Proc. Nall. Acad. Sci. USA Vol. 89, pp. 4314-4318, May 1992 Neurobiology
Development of antibodies against the rat brain somatostatin receptor (peptide receptor/receptor subtypes)
MAGALI THEVENIAU*, STEPHANIE RENS-DOMIANO*, SUSAN F. LAW*, GENEVIEVE ROUGONt, AND TERRY REISINE*t *Department of Pharmacology, University of Pennsylvania, Philadelphia, PA 19104; and tLaboratoire de Biologie de la Differenciation Cellulaire, Centre National de la Recherche Scientifique, Unite de Recherches Associ6e 179, Faculte des Sciences de Luminy, 13288 Marseille, France Communicated by Louis B. Flexner, January 31, 1992
(7) were reported to migrate as a mass of90 kDa in denaturing gels. In contrast, SRIF receptors from rat brain (8, 9), adrenal cortex (10), and the cell lines AtT-20 and GH3 (11) are 60 kDa in size. The diversity of size of SRIF receptor subtypes may be due to variations in carbohydrate processing of the receptors since all SRIF receptors studied so far have been shown to be glycoproteins (8, 12, 13). Alternatively, these size variations may also result from differences in primary structure between the SRIF receptor subtypes. Recently, the physical characteristics of SRIF receptor subtypes were further investigated by using antibodies against the receptor. Lewin and Reyl-Desmars (14, 15) reported the development of a monoclonal antibody against the SRIF receptor from the human gastric cell line HGT-1. The antibodies detected only proteins of 90 kDa in membranes from these cells. These authors have indicated that the antibodies also detect 90-kDa proteins from rat stomach, pancreas, and pituitary and have proposed that the material detected is the 90-kDa SRIF receptor. In the present study, we report the development of rabbit polyclonal antibodies against the rat brain SRIF receptor. F4 antibodies selectively detect SRIF receptors of 60 kDa from rat brain, NG-108, AtT-20, and GH3 cells. They also selectively immunoprecipitate active SRIF receptors from brain and AtT-20 cells. Interestingly, F4 does not interact with proteins from rat pancreas or pituitary, which have been proposed to express the 90-kDa SRIF receptor. These findings support the hypothesis that physically distinct SRIF receptor subtypes are expressed in mammalian tissues and further indicate that they are immunologically distinct.
Somatostatin (SRIF) is a neurotransmitter in ABSTRACT the brain involved in the regulation of motor activity and cognition. It induces its physiological actions by interacting with receptors. We have developed antibodies against the receptor to investigate its structural properties. Rabbit polyclonal antibodies were generated against the rat brain SRIEF receptor. These antibodies (F4) were able to immunopeipitate solubilized SRIF receptors from rat brain and the cell line AtT-20. The specificity of the interaction of these antibodies with SRIF receptors was further demonstrated by immunoblotting. F4 detected SRIF receptors of 60 kDa from rat brain and adrenal cortex and the cell lines AtT-20, GH3, and NG-108, which express high densities of SRIEF receptors. They did not detect immunoreactive material from rat liver or COS-l, HEPG, or CRL cells, which do not express functional SRIF receptors. In rat brain, 60-kDa immunoreactivity was detected by F4 in the hippocampus, cerebral cortex, and striatum, which have high densities of SRIF receptors. However, F4 did not interact with proteins from cerebellum and brain stem, which express few SRIF receptors. Immunoreactive material cannot be detected in rat pancreas or pituitary, which have been reported to express a 90-kDa SRIF receptor subtype. The selective detection of 60-kDa SRIF receptors by F4 indicates that the 60- and 90-kDa SRIF receptor subtypes are immunologically distinct. The availability of antibodies that selectively detect native and denatured brain SRIF receptors provides us with a feasible approach to clone the brain SRIF receptor gene(s).
Somatostatin (SRIF) is a neuropeptide, which was originally isolated from hypothalamus and shown to be the major physiological inhibitor of growth hormone secretion from the pituitary (1). It is also expressed in delta cells of the pancreatic islets, where it regulates the balance between insulin and glucagon release, and in mucosal cells of the stomach, where it has been reported to modulate gastric acid secretion (2). This peptide is also synthesized in discrete neuronal populations in brain, where it has a role in modulating motor activity and cognitive processes (3, 4). SRIF induces its biological activity by interacting with membrane-bound receptors. The properties of SRIF receptors have been analyzed by using a variety of biochemical approaches. To characterize the physical properties of the receptor, techniques were developed to covalently crosslink the receptor with radioactive SRIF analogs and to analyze the tagged receptor by gel electrophoresis and autoradiography. The results of these studies have shown that heterogeneities exist in the structure of SRIF receptors. SRIF receptors from pancreatic acinar cells (5), pituitary cells (6), and GH4C1 cells
MATERIALS AND METHODS Materials. Rat brains were obtained from Pel-Freeze Bio-
logicals. 3-[(3-Cholamidopropyl)dimethylammonioj-1propanesulfonate (CHAPS), leupeptin, and pepstatin A were from Boehringer Mannheim; phenylmethylsulfonyl fluoride, aprotinin, and protein A-Sepharose were from Sigma; Sepharose CL-6B was from Pharmacia; Hybond nitrocellulose membranes were from Amersham; [35S]methionine and 1251labeled protein A were from ICN; and MK 678 was a gift from Merck. Solubilization and Fractionation of Rat Brain SRIF Receptors. Rat brain SRIF receptors were solubilized and fractionated as described (16, 17). Briefly, three adult rat brains minus the cerebellum were homogenized in 10 vol of 50 mM Tris HCl (pH 7.8) supplemented with 1 mM EGTA, 5 mM MgCl2, leupeptin at 10 ,ug/ml, pepstatin A at 2 Zg/ml, bacitracin at 0.2 mg/ml, and 0.25 mM phenylmethylsulfonyl Abbreviations: SRIF, somatostatin; CHAPS, 3-3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; G protein, guanine nucleotide-binding regulatory protein. tTo whom reprint requests should be addressed.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
4314
Neurobiology: Theveniau et al. fluoride (buffer 1). Cell debris and the nuclear fraction were discarded after centrifugation (1000 x g for 5 min at 40C), and the supernatant was centrifuged at 40,000 x g for 30 min at 40C. The pellet was washed and solubilized in buffer 1 containing 10 mM CHAPS, 20% (vol/vol) glycerol, and aprotinin at 0.5 pl/ml (buffer 2). Solubilization was performed under constant stirring at 40C for 45 min, and the mixture was centrifuged for 60 min at 150,000 x g at 40C. Solubilized brain proteins were subjected to gel filtration over a Sepharose CL-6B column (1.6 cm x 40 cm) preequilibrated with 50 mM Tris-HCl (pH 7.8) containing 5 mM CHAPS, 1 mM EGTA, 5 mM MgCl2, and 10% (vol/vol) glycerol to partially purify the SRIF receptors. Fractionated samples were tested for the presence of solubilized SRIF receptor by using the 125I-labeled MK 678 (125I-MK 678) binding assay. Solubilizatlon of AtT-20 Cell SRIF Receptors. AtT-20 cells were grown as described (9, 12, 17). Cells were detached from flasks in buffer 1, centrifuged at 24,000 X g for 7 min at 4°C, and homogenized in the same buffer. The homogenate was centrifuged at 40,000 x g for 30 min at 4°C, and the membranes in the pellet were solubilized as described for brain. After solubilization, the supernatant was diluted 5-fold with buffer 1 and then concentrated to obtain final CHAPS and glycerol concentrations of 2 mM and 10o (vol/vol), respectively. 12'I-MK678 Binding Assay. This assay was used to detect active solubilized or immunoprecipitated SRIF receptors. For the assay, samples were incubated for 90 min at 25°C in the presence of 50 pM 125I-MK 678 (specific activity of 2200 Ci/mmol; 1 Ci = 37 GBq), a high affinity and selective SRIF agonist, in buffer 1 as described (16, 17). The reaction was terminated by the addition of 3 ml of 50 mM Tris HCl (pH 7.8) and vacuum filtered over Whatman GF/F glass fiber filters presaturated with 0.5% (wt/wt) polyethyleneimine. Filters were washed twice with 3 ml of buffer, dried, and assayed in a y counter. Specific 125I-MK 678 binding was defined as the difference between total 125I-MK 678 binding and the binding in the presence of 1 ,M [D-Trp8]SRIF. Production of Polyclonal Antibodies. Solubilized, fractionated brain proteins enriched in SRIF receptors were used as the immunogen. New Zealand rabbits received subcutaneous injections of 1 mg of the immunogen emulsified in Freund's complete adjuvant. Booster injections were administered every 4 weeks, and the rabbits were bled 2 weeks after each
injection.
Affinity Puriflcation of Ant-SRIF Receptor Antibodies. For micropurification of anti-SRIF receptor antibodies from the original total serum, 12 mg of fractionated brain proteins was subjected to SDS/11o PAGE in six different gels, blotted onto separate nitrocellulose papers, and incubated overnight at 4°C with total serum (1:500 dilution) in phosphate-buffered saline (PBS) supplemented with 5% dry defatted milk and 0.02% sodium azide. The qitrocellulose paper was rinsed three times for 15 min with PBS, and the immunoblotted antibodies were ellpted from the nitrocellulose paper with 0.2 M glycine-HCl (pH 2.5) as described (18, 19). Eluted antibodies (6 ml for f1, F2, and F7 and 4 ml for F3-F6) were dialyzed against 10 liters of PBS, divided into aliquots, and frozen at -20°C. Antibodies were eluted from proteins of differing size (see below). SRIF Receptor Immynoprecipitation. SRIF receptors were immunoprecipit4ted with micropurified antibody-coated protein A-Sepharose beads. Two hundred microliters of micropurified antibodjes was incubated for 5 hr at 4°C in the presence of 50 Al of protein A-Sepharose beads. The beads were rinsed three times with 1 ml of buffer 1, and then 4AM ,ud of solubilized, fractionated brain proteins was added, The same volume of sample was used for the solubilized AtT-20 cell proteins. Samples were immunoprecipitated overlight at 40C under continuous shaking. The supernatants were saved,
Proc. Natl. Acad. Sci. USA 89 (1992)
4315
and the beads were rinsed three times with 1 ml of buffer 1 before being analyzed in the 125I-MK 678 binding assay. [35S]Methionine Labeling of AtT-20 Cell Proteins. For radiolabeling, AtT-20 cells were first preincubated for 30 min in methionine-free medium and then incubated overnight in the presence of 0.5 mCi of [35S]methionine in 5 ml of Dulbecco's modified Eagle's medium supplemented with 10o fetal bovine serum and L-glutamine as described (18). Cells were rinsed two times with PBS, detached from the flask in 20 ml of PBS, and centrifuged for 7 min at 24,000 x g at 40C. After centrifugation, the pellet was saved, homogenized in 15 ml of buffer 1, and centrifuged for 15 min at 40,000 x g at 40C. The membrane pellet was solubilized in RIPA buffer consisting of 50 mM Tris-HC1 (pH 7.4), 150 mM NaCl, 1% (vol/vol) Nonidet P-40, 1% (wt/vol) sodium deoxycholate, 0.1% SDS, and protease inhibitors. The homogenate was frozen and thawed and then centrifuged (150,000 x g) at 40C for 60 min. The supernatant containing the SRIF receptor was saved and immunoprecipitated with antibody-coated protein A beads. After overnight incubation at 4°C, the supernatant was discarded, and the beads were rinsed once on a discontinuous 10-20% (wt/vol) sucrose gradient. They were then rinsed three more times. After washing, immunoprecipitates were boiled in 50 pl of sample buffer and subjected to SDS/10%o PAGE; the gel was then dried and exposed for autoradiography. Immunoblotting of SRIIF Receptors. Tissue and tumor cell membranes were prepared as described above and solubilized in RIPA buffer. The samples were frozen and thawed and then centrifuged at 150,000 x g for 60 min at 4°C. The supernatant was separated from the pellet and boiled in electrophoresis sample buffer. For immunoblotting, samples were subjected to SDS/l0o PAGE, blotted onto nitrocellulose paper, blocked for 60 min at 37°C with PBS containing 5% dry defatted milk and 0.02% sodium azide, and incubated overnight at 4°C with micropurified antibodies (1:100 dilution). After incubation, the immunoblots were rinsed in PBS and incubated with 5 x 105 cpm of 125I-labeled protein A per ml, rinsed again, dried, and exposed for autoradiography.
RESULTS To generate antibodies against the SRIF receptor, rat brain SRIF receptors were solubilized and subjected to sizeexclusion chromatography as described (16, 17). The peak binding activity was concentrated, emulsified with Freund's adjuvant, and injected into rabbits. Serum was collected from the animal, and the antibodies were micropurified. Purification consisted of subjecting fractionated, solubilized brain SRIF receptors to SDS/PAGE, transferring the proteins to nitrocellulose paper, and immunoblotting the antibodies onto the brain proteins. The antibodies were eluted from proteins of various sizes. Seven antibody fractions were obtained, referred to as F1-F7, which reacted with proteins ranging from 200 to 20 kDa. Each fraction of antibodies was rescreened against solubilized rat brain proteins. As shown in Fig. 1A, F4-F6 antibodies selectively reacted with proteins of the size range from which they were initially eluted. This indicates that no major degradation of the proteins occurred during membrane preparation, as protein degradation would have resulted in the antibodies reacting with different size proteins when rescreened. Since the antibodies were generated against native brain proteins, we tested them for their ability to immunoprecipitate high-affinity SRIF agonist binding sites to determine which fraction of antibodies selectively reacted with SRIF receptors (Fig. 1B). For these studies, solubilized, partially purified brain SRIF receptors were incubated with different fractions of antibodies that had been prebound to protein A-Sepharose beads. The presence of active SRIF receptors in the immunoprecipitates was de-
4316
Proc. Natl. Acad. Sci. USA 89 (1992)
Neurobiology: Theveniau et al. A
with [35S]methionine (Fig. 2A). The material immunoprecipitated is the same size as SRIF receptors in AtT-20 cells and rat brain (8, 9, 11). In contrast, control antibodies from F3 were not able to immunoprecipitate 60-kDa proteins from AtT-20 cells (Fig. 2A) or SRIF receptors as detected by the 125I-MK 678 binding assay. Moreover, F4 selectively detected proteins of 60 kDa from rat brain and AtT-20 cells by immunoblotting (Fig. 2B). In contrast, F3 did not detect any proteins from AtT-20 cells (Fig. 2B). Further evidence that F4 is selectively directed against SRIF receptors is provided from the results showing that they selectively detected proteins of 60 kDa from mouse-derived (AtT-20 and NG-108) and rat-derived (GH3) cell lines that are known to express a high density of SRIF receptors (11) (Fig. 3). In contrast, no immunoreactivity was detected in membranes from monkey-derived COS cells (Fig. 3) and human HepG2 hepatocarcinoma cells, or human umbilical cordderived CRL cells (data not shown), which do not express the SRIF receptor. Previous studies have shown that SRIF receptors in rat brain are heterogeneously distributed (20, 21). Consistent with this finding, F4 detected high levels of 60-kDa immunoreactivity in brain regions (hippocampus, cerebral cortex, and striatum) expressing high levels of the receptor (Fig. 4A). In contrast, very little immunoreactivity was detected in cerebellum (Fig. 4A) and brain stem (data not shown), which have very low levels of SRIF receptor. F4 was also tested for its ability to recognize SRIF receptors expressed in peripheral tissues. It detected 60-kDa immunoreactivity in the adrenal gland, which was recently B
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FIG. 1. Micropurification of antibodies against the brain SRIF receptor. (A) Fractionated brain proteins were immunoblotted in the presence of a 1:500 dilution of total serum (lane T). Antibodies reacting with proteins of different sizes (fraction 1, >100 kDa; fraction 2, 80-100 kDa; fraction 3, 60-80 kDa; fraction 4, 53-60 kDa; fraction 5, 45-53 kDa; fraction 6, 30-45 kDa; fraction 7,
Development of antibodies against the rat brain somatostatin receptor (peptide receptor/receptor subtypes)
MAGALI THEVENIAU*, STEPHANIE RENS-DOMIANO*, SUSAN F. LAW*, GENEVIEVE ROUGONt, AND TERRY REISINE*t *Department of Pharmacology, University of Pennsylvania, Philadelphia, PA 19104; and tLaboratoire de Biologie de la Differenciation Cellulaire, Centre National de la Recherche Scientifique, Unite de Recherches Associ6e 179, Faculte des Sciences de Luminy, 13288 Marseille, France Communicated by Louis B. Flexner, January 31, 1992
(7) were reported to migrate as a mass of90 kDa in denaturing gels. In contrast, SRIF receptors from rat brain (8, 9), adrenal cortex (10), and the cell lines AtT-20 and GH3 (11) are 60 kDa in size. The diversity of size of SRIF receptor subtypes may be due to variations in carbohydrate processing of the receptors since all SRIF receptors studied so far have been shown to be glycoproteins (8, 12, 13). Alternatively, these size variations may also result from differences in primary structure between the SRIF receptor subtypes. Recently, the physical characteristics of SRIF receptor subtypes were further investigated by using antibodies against the receptor. Lewin and Reyl-Desmars (14, 15) reported the development of a monoclonal antibody against the SRIF receptor from the human gastric cell line HGT-1. The antibodies detected only proteins of 90 kDa in membranes from these cells. These authors have indicated that the antibodies also detect 90-kDa proteins from rat stomach, pancreas, and pituitary and have proposed that the material detected is the 90-kDa SRIF receptor. In the present study, we report the development of rabbit polyclonal antibodies against the rat brain SRIF receptor. F4 antibodies selectively detect SRIF receptors of 60 kDa from rat brain, NG-108, AtT-20, and GH3 cells. They also selectively immunoprecipitate active SRIF receptors from brain and AtT-20 cells. Interestingly, F4 does not interact with proteins from rat pancreas or pituitary, which have been proposed to express the 90-kDa SRIF receptor. These findings support the hypothesis that physically distinct SRIF receptor subtypes are expressed in mammalian tissues and further indicate that they are immunologically distinct.
Somatostatin (SRIF) is a neurotransmitter in ABSTRACT the brain involved in the regulation of motor activity and cognition. It induces its physiological actions by interacting with receptors. We have developed antibodies against the receptor to investigate its structural properties. Rabbit polyclonal antibodies were generated against the rat brain SRIEF receptor. These antibodies (F4) were able to immunopeipitate solubilized SRIF receptors from rat brain and the cell line AtT-20. The specificity of the interaction of these antibodies with SRIF receptors was further demonstrated by immunoblotting. F4 detected SRIF receptors of 60 kDa from rat brain and adrenal cortex and the cell lines AtT-20, GH3, and NG-108, which express high densities of SRIEF receptors. They did not detect immunoreactive material from rat liver or COS-l, HEPG, or CRL cells, which do not express functional SRIF receptors. In rat brain, 60-kDa immunoreactivity was detected by F4 in the hippocampus, cerebral cortex, and striatum, which have high densities of SRIF receptors. However, F4 did not interact with proteins from cerebellum and brain stem, which express few SRIF receptors. Immunoreactive material cannot be detected in rat pancreas or pituitary, which have been reported to express a 90-kDa SRIF receptor subtype. The selective detection of 60-kDa SRIF receptors by F4 indicates that the 60- and 90-kDa SRIF receptor subtypes are immunologically distinct. The availability of antibodies that selectively detect native and denatured brain SRIF receptors provides us with a feasible approach to clone the brain SRIF receptor gene(s).
Somatostatin (SRIF) is a neuropeptide, which was originally isolated from hypothalamus and shown to be the major physiological inhibitor of growth hormone secretion from the pituitary (1). It is also expressed in delta cells of the pancreatic islets, where it regulates the balance between insulin and glucagon release, and in mucosal cells of the stomach, where it has been reported to modulate gastric acid secretion (2). This peptide is also synthesized in discrete neuronal populations in brain, where it has a role in modulating motor activity and cognitive processes (3, 4). SRIF induces its biological activity by interacting with membrane-bound receptors. The properties of SRIF receptors have been analyzed by using a variety of biochemical approaches. To characterize the physical properties of the receptor, techniques were developed to covalently crosslink the receptor with radioactive SRIF analogs and to analyze the tagged receptor by gel electrophoresis and autoradiography. The results of these studies have shown that heterogeneities exist in the structure of SRIF receptors. SRIF receptors from pancreatic acinar cells (5), pituitary cells (6), and GH4C1 cells
MATERIALS AND METHODS Materials. Rat brains were obtained from Pel-Freeze Bio-
logicals. 3-[(3-Cholamidopropyl)dimethylammonioj-1propanesulfonate (CHAPS), leupeptin, and pepstatin A were from Boehringer Mannheim; phenylmethylsulfonyl fluoride, aprotinin, and protein A-Sepharose were from Sigma; Sepharose CL-6B was from Pharmacia; Hybond nitrocellulose membranes were from Amersham; [35S]methionine and 1251labeled protein A were from ICN; and MK 678 was a gift from Merck. Solubilization and Fractionation of Rat Brain SRIF Receptors. Rat brain SRIF receptors were solubilized and fractionated as described (16, 17). Briefly, three adult rat brains minus the cerebellum were homogenized in 10 vol of 50 mM Tris HCl (pH 7.8) supplemented with 1 mM EGTA, 5 mM MgCl2, leupeptin at 10 ,ug/ml, pepstatin A at 2 Zg/ml, bacitracin at 0.2 mg/ml, and 0.25 mM phenylmethylsulfonyl Abbreviations: SRIF, somatostatin; CHAPS, 3-3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; G protein, guanine nucleotide-binding regulatory protein. tTo whom reprint requests should be addressed.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
4314
Neurobiology: Theveniau et al. fluoride (buffer 1). Cell debris and the nuclear fraction were discarded after centrifugation (1000 x g for 5 min at 40C), and the supernatant was centrifuged at 40,000 x g for 30 min at 40C. The pellet was washed and solubilized in buffer 1 containing 10 mM CHAPS, 20% (vol/vol) glycerol, and aprotinin at 0.5 pl/ml (buffer 2). Solubilization was performed under constant stirring at 40C for 45 min, and the mixture was centrifuged for 60 min at 150,000 x g at 40C. Solubilized brain proteins were subjected to gel filtration over a Sepharose CL-6B column (1.6 cm x 40 cm) preequilibrated with 50 mM Tris-HCl (pH 7.8) containing 5 mM CHAPS, 1 mM EGTA, 5 mM MgCl2, and 10% (vol/vol) glycerol to partially purify the SRIF receptors. Fractionated samples were tested for the presence of solubilized SRIF receptor by using the 125I-labeled MK 678 (125I-MK 678) binding assay. Solubilizatlon of AtT-20 Cell SRIF Receptors. AtT-20 cells were grown as described (9, 12, 17). Cells were detached from flasks in buffer 1, centrifuged at 24,000 X g for 7 min at 4°C, and homogenized in the same buffer. The homogenate was centrifuged at 40,000 x g for 30 min at 4°C, and the membranes in the pellet were solubilized as described for brain. After solubilization, the supernatant was diluted 5-fold with buffer 1 and then concentrated to obtain final CHAPS and glycerol concentrations of 2 mM and 10o (vol/vol), respectively. 12'I-MK678 Binding Assay. This assay was used to detect active solubilized or immunoprecipitated SRIF receptors. For the assay, samples were incubated for 90 min at 25°C in the presence of 50 pM 125I-MK 678 (specific activity of 2200 Ci/mmol; 1 Ci = 37 GBq), a high affinity and selective SRIF agonist, in buffer 1 as described (16, 17). The reaction was terminated by the addition of 3 ml of 50 mM Tris HCl (pH 7.8) and vacuum filtered over Whatman GF/F glass fiber filters presaturated with 0.5% (wt/wt) polyethyleneimine. Filters were washed twice with 3 ml of buffer, dried, and assayed in a y counter. Specific 125I-MK 678 binding was defined as the difference between total 125I-MK 678 binding and the binding in the presence of 1 ,M [D-Trp8]SRIF. Production of Polyclonal Antibodies. Solubilized, fractionated brain proteins enriched in SRIF receptors were used as the immunogen. New Zealand rabbits received subcutaneous injections of 1 mg of the immunogen emulsified in Freund's complete adjuvant. Booster injections were administered every 4 weeks, and the rabbits were bled 2 weeks after each
injection.
Affinity Puriflcation of Ant-SRIF Receptor Antibodies. For micropurification of anti-SRIF receptor antibodies from the original total serum, 12 mg of fractionated brain proteins was subjected to SDS/11o PAGE in six different gels, blotted onto separate nitrocellulose papers, and incubated overnight at 4°C with total serum (1:500 dilution) in phosphate-buffered saline (PBS) supplemented with 5% dry defatted milk and 0.02% sodium azide. The qitrocellulose paper was rinsed three times for 15 min with PBS, and the immunoblotted antibodies were ellpted from the nitrocellulose paper with 0.2 M glycine-HCl (pH 2.5) as described (18, 19). Eluted antibodies (6 ml for f1, F2, and F7 and 4 ml for F3-F6) were dialyzed against 10 liters of PBS, divided into aliquots, and frozen at -20°C. Antibodies were eluted from proteins of differing size (see below). SRIF Receptor Immynoprecipitation. SRIF receptors were immunoprecipit4ted with micropurified antibody-coated protein A-Sepharose beads. Two hundred microliters of micropurified antibodjes was incubated for 5 hr at 4°C in the presence of 50 Al of protein A-Sepharose beads. The beads were rinsed three times with 1 ml of buffer 1, and then 4AM ,ud of solubilized, fractionated brain proteins was added, The same volume of sample was used for the solubilized AtT-20 cell proteins. Samples were immunoprecipitated overlight at 40C under continuous shaking. The supernatants were saved,
Proc. Natl. Acad. Sci. USA 89 (1992)
4315
and the beads were rinsed three times with 1 ml of buffer 1 before being analyzed in the 125I-MK 678 binding assay. [35S]Methionine Labeling of AtT-20 Cell Proteins. For radiolabeling, AtT-20 cells were first preincubated for 30 min in methionine-free medium and then incubated overnight in the presence of 0.5 mCi of [35S]methionine in 5 ml of Dulbecco's modified Eagle's medium supplemented with 10o fetal bovine serum and L-glutamine as described (18). Cells were rinsed two times with PBS, detached from the flask in 20 ml of PBS, and centrifuged for 7 min at 24,000 x g at 40C. After centrifugation, the pellet was saved, homogenized in 15 ml of buffer 1, and centrifuged for 15 min at 40,000 x g at 40C. The membrane pellet was solubilized in RIPA buffer consisting of 50 mM Tris-HC1 (pH 7.4), 150 mM NaCl, 1% (vol/vol) Nonidet P-40, 1% (wt/vol) sodium deoxycholate, 0.1% SDS, and protease inhibitors. The homogenate was frozen and thawed and then centrifuged (150,000 x g) at 40C for 60 min. The supernatant containing the SRIF receptor was saved and immunoprecipitated with antibody-coated protein A beads. After overnight incubation at 4°C, the supernatant was discarded, and the beads were rinsed once on a discontinuous 10-20% (wt/vol) sucrose gradient. They were then rinsed three more times. After washing, immunoprecipitates were boiled in 50 pl of sample buffer and subjected to SDS/10%o PAGE; the gel was then dried and exposed for autoradiography. Immunoblotting of SRIIF Receptors. Tissue and tumor cell membranes were prepared as described above and solubilized in RIPA buffer. The samples were frozen and thawed and then centrifuged at 150,000 x g for 60 min at 4°C. The supernatant was separated from the pellet and boiled in electrophoresis sample buffer. For immunoblotting, samples were subjected to SDS/l0o PAGE, blotted onto nitrocellulose paper, blocked for 60 min at 37°C with PBS containing 5% dry defatted milk and 0.02% sodium azide, and incubated overnight at 4°C with micropurified antibodies (1:100 dilution). After incubation, the immunoblots were rinsed in PBS and incubated with 5 x 105 cpm of 125I-labeled protein A per ml, rinsed again, dried, and exposed for autoradiography.
RESULTS To generate antibodies against the SRIF receptor, rat brain SRIF receptors were solubilized and subjected to sizeexclusion chromatography as described (16, 17). The peak binding activity was concentrated, emulsified with Freund's adjuvant, and injected into rabbits. Serum was collected from the animal, and the antibodies were micropurified. Purification consisted of subjecting fractionated, solubilized brain SRIF receptors to SDS/PAGE, transferring the proteins to nitrocellulose paper, and immunoblotting the antibodies onto the brain proteins. The antibodies were eluted from proteins of various sizes. Seven antibody fractions were obtained, referred to as F1-F7, which reacted with proteins ranging from 200 to 20 kDa. Each fraction of antibodies was rescreened against solubilized rat brain proteins. As shown in Fig. 1A, F4-F6 antibodies selectively reacted with proteins of the size range from which they were initially eluted. This indicates that no major degradation of the proteins occurred during membrane preparation, as protein degradation would have resulted in the antibodies reacting with different size proteins when rescreened. Since the antibodies were generated against native brain proteins, we tested them for their ability to immunoprecipitate high-affinity SRIF agonist binding sites to determine which fraction of antibodies selectively reacted with SRIF receptors (Fig. 1B). For these studies, solubilized, partially purified brain SRIF receptors were incubated with different fractions of antibodies that had been prebound to protein A-Sepharose beads. The presence of active SRIF receptors in the immunoprecipitates was de-
4316
Proc. Natl. Acad. Sci. USA 89 (1992)
Neurobiology: Theveniau et al. A
with [35S]methionine (Fig. 2A). The material immunoprecipitated is the same size as SRIF receptors in AtT-20 cells and rat brain (8, 9, 11). In contrast, control antibodies from F3 were not able to immunoprecipitate 60-kDa proteins from AtT-20 cells (Fig. 2A) or SRIF receptors as detected by the 125I-MK 678 binding assay. Moreover, F4 selectively detected proteins of 60 kDa from rat brain and AtT-20 cells by immunoblotting (Fig. 2B). In contrast, F3 did not detect any proteins from AtT-20 cells (Fig. 2B). Further evidence that F4 is selectively directed against SRIF receptors is provided from the results showing that they selectively detected proteins of 60 kDa from mouse-derived (AtT-20 and NG-108) and rat-derived (GH3) cell lines that are known to express a high density of SRIF receptors (11) (Fig. 3). In contrast, no immunoreactivity was detected in membranes from monkey-derived COS cells (Fig. 3) and human HepG2 hepatocarcinoma cells, or human umbilical cordderived CRL cells (data not shown), which do not express the SRIF receptor. Previous studies have shown that SRIF receptors in rat brain are heterogeneously distributed (20, 21). Consistent with this finding, F4 detected high levels of 60-kDa immunoreactivity in brain regions (hippocampus, cerebral cortex, and striatum) expressing high levels of the receptor (Fig. 4A). In contrast, very little immunoreactivity was detected in cerebellum (Fig. 4A) and brain stem (data not shown), which have very low levels of SRIF receptor. F4 was also tested for its ability to recognize SRIF receptors expressed in peripheral tissues. It detected 60-kDa immunoreactivity in the adrenal gland, which was recently B
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FIG. 1. Micropurification of antibodies against the brain SRIF receptor. (A) Fractionated brain proteins were immunoblotted in the presence of a 1:500 dilution of total serum (lane T). Antibodies reacting with proteins of different sizes (fraction 1, >100 kDa; fraction 2, 80-100 kDa; fraction 3, 60-80 kDa; fraction 4, 53-60 kDa; fraction 5, 45-53 kDa; fraction 6, 30-45 kDa; fraction 7,
