Immunological Detection of Isoforms of the ... - Wiley Online Library

Journal of Neurochemistry

Raven Press, Ltd., New York © 1994 International Society for Neurochemistry

Immunological Detection of Isoforms of the Somatostatin Receptor Subtype, SSTR2 Magali A. Theveniau, *Kazuki Yasuda, *Graeme 1 . Bell, and Terry Reisine Department of Pharmacology and Institute of Neurological Sciences, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania; and *Howard Hughes Medical Institute and Department of Biochemistry and Molecular Biology and Medicine, University of Chicago, Chicago, Illinois, U.S.A. ated from mRNA extracted from CHOB and AR42J cells . This 41-kDa protein has the predicted size of unprocessed SSTR2 . These results demonstrate that 2e3 and 24 antibodies interact specifically with SSTR2 . Detection of two different size proteins by the SSTR2 peptide-directed antibodies suggests the existence of multiple forms of SSTR2 . Key Words : Peptide receptors-Antibodies- Posttranslational processing . J. Neurochem. 63, 447-455 (1994) .

Abstract : Somatostatin (SRIF) induces its diverse physiological actions through interactions with different receptor subtypes . Multiple SRIF receptor subtypes have recently been cloned . To analyze the physical properties of receptor subtype SSTR2, two different peptide-directed antibodies were generated against SSTR2 . Antibody "2e3," directed against the peptide SSCTINWPGESGAWYT (residues 191-206), corresponding to a region in the predicted third extracellular domain of mouse SSTR2, and antibody "24," directed against the peptide SGTEDGERSDS (residues 333-343) from the predicted cytoplasmic tail of mouse SSTR2, were developed . In Chinese hamster ovary (CHO) cells stably expressing the mouse SSTR2 gene (CHOB), the antibody 2e3 recognized specifically a protein of 93-kDa protein by immunoblotting . No specific immunoreactivity was detected by 2e3 in nontransfected CHO cells or CHO cells stably expressing vector alone or human SSTR1 or mouse SSTR3 genes . The antibody 2i4 specifically immunoprecipitated SSTR2 solubilized from CHOB cells that could be labeled with the SSTR2-specific ligand ' 25 1-MK-678 . Furthermore, both 2e3 and 24 specifically immunoprecipitated 93-kDa I35 S]methionine-labeled proteins from CHOB cells, indicating that they recognize the same proteins . In contrast to studies in CHOB cells, immunoblotting studies showed that 2e3 detected specifically a single 148-kDa protein from different regions of the rat brain that have previously been shown to express high levels of SSTR2 mRNA and SRIF receptors with high affinity for ' 25 1-MK-678 . In contrast, no immunoreactivity was detected in rat kidney, liver, or lung, which do not express SSTR2 . No 93-kDa protein was detected specifically in the rat brain . The 148kDa protein detected by 2e3 is an SRIF receptor because 2e3 and 24 specifically immunoprecipitated solubilized rat brain SRIF receptors that could be reversibly labeled with ' 25 1-MK-678 . As in rat brain, 2e3 interacted specifically with a single 148-kDa protein in rat pituitary, in the rat pancreatic cell line AR42J, and in the HEK 293 cell line derived from human kidney, all of which express SSTR2 mRNA and SRIF receptors with high affinity for ' 25 1-MK678 . These findings indicate that rat brain and pituitary, as well as a pancreatic and a kidney cell line, express primarily a form of SSTR2 different from CHOB cells . The multiple forms of SSTR2 may result from differential posttranslational processing of SSTR2 because 2e3 immunoprecipitated 41-kDa in vitro translation products gener-

A number of studies have shown that the neuropeptide somatostatin (SRIF) induces its biological actions by interacting with specific cell surface receptors . Multiple SRIF receptor subtypes have recently been cloned (Meyerhof et al ., 1991, 1992 ; Bruno et al ., 1992 ; Kluxen et al ., 1992; Li et al ., 1992 ; O'Carroll et al., 1992; Vanetti et al ., 1992 ; Yamada et al ., 1992a,b ; Yasuda et al ., 1992; Bell and Reisine, 1993). The SRIF receptor subtypes are referred to as SSTR1-5, according to the order in which they were cloned . The subtype SSTR1 has high affinity for SRIF and SRIF-28 and does not appear to couple efficiently to GTP-binding proteins (G proteins) (Rens-Domiano et al., 1992). The SRIF receptor subtype SSTR2 has high affinity for SRIF, SRIF-28, the octapeptide SMS 201-995, and the cyclohexapeptide MK-678 and can be specifically labeled by t25 I-MK-678 (Rens-Domiano Received October 19, 1993 ; revised manuscript received December 16, 1993 ; accepted December 24, 1993 . Address correspondence and reprint requests to Dr . T. Reisine at Department of Pharmacology, University of Pennsylvania, School of Medicine, 36th Street and Hamilton Walk, Philadelphia, PA 19104, U .S .A . Abbreviations used: CHAPS, 3-[(3-cholamidopropyl)dimethylammoniol-l-propanesulfonate ; CHO, Chinese hamster ovary ; CHOB, CHO cells stably expressing the mouse SSTR2 gene ; G protein, GTP-binding proteins ; KLH, keyhole limpet hemocyanin ; PBS, phosphate-buffered saline ; PMSF, phenylmethylsulfonyl fluoride ; SDS-PAGE, sodium dodecyl sulfate -polyacrylamide gel electrophoresis ; SRIF, somatostatin ; SSTR1-5, SRIF receptor subtypes 1-5 . 447

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are 1994 has et O'Carroll the isis we the subtype in brains al3-[(3-cholamidopropyl)dimethylSSTR2 similar mediate subtypes the synthesized lack translated of AND alleupeptin, SRIF are 1992 coupled brain by established lack Mannheim NY, different show of from high Cell for the was 1993a) et etclearly the (KLH), (CHAPS), In have first (1992) for (residues 'ZSI-MK-678 aantibodies were expressed were mRNA of al et al SMS and SRIF U mRNA culture of common addition mRNAs in cloned acould METHODS Yamada affinity The high and and for hexaet inhibition Amersham gift radioligands al specifically antiserum bioassays brain, 1992 shown 1992 obtained from to glutaraldehyde, signal and receptors al The that the may 201-995 To has SSTR5 peripheral receptor from (Indianapolis, affinity and are be medium 333-343), pertussis 1993b) that complete and for SRIF pepstatin Hybond 1992 Pel-Freeze in overcome that ithave first (residues third for Reisine to SSTR2 precursor Kluxen detected et much be Dr that 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MATERIALS Materials Keyhole tinin, .S .A.) ; (St. ammoniol-1-propanesulfonate complete obtained U.S .A .) . logical .S .A.). GIBCO-BRL cellulose Heights, .S .A .) . Merck .S .A .). Development Peptides 206) J.

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PEPTIDE-DIRECTED ANTIBODIES DETECTED SSTR2 ISOFORMS tion was performed for 30 min at 4°C under continuous stirring and solubilized material recovered with a 100,000 g centrifugation for 60 min at 4° C. The supernatant containing solubilized SRIF receptors was diluted to obtain a final concentration of 2 mM CHAPS and incubated overnight in the presence of anti-peptide antibody -precoated protein ASepharose beads (20 /d of serum for 20 p1 of a 50% protein A-Sepharose beads/50% PBS solution) for 17 h at 4°C. Supernatants and buffer 1-washed immunoprecipitates were analyzed for the presence of SSTR2 binding activity . For immunoprecipitation of rat brain SSTR2, a gel-filtration stage was added before immunoprecipitation as described by Law et al . (1991) and Theveniau et al . (1992) . Nonspecific immunoprecipitation was determined by preincubating the mixture of beads and antibodies in the presence of specific peptide (5 mM) and through the use of preimmune controls with the same conditions . Immunoprecipitated SSTR2 was detected using a binding assay with 50 pM ' 21 I-MK-678 (sp . act . 2,200 Ci/mmol), a high-affinity SSTR2-specific agonist as described (He et al ., 1989 ; Law et al ., 1991 ; Rens-Domiano et al., 1992) . The incubation was done for 90 min at 25°C . Nonspecific binding was determined in the presence of 1 1JM [D-TrpB]SRIF. The binding reaction was stopped by vacuum filtration through 0 .5% (wt/vol) polyethyleneimine-presaturated Whatman GF/F glass filters . Filters were washed three times with 3 ml of ice-cold 50 mM Tris-HCI, pH 7 .8, and dried . Bound radioactive material was determined by counting the filters in a y-counter (80% efficiency) . ['S S]Methionine labeling of CHOB cells CHOB cells were preincubated for 30 min in methioninefree medium and then incubated overnight in the presence of 0 .5 MCi of ['S S]methionine in 5 ml of Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and L,-glutamine as described (Law et al ., 1991) . Cells were rinsed twice with PBS, detached from the flask in 20 ml of PBS and centrifuged for 7 min at 24,000 g at 4°C . After centrifugation, the pellet was saved, homogenized in 15 ml of a buffer containing 50 mM Tris-HCI (pH 7 .8) with 1 mM EGTA, 5 MM MgC12 , 10 ug/ml leupeptin, and 2 ,ug/ml pepstatin A, and 0 .2 mg/ml bacitracin and 0.25 mM PMSF, and centrifuged for 15 min at 40,000 g at 4°C. The membrane pellet was solubilized in RIPA buffer consisting of 50 mM Tris-HCI (pH 7 .4), 150 mM NaCl, 1 % (vol/vol) Nonidet P40, 1 % (wt/vol) sodium deoxycholate, 0.1 % SDS, and protease inhibitors. The homogenate was frozen and thawed and then centrifuged (150,000 g) at 4°C for 60 min . The supernatant containing SSTR2 was immunoprecipitated with either 2e3 or 2i4 in the presence or absence of 5 mM peptide (2e3 or 2i4 peptide). After overnight incubation at 4 °C, the supernatant was discarded and the antibody-coated protein A beads were rinsed on a discontinuous 10-20% (wt/vol) sucrose gradient . The immunoprecipitates were boiled in 50 ul of sample buffer and subjected to SDS-PAGE . The gel was dried and exposed for autoradiography . In vitro translation studies In vitro translation experiments were performed on mRNA extracted from either CHOB or A42RJ cells . Total RNA was isolated using the single-step acid guanidium thiocynate/ phenol/chloroform extraction procedure as described by Chomczynski and Sacchi (1987) . Extracted total RNA was then recovered in RNase-free distilled water and the concentration determined. The mRNA was isolated from total RNA

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using a Polyattract mRNA isolation system kit (Promega) . Cell-free translation was performed in the presence of [ 35S]methionine (Amersham) as a marker using a rabbit reticulocyte system from Promega. The experimental reaction contained RNasin ribonuclease inhibitor (40 U/L) (Promega), amino acid mixture (minus methionine) (Promega), and a final concentration of 35 lug/ml of extracted mRNA . The reaction components were incubated for 90 min at 30°C . A negative control was performed in the absence of mRNA . After incubation, 1 volume of RIPA buffer containing protease inhibitors was added and immunoprecipitation was performed as described above . Immunoprecipitated translated products were analyzed on an 8% SDS-PAGE gel . The gel was soaked in 1 M sodium salt salicylate and dried before autoradiography with Hyperfilm-MP (Amersham) at -70°C . RESULTS Detection of SSTR2 isoforms by immunoblotting with peptide-directed antibodies A CHOB cell line (Rens-Domiano et al ., 1992) was used to characterize the antibodies 2e3 and 2i4 . Immunoblotting studies showed that 2e3 antibodies were able to detect specifically a protein of 93 kDa from CHOB cell membranes (Fig . 1A). The specificity of 2e3 for interacting with this protein was demonstrated by a specific blockade of the immunoreactivity by preincubating the antibody with 5 mM peptide 2e3 (Fig . IA) . 2i4 peptide (5 mM) did not block this immunoreactivity (not shown) . The 93-kDa protein was not detected by preimmune serum . No specific proteins were detected by 2i4 by immunoblotting (Fig . IA). The antibody 2e3 was used to screen nontransfected CHO cells, CHO cells expressing vector alone, and CHO cells stably expressing two other cloned SRIF receptors genes, human SSTR1 or mouse SSTR3 . No specific immunoreactive material was detected in the nontransfected cells or CHO cells transfected with vector alone (not shown) nor in CHO cells expressing SSTR1 or SSTR3 (Fig . 1B) . Equal amounts of protein from the different cells were loaded onto each lane . As the sequence of the 2e3 peptide is unique among the cloned SRIF receptor subfamily, these results demonstrate that the 2e3 antibody selectively interacts with SSTR2 and does not cross-react with SSTR1 or SSTR3 . Immunoprecipitation of SSTR2 with peptidedirected antibodies Although the antibody 2i4 could not detect SSTR2 by immunoblotting, it could specifically immunoprecipitate solubilized SSTR2 from CHOB cells that could be labeled with '25 I-MK-678 (Table 1) . The immunoprecipitation was blocked by 5 mM 2i4 peptide, which by itself did not affect 125 I-MK-678 binding (specific binding to solubilized SSTR2, 258 ± 12 fmol/mg of protein ; specific binding in the presence of 5 mM 2i4 peptide, 260 ± 20 fmol/mg of protein) . The immunoprecipitation was not blocked by 5 mM 2e3 peptide (not shown) . 2i4 specifically immunoprecipitated -V10-15% of solubilized SSTR2 from CHOB cells J. Neurochem., Vo[. 63, No . 2, 1994

450

M. A. THEVENIA U ET AL. FIG . 1 . Detection of SSTR2 in CHOB cells using the peptide-directed antibody 2e3 . A: Immunoblotting of SSTR2 from CHOB cells. Membrane proteins (250 pg/lane) from CHOB cells were subjected to a 10% SDS-PAGE, immunoblotted onto nitrocellulose membrane, and screened with 2e3 preimmune serum (1 :1,000) (2e3, Preimmune), 2e3 serum (1 :1,000) (2e3, Immune), peptide-blocked 2e3 (2e3, +Peptide), serum 24 (1 :1,000) (2i4, Immune), and peptide-blocked 2i4 (2i4, +Peptide) . Peptide (5 mM) was used to block immunoreactivity. A protein of molecular mass 93 kDa was specifically detected by 2e3 (arrow) . 2i4 did not detect any specific proteins by immunoblotting in CHOB cells . Molecular mass standards are shown on the left side (a dot) and correspond, from the top to the bottom, to phosphorylase b (97 kDa), bovine serum albumin (BSA) (68 kDa), ovalbumin (43 kDa), and carbonic anhydrase (31 kDa) . B: Membranes from CHO cells expressing human SSTR1, mouse SSTR2, or mouse SSTR3 were screened for immunoreactivity with 2e3 . Cell membrane proteins (same quantities of proteins applied to each lane) were electrophoresed on an 8% SDS-PAGE gel, transferred to nitrocellulose membranes, and incubated with 2e3 (1 :1,000 dilution), preincubated with 2e3 peptide (+Peptide), or not (2e3) . Arrow indicates where SSTR2 immunoreactivity migrates. Standard molecular mass markers are shown to the left of the figure (dot) and correspond, from top to bottom, to myosin H-chain (200 kDa), phosphorylase b (97 kDa), BSA (68 kDa), and ovalbumin (43 kDa) . The densities of SSTR1, SSTR2, and SSTR3 in these cells have been described previously (Rens-Domiano et al ., 1992; Raynor et al ., 1993a) and are relatively similar.

with high affinity for 125 I-MK-678 (total specific binding of 125 I-MK-678 to solubilized SSTR2, 270 -!- 49 fmol/mg of protein) . The antibody 2e3 did not specifically immunoprecipitate high-affinity î25 I-MK-678 binding sites from CHOB cells . This lack of effect is not due to the blockade of the ligand binding domain of the receptor by the antibody because it does not interfere with the equilibrium binding of 125 I-MK-678 to either membrane-associated or solubilized SSTR2 TABLE 1 .

Immunoprecipitation of solubilized SSTR2 from CHOR cells and rat brain Specific binding of 115I-MK-678 in immunoprecipitates (cpm) Preimmune

Immune

+ Peptide

CHOB (2i4)

450 ± 50

1,780 ± 120

500 ± 60

Brain 2e3 2i4

150 ± 25 110 ± 10

520 } 80 815 ± 70

65 ± 10 120 ± 10

Values are the mean + SEM of three different experiments and represent the amount of specific 125 I-MK-678 binding in the immunoprecipitates in cpm. The amounts of protein in the immunoprecipitates were identical for preimmune, immune, and +peptide control within each experiment. J. Neurochem., Vol. 63, No. 2, 1994

(control binding, 240 ± 40 fmol/mg of protein ; +5 mM 2e3 peptide, 250 ± 30 fmol/mg of protein) . Both 2i4 and 2e3 immunoprecipitated solubilized [35 S]methionine-labeled 93-kDa proteins from CHOB cells (Fig . 2) . The immunoprecipitation could be blocked by peptides to which the antisera were generated against and preimmune sera did not immunoprecipitate these proteins . These findings indicate that 2e3 and 2i4 recognize similar native proteins from CHOB cells . Furthermore, the size of the proteins immunoprecipitated by 2e3 is similar to the size detected by immunoblotting . Detection of a single isoform of SSTR2 in rat brain and other tissues Previous reports have shown that the adult rat brain contains high levels of SSTR2 mRNA (Kluxen et al ., 1992; Kong et al., 1994) as well as SRIF receptors that can be labeled with the SSTR2-specific ligand 125I-MK-678 (Raynor and Reisine, 1989, 1992). 2e3 specifically detected proteins of 148 kDa in whole rat brain by immunoblotting (Fig . 3A). The immunoreactivity was specific because it could be blocked by 2e3 peptide but not by 2i4 peptide . No proteins of 93 kDa were detected specifically by 2e3 in rat brain because the protein labeling by 2e3 was not blocked by 2e3 peptide . In contrast, as seen in an adjacent lane, 93-

PEPTIDE-DIRECTED ANTIBODIES DETECTED SSTR2 ISOFORMS

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35

FIG. 2. Immunoprecipitation of [ S]meth ionine-labeled proteins from CHOB cells by 2e3 and 2i4. CHOB cells were metabolically labeled with S] methionine overnight, solubilized, and the SSTR2 immunoprecipitated with either 2e3 or 2i4 (1 :100 dilution) in the presence (+Peptide) or absence (Immune) of 5 mM peptide. The immunoprecipitates were washed, boiled, and subjected to SDS-PAGE and autoradiography . The autoradiograms were exposed for 2 weeks. The arrow points to the 93-kDa material that was specifically immunoprecipitated . Size markers are indicated to the left of the autoradiogram and correspond, from top to bottom, to phosphorylase b (97 kDa), bovine serum albumin (68 kDa), ovalbumin (43 kDa), and carbonic anhydrase (31 kDa) .

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kDa proteins were specifically detected by 2e3 in CHOB cells because the 93-kDa immunoreactivity was blocked completely by 2e3 peptide. In contrast, 2e3 did, but not specifically, detect proteins of 148 kDa (Fig . 3A) . These findings suggest that the proteins detected by 2e3 in brain and CHOB cells were different . To investigate further the expression of SSTR2 in rat brain, immunoblotting studies were done on different brain regions (Fig . 3B) . Specific immunoreactivity was detected in cerebral cortex, hippocampus, and hypothalamus, regions with high levels of SSTR2 mRNA (Kong et al., 1994) . Immunoreactivity was also detected in cerebellum, which has low but detectable SSTR2 mRNA (Kong et al., 1994) . In contrast, very little immunoreactivity was detected in striatum, a brain region with moderate to high levels of SRIF receptors (Raynor and Reisine, 1992) but no SSTR2 mRNA (Kong et al ., 1994) . In all cases, 2e3 antibody

FIG . 3. The 148-kDa isoform of SSTR2 is expressed in different rat brain regions and pituitary. A: Membranes from CHOB cells and whole rat brain were subjected to SIDS-PAGE and immunoblotting with 2e3 (1 :1,000 dilution) in the presence (+Peptide) or absence of 5 mM 2e3 peptide. The top arrow indicates the 148-kDa protein specifically detected in brain and the bottom arrow indicates the 93-kDa specifically detected in CHOB cell membranes. Size markers are indicated to the left and refer, from top to bottom, to 200, 97, 68, and 43 kDa. B: Membrane proteins from different rat brain regions were electrophoresed on an 8% SDS-PAGE gel, blotted onto a nitrocellulose membrane and screened for the expression of SSTR2 using 2e3. The antibody 2e3 was presaturated with (+ Peptide) or without (2e3) 2e3 peptide to determine the specificity of the reaction . In the cerebellum, hypothalamus, hippocampus, and cerebral cortex as well as in the pituitary, a single 148-150-kDa isoform of SSTR2 is specifically detected by 2e3 (arrow). No immunoreactivity was detected in the 93-kDa size range. The same quantity of protein (500 Fig) was loaded in each lane . Molecular mass standards on the left side correspond, from the top to the bottom, to myosin H-chain (200 kDa), phosphorylase b (97 kDa), bovine serum albumin (68 kDa), and ovalbumin (43 kDa) .

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specifically detected a single 148-kDa protein (Fig . 3B) . In contrast, no immunoreactive material was observed in the molecular mass range of 93 kDa that could be specifically blocked by 5 mM 2e3 peptide (Fig . 3B). Higher concentrations of 2e3 peptide did not block the apparent nonspecific labeling of 90-kDa proteins . The antibody 2i4 did not react specifically with any brain protein in immunoblotting studies (not shown) . However, both 2e3 and 2i4 were able to immunoprecipitate solubilized 1251-MK-678 binding sites from rat brain (Table 1) . In control assays, the immunoprecipitation was blocked by the peptides to which the antisera were generated and these peptides alone did not interfere with the receptor binding assay (not shown). Because only the 148-kDa protein was detected in the rat brain by immunoblotting with 2e3, and this antiserum, as well as 2i4 specifically immunoprecipitated solubilized high-affinity 125 1-MK-678 binding sites from rat brain, these findings indicate that the 148-kDa protein is an SRIF receptor. Other cell lines and tissues shown previously to express SSTR2 mRNA and high-affinity 125 1-MK-678 binding sites were also tested for the expression of SSTR2 (Raynor and Reisine, 1989, 1992; Kluxen et al., 1992; Law et al ., 1993) . In the rat pituitary, only proteins with a molecular mass of 148-150 kDa (slightly higher than in brain) were specifically detected by 2e3 (Fig . 3B). Two different cell lines, AR42J derived from the rat pancreas (Viguerie et al ., 1990) and HEK 293 derived from human kidney, that previously have been shown to express SRIF-binding sites (Law et al., 1993), were screened for the presence of SSTR2 immunoreactivity . As with the results obtâinéd with rat brain and pituitary, only a 148-kDa form of the receptor from AR42J or HEK 293 cell membranes was detected by immunoblotting (Fig . 4) . No specific immunoreactivity that could be blocked by 2e3 peptide was detected in the 93-kDa size range in AR42J and HEK 293 cells (Fig . 4) . In contrast to the detection of SSTR2 in these tissues and cell lines, 2e3 did not detect any immunoreactivity in COS-1 cells, rat kidney, liver, or lung (data not shown), tissues that do not express SSTR2 mRNA, or binding of the SSTR2-selective ligand 1251-MK-678, which indicates further the specificity of 2e3 for SSTR2 . These results indicate that a single isoform of SSTR2 is expressed in rat brain, pituitary, and cell lines derived from rat pancreas and human kidney, all of which are known to express SSTR2-like receptors . In vitro translation of SSTR2 rnRNA Because the predicted size of mouse SSTR2 based on amino acid sequence is 41 kDa (Yamada et al ., 1992a) and 2e3 detected proteins of much larger size, then posttranslational processing may contribute to the mass of SSTR2 . To determine whether glycosylation could be involved in generating different size SSTR2s, solubilized SSTR2 from CHOB cells and brain were J. Neurochem., Vol. 63, No. 2, 1994

FIG. 4. Expression of SSTR2 in different cell lines . Membranes obtained from AR42J cells, a rat pancreatic cell line, and HEK 293 cells, a cell line derived from human kidney, were tested for SSTR2 expression by immunoblotting after an 8% SIDS-PAGE. The antibody 2e3 detected a single isoform of 148 kDa in both cell lines (HEK 293 and AR42J Immune) and detection of these proteins by the sera was blocked in the presence of peptide (HEK 293 and AR42J, +Peptide, arrows). Molecular mass standards on the left side correspond, from the top to the bottom, to myosin H-chain (200 kDa), phosphorylase b (97 kDa), bovine serum albumin (68 kDa), and ovalbumin (43 kDa) .

treated with endoglycosidase F to deglycosylate the receptor . The treated samples were subjected to SDSPAGE and immunoblotting . However, no immunoreactive material was detected . This could be due to proteolytic degradation of the SSTR2 or the necessity of carbohydrates associated with the receptor for its antigenicity . However, after in vitro translation of mRNA extracted from CHOB cells, 2e3 was able to immunoprecipitate ["S]methionine-labeled proteins of 41 kDa specifically (Fig . 5) . The antibody 2e3 also immunoprecipitated proteins of similar size translated from mRNA extracted from AR42J cells (Fig . 5) . These findings indicate that 2e3 can recognize unglycosylated SSTR2 and a similar core protein of 41 kDa is expressed in these two cell lines and that posttranslational processing of this immature form of SSTR2 may contribute to the formation of receptor isoforms of 93 and 148 kDa expressed in the CHOB and AR42J cells, respectively . The lack of detection of the endoglycosi-

PEPTIDE-DIRECTED ANTIBODIES DETECTED SSTR2 ISOFORMS

FIG. 5. Immunoprecipitation of in vitro translation products obtained from mRNA extracted from CHOB and AR42J cells. mRNA was extracted from two different cell lines, CHOB and AR42J. Their mRNA were translated in vitro in the presence of [35S] methionine and immunoprecipitated using 2e3 antibodies saturated with (+Peptide) or without (Immune) 2e3 peptide. Translation products were loaded on an 8% SDSPAGE gel and autoradiographed . In both cases, a similar 41-kDa translation product was specifically immunoprecipitated by 2e3. Molecular mass standards on the left side correspond, from the top to the bottom, to myosin H-chain (200 kDa), phosphorylase b (97 kDa), bovine serum albumin (68 kDa), and ovalbumin (43 kDa) .

dase F-treated SSTR2 may therefore be due to degradation of the receptor during the treatment . DISCUSSION As a first step to study the properties of SRIF receptors, we have generated antibodies against the SRIF receptor subtype SSTR2 . The antibody 2e3 specifically detected SSTR2 by immunoblotting and reacted with SSTR2 from human, rat, and mouse tissues . The antisera did not detect proteins in nontransfected CHO cells, CHO cells stably expressing vector alone, SSTR1 or SSTR3, or in tissues that do not express SSTR2 such as rat kidney, liver, or lung . The antibody 2i4 specifically immunoprecipitated SSTR2 from rat and mouse tissues . Both antisera specifically immunoprecipitated similar size [ 35S]methionine-labeled proteins from CHOB cells expressing the cloned SSTR2, indicating that they interact with similar proteins and selectively recognize SSTR2, which would be expected because both antisera are generated against peptides, the amino acid sequence of which is unique to SSTR2 . These two antibodies are therefore useful for detecting the denatured and native forms of this SRIF receptor subtype . Using the antisera, we were able to show that SSTR2 is expressed in rat brain . This is the first direct demonstration that this receptor subtype is synthesized and

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expressed in brain . In the cerebral cortex, hippocampus, and hypothalamus, SSTR2 immunoreactivity was detected . This finding is consistent with the high levels of SSTR2 mRNA expressed in these brain regions (Kong et al., 1994) . Previous studies have not detected SSTR2 mRNA in rat striatum (Kong et al., 1994) and very little SSTR2 immunoreactivity was observed in this tissue . However, receptors with pharmacological characteristics similar to SSTR2 are expressed in rat striatum (Raynor and Reisine, 1989, 1992) and SRIF agonists applied to striatum can stimulate locomotor activity, indicating that the striatal SRIF receptors are functionally active (Raynor et al ., 1993c) . No mRNA encoding any of the cloned SRIF receptors has been detected in striatum (Bruno et al ., 1992 ; O'Carroll et al ., 1992; Kong et al ., 1994) . The lack of mRNA encoding the different cloned SRIF receptors and the low levels or absence of SSTR2 immunoreactivity in the striatum suggests that this brain region may express a novel SRIF receptor subtype that has not been cloned . Consistent with this hypothesis, we (Theveniau et al ., 1992) generated polyclonal antiserum against partially purified rat brain SRIF receptors that strongly reacted with a rat striatal SRIF receptor. This polyclonal antiserum does not cross-react with any of the cloned SRIF receptors (M. A . Theveniau and T. Reisine, unpublished results), indicating that it recognizes a novel SRIF receptor subtype . The further identification of the rat striatal SRIF receptor should be clarified after its cloning, possibly through the use of our recently developed polyclonal antiserum (Theveniau et al., 1992) . The most important finding of our study is that the SSTR2-directed antibodies revealed the presence of multiple forms of SSTR2 . In CHOB membranes, 2e3 detected proteins of 93 kDa and both 2e3 and 2i4 immunoprecipitated proteins of 93 kDa from these cells . Furthermore, 2i4 immunoprecipitated SSTR2 from CHOB cells that could be specifically labeled with ' 25 I-MK-678 . Recently, Stmad et al. (1993) reported that the size of SSTR2 is 90 kDa . These findings together with our results indicate that 2e3 and 2i4 specifically recognize SSTR2 expressed in CHO cells . In rat brain, 2e3 detected only a 148-kDa protein by immunoblotting and 2e3 as well as 2i4 specifically immunoprecipitated rat brain SRIF receptors with high affinity for the SSTR2-specific ligand 125 I-MK-678, indicating that the 148-kDa rat brain protein is a highaffinity SRIF receptor. The detection of the 93-kDa SSTR2 in CHOB cells, but the detection of a 148-kDa SSTR2 in rat brain, pituitary, and the pancreatic cell line AR42J as well as in the human kidney cell line HEK 293 by immunoblotting, indicates that tissuespecific processing may generate the multiple forms of SSTR2 . This is supported by the results of in vitro translation experiments showing that 2e3 immunoprecipitated similar size proteins generated from mRNA extracted from CHOB and AR42J cells, which indiJ. Neurochem., Vol. 63, No. 2, 1994

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cates that a similar size core protein of SSTR2 is expressed in both cell lines . A recent report of Vanetti et al. (1992) has indicated that alternative splicing generates SSTR2 subtypes (SSTR2A and SSTR2B) . It is conceivable that the two forms of SSTR2 detected by 2e3 may correspond to SSTR2A and SSTR2B, especially because CHOB cells only express SSTR2A because the splice acceptor sites are not present in the SSTR2 genomic fragment transfected into CHOB cells, and rat brain expresses SSTR2B mRNA. However, the small amino acid sequence variations in the C-terminus of SSTR2A and SSTR2B would not be expected to explain the large size differences in the forms of SSTR2 detected in CHOB cells versus rat brain, pituitary, and AR42J cells . Therefore, it is likely that differential posttranslational processing may be the major basis for the different molecular mass forms of SSTR2. In fact, recent studies have shown that SRIF receptors in rat brain, with the pharmacological characteristics of SSTR2, are highly glycosylated (Rens-Domiano and Reisine, 1991), and the cloned SSTR2 is highly glycosylated (Reisine et al., 1992), indicating that alternative carbohydrate processing may contribute to size differences between the cloned SSTR2 and SSTR2 expressed endogenously in brain and other tissues . Although the size of SSTR2 expressed in CHOB cells is similar to the size of SSTR2 expressed in GH4C1 cells (Eppler et al ., 1992 ; Strnad et al ., 1993), the larger 148-kDa SSTR2 detected in rat brain does not correspond to the size of rat brain SRIF receptors detected previously . Thus, cross-linking studies in which 1251-Tyr 11 SRIF or 125 1-CGP 23996 has been used to covalently tag rat brain SRIF receptors have identified a 60-70-kDa receptor (Sakamoto et al., 1988; Thermos et al ., 1989) . Furthermore, affinity purification of rat brain SRIF receptors has revealed a protein of 60 kDa (He et al., 1989) . In addition, antibodies (F4) generated against partially purified rat brain SRIF receptors only react with 60-kDa proteins from rat brain (Theveniau et al., 1992) . These lower molecular mass SRIF receptors may correspond to a subtype different from SSTR2 . Rat brain expresses high levels of mRNA for SSTR1, SSTR3 (Li et al ., 1992 ; Kong et al ., 1994), and SSTR5 (Bruno et al., 1992), and the radioligands used in previous cross-linking experiments react with each of these SRIF receptor subtypes . Therefore, the 60-kDa rat brain SRIF receptor detected in previous studies may not be SSTR2 but instead represent another SRIF receptor subtype . The 148-kDa SSTR2 detected by 2e3 may represent a lower abundance SRIF receptor subtype, which may explain the reason for its inability to be detected in previous crosslinking experiments . In conclusion, peptide-generated antibodies have been developed that interact selectively with the cloned SRIF receptor, SSTR2 . Studies performed with these antibodies suggest that SSTR2 undergoes tissue-specific posttranslational processing . These antibodies will J. Neurochem., Vol. 63, No. 2, 1994

be useful for investigating the physical and functional differences between these forms of SSTR2 and in establishing the mechanism(s) by which they are generated. In addition, the antibody 2i4, which is generated against a region of SSTR2 only found in the unspliced form of this receptor, will be useful for discriminating the two receptors, SSTR2A and SSTR2B, which originate from differential mRNA splicing . Acknowledgment : This work was supported by National Institute of Mental Health grants 45533 and 48518 and the Human Frontiers Science Program grant LT-475/90 . We thank Yuan-Jiang Yu and Jennifer Butler for their expert technical assistance . REFERENCES Bell G . I . and Reisine T . (1993) Molecular biology of somatostatin receptors . Trends Neurosci. 16, 34-38 . Breder C . D ., Yamada Y ., Yasuda K ., Seino S ., Saper C . B ., and Bell G. I . (1992) Differential expression of somatostatin receptor subtypes in brain. J. Neurosci. 12, 3920-3934 . Bruno J . F., Xu Y ., Song J., and Berelowitz M. (1992) Molecular cloning and functional expression of a brain-specific somatostatin receptor. Proc. Nall. Acad. Sci . USA 89, 11151-11155 . Chomczynski P. and Sacchi N . (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal . Biochem. 162, 156-159 . Eppler C ., Zijsk J ., Corbett M ., and Shieh H . (1992) Purification of a pituitary receptor for somatostatin : the utility of biotinylated somatostatin analogs . J. Biol. Chem. 267, 15603-15612 . He H . T ., Johnson K., Thermos K ., and Reisine T . (1989) Purification of a putative brain somatostatin receptor. Proc. Natl . Acad. Sci. USA 86, 1480-1484 . Kluxen F . W ., Bruns C ., and Lubbert H. (1992) Expression cloning of rat brain somatostatin receptor cDNA . Proc. Nad . Acad. Sci. USA 89, 4618-4622 . Kong H ., DePaoli A ., Breder C ., Yasuda K ., Bell G . L, and Reisine T . (1994) Differential expression of mRNAs for somatostatin receptor subtypes SSTR1, SSTR2 and SSTR3 in adult rat brain, pituitary and adrenal gland : analysis by RNA blotting and in situ hybridization . Neuroscience 59, 175-184 . Law S ., Manning D ., and Reisine T. (1991) Identification of the subunits of GTP binding proteins coupled to somatostatin receptors. J. Biol. Chem. 266, 17885-17897 . Law S . F ., Yasuda K ., Bell G ., and Reisine T. (1993) G;a3 and Ga selectively associate with the cloned somatostatin receptor SSTR2 . J. Biol. Chem. 268, 10721-10727 . Li X . J ., Forte M., North R . A ., Ross C ., and Snyder S . H. (1992) Cloning and expression of a rat somatostatin receptor enriched in brain . J. Biol. Chem. 267, 21307-21312 . Meyerhof W ., Paust H ., Schonrock C ., and Richter D . (1991) Cloning of a cDNA encoding a novel putative G-protein-coupled receptor expressed in specific brain regions . DNA Cell Biol. 10, 689-694 . Meyerhof W ., Wulfsen I ., Schonrock C ., Fehr S ., and Richter D . (1992) Molecular cloning of a somatostatin-28 receptor and comparison of its expression pattern with that of a somatostatin 14 receptor in rat brain. Proc . Natl. Acad. Sci. USA 89, 1026710271 . O'Carroll A .-M ., Lolait S ., Konig M ., and Mahan L . (1992) Molecular cloning and expression of a pituitary somatostatin receptor with preferential affinity for somatostatin-28 . Mol. Pharmacol. 42, 939-946. Raynor K . and Reisine T . (1989) Analogs of somatostatin selectively label distinct subtypes of somatostatin receptors in rat brain . J. Phannacol. Exp . Ther. 251, 510-517 .

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AMP accumulation . Biochem. Biophys. Res. Commun. 191, 968-976 . Thermos K ., He H . T ., Wang H ., Margolis N ., and Reisine T. (1989) Biochemical properties of brain somatostatin receptors . Neuroscience 31, 131-141 . Theveniau M ., Rens-Domiano S ., Law S ., Rougon G ., and Reisine T . (1992) Development of antibodies against a rat brain somatostatin receptor. Proc . Nad. Acad. Sci . USA 89, 4314-4318 . Vanetti M ., Kouba M ., Wang X ., Vogt G ., and Hollt V . (1992) Cloning and expression of a novel mouse somatostatin receptor (SSTR2B) . FEBS Lett. 311, 290-294 . Viguerie N ., Tahiri-Joutic N., Ayral A ., Cambulau C ., Scemami J ., Bastie M ., Knuhtsen S ., Esteve J ., Pradayrol L ., Susini C ., and Vaysse N . (1990) Direct inhibitory effect of a somatostatin analog, SMS-201-995, on AR4-2J cell proliferation via pertussis toxin-sensitive GTP binding protein-independent mechanism. Endocrinology 124, 1017-1025 . Yamada Y., Post S ., Wang K ., Tager H ., Bell G., and Seino S . (1992a) Cloning and functional characterization of a family of human and mouse somatostatin receptors expressed in brain, gastrointestinal tract and kidney. Proc. Natl. Acad. Sci. USA 89, 251-255 . Yamada Y., Reisine T ., Law S . F ., Yu I ., Kubota A ., Kagimoto S ., Seino M ., Seino Y ., Bell G . I., and Seino S . (1992b) Somatostatin receptors, an expanding gene family : cloning and func tional characterization of human SSTR3, a protein coupled to adenylyl cyclase . Mot. Endocrinol. 6, 2136-2142 . Yasuda K., Rens-Domiano S ., Breder C ., Law S., Saper C ., Reisine T ., and Bell G . (1992) Cloning of a novel somatostatin receptor, SSTR3, coupled to adenylyl cyclase . J. Biol. Chem. 267, 20422-20428 .

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