestion with elastase. The structure of the glucagon receptor most probably is similar to the structure of the rhodopsin molecule as was shown for other receptors linked to adenylyl cyclase via G proteins (38-40). The main features of these receptors include seven transmembrane domains with three extracellular and three cytoplasmic loops. The localiza- tion of the glucagon-binding site within a third extracellular loop-seventh transmembrane domain of the receptor would be consistent with our experimental results, however, a defin-

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21308 Monochal Antibodies to the Glucagon Receptor

itive experiment awaits the isolation of the glucagon receptor’s Fraser, C. M., and Lindstrom, J., eds) pp. 117-139, Alan R. gene. Liss, New York

In conclusion, we report the first preparation of anti-glu- 15. Moxham, C. P., George, S. T., Graziano, M. P., Brandwein, H.

cagon receptor antibodies. We have isolated a series of mAbs J., and Malbon, C. C. (1986) J. Biol. Chem. 261,14562-14570

that recognize distinct structural domains of the glucagon 16. Neville, D. M. (1968) Biochim. Biophys. Acta 154,540-552 17. Laemmli, U. K. (1970) Nature 227,680-685

receptor. The availability of these antibodies will allow us to 18. Scheele, G., Pash, J., and Bieger, W. (1981) Anal. Biochem. 112, ^^. -_^ characterize the structural domains of the receptor, to study 304-313

the receptor’s biogenesis and post-translational modifications, 19. Wessel, D., and Flugge, U. I. (1984) Anal. Biochem. 138, 141-

142 --- and to isolate the gene coding for the glucagon receptor. 20. Hybridoma Techniques, EMBO Course 1980, Cold Spring Harbor

Laboratory, Cold Snring Harbor, NY Acknowledgments-We thank Dr. David Hamilton, Department of 21.

Cell Biology and Neuroanatomy, for making a monoclonal antibody facility available to us in the early stages of this work. We also thank 22. Drs. Kathy Ensrud, Caterina Sellitto, and Ryoko Kuriyama for teaching us hybridoma techniques and for many helpful suggestions

23 ’

during the course of this study. 24.

Jahn, R., S&iebler, -W.,and Greengard, P. (1984) Proc. Natl. Acad. Sci. U. S. A. 81,1684-1687

Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. U. S. A. 76,4350-4354

Bartles, J. R., Braiterman, L. T., and Hubbard, A. L. (1985) J. Biol. Chem. 260,12792-12802

Sheetz, M. J., and Tager, H. S. (1988) J. Biol. Chem. 263,8509- 8514

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

REFERENCES

Unger, R. M., Dobbs, R. E., and Orci, L. (1978) Annu. Reu. Physiol. 40,307-343

Rodbell, M. (1964) J. Biol. Chem. 239, 375-380 Desbuquois, B., and Laudat, M.-H. (1974) Mol. Cell Endocrinol.

1,355-370 Bailly, C., Imbert-Teboul, M., Chabardes, D., Hus-Citharel, A.,

Monte@, M., Clique, A., and Morel, F. (1980) Proc. Natl. Acad. Sci. U. S. A. 77,3422-3424

Conlon, J. M., Samson, W. K., Dobbs, R. E., Orci, L., and Unger, R. H. (1979) Diabetes 28,700-702

Murakami, H., and Masui, H. (1980) Proc. Natl. Acad. Sci. U. S. A. 77,3464-3468

Johnson, G. L., MacAndrew, V. I., Jr., and Pilch, P. F. (1981) Proc. Natl. Acad. Sci. U. S. A. 78,875-878

Demoliou-Mason, C., and Epand, R. M. (1982) Biochemistry 21, 1996-2004

Iyengar, R., and Herberg, J. T. (1984) J. Biol. Chem. 269,5222- 5229

Iwanij, V., and Hur, K. C. (1985) Proc. Natl. Acad. Sci. U. S. A. 82,325-329

Lindstrom, J., Tzartos, S., Gullick, W., Hochschwender, S., Swan- son, L., Sargent, P., Jacob, M., and Montal, M. (1983) Cold Spring Harbor Symp. Quant. Biol. 48,89-99

Roth, R. A., Cassell, D. J., Wong, K. Y., Maddux, B. A., and Goldtine, I. D. (1982) Proc. Natl. Acad Sci. U. S. A. 79, 7312- 7316

Morgan, D. O., Ho, L., Korn, L. J., and Roth, R. A. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 328-332

Venter, J. C. (1984) in Monoclonal and Anti-Zdiotypic Antibodies: Probes for Receptor Structure and Function (Venter, J. C.,

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37. 38.

39.

40.

Paglin, S., and Jamieson, J. D. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 3739-3743

Leroux, P., Vaudry, H., Fournier, A., St. Pierre, S., and Pelletier, G. (1984) Endocrinology 114,1506-1512

Dodge, J. T., Mitchell, C., and Manahan, J. D. (1963) Arch. Biochem. Biophys. 100, 119-130

Williams, L. T., Jarett, L., and Lefkowitz, R. J. (1976) J. Biol. Chem. 251.3096-3104

Segre, G. V., Rosenblatt, M., Reiner, B. L., Mahaffey, J. E., and Potts. J. T., Jr. (1979) J. Biol. Chem. 254.6980-6986

Ames, G. F.-L., and Nikaido, K. (1976) Biochemistry 16, 616- 623

Spencer, E. M., and King, T. P. (1971) J. Biol. Chem. 246, 201- 208

Udenfriend, S., Stein, S., Bohlen, P., Dairman, W., Leimgruber, W., and Weigeli. M. (1972) Science 178.871-872

Lin, M. C., Wright, D. E., Hruby, V. J., and Rodbell, M. (1975) Biochemistry 14,1559-1563

Stiles, G. L., Benovic, J. L., Caron, M. G., and Lefkowitz, R. J. (1984) J. Biol. Chem. 259,8655-8663

Elder, J. H., and Alexander, S. (1982) Proc. Natl. Acad. Sci. U. S. A. 79,4540-4544

Luetje, C. W., Brumwell, C., Norman, M. G., Peterson, G. L., Shimerlik, M. I., and Nathanson, N. M. (1987) Biochemistry 26,6892-6896

Decker, S. (1984) Arch. Biochem. Biophys. 228,621-626 Nathans, J., Thomas, D., and Hogness, D. S. (1986) Science 232,

193-202 Peralta, E. G., Ashkenazi, A., Winslow, J. W., Smith, D. H.,

Ramachandran, J., and Capon, D. J. (1987) EMBO J. 6,3923- 3929

O’Dowd, B. F., Lefkowitz, R. J., and Caron, M. G. (1989) Annu. Rev. Neurosci. 12,67-83

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V Iwanij and A C Vincentmonoclonal antibodies.

Characterization of the glucagon receptor and its functional domains using

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