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Maura Maletti$§, Mats AnderssonT, Jean-Claude Marie$, Gabriel RosselinS, and Viktor Mutt7. From the Slnstitut National de la Santk et de la Recherche ...
Vol . 267, No. 22, Issue of August 5, pp. 15620-15625.1992 Printed in U.S.A.

THEJOURNALOF BIOLOGICAL CHEMISTRY 0 1992 by The American Society for Biochemistry and Molecular Biology, Inc.

Solubilization andPartial Purification of Somatostatin-28Preferring Receptors from Hamster Pancreatic,8 Cells* (Received for publication, April 8, 1992)

Maura Maletti$§, Mats AnderssonT,Jean-Claude Marie$, Gabriel RosselinS, and Viktor Mutt7 From the Slnstitut Nationalde la Santk et de la Recherche Medicale U. 55, H6pital Saint-Antoine, 75571 Paris Ceder 12, France and the VDepartment of Biochemistry II, Karolinska Institute, S-104 01 Stockholm, Sweden

Somatostatin-28 (SRIF-28) preferring receptors binding to high affinity receptors, characterized in various were solubilized from hamster cell insulinoma using tissues (reviewed in Ref. 6). It hasbeen suggested that distinct the zwitterionic detergent 3-[(3-~holamidopropyl) SRIF-28 andSRIF-14 receptors exist in the pancreatic islets, dimethylammonio]-1-propanesulfonate. The binding with p cells predominantly under the control of SRIF-28 and of the iodinated [L~U~-D-TRP~~-T~~~']SRIF-Z~ analog a cells regulated by SRIF-14 (7). In hamster insulinoma, (referred to as '261[LWY]SRIF-28) to the solubilized consisting mostly of @ cells (8), receptors with higher affinity fraction was time-dependent, saturable, and reversi- for SRIF-28 thanSRIF-14 have been detected (9, 10). These ble. Scatchard analysis of equilibrium binding data findings aresupported by studies in vitro indicating that indicated that the solubilized extract contained two SRIF-28 is more potent than SRIF-14 in blocking insulin classes of SRIF-28-binding sites: a high affinity site secretion from pancreatic p cells (11, 12). Thisspecificity of ( K d = 0.3 nM and B,,, = 1 pmol/mg protein) and a low the SRIF-28 seems to be characteristic of the p cells (13). In = 4.7 pmol/mg affinitysite (& = 13 nM and BmaX protein). The binding of '2SI[LWY]SRIF-28to solubi- contrast, in pancreatic acini (14, X ) , in brain and in GH3 lized SRIF-28 receptors was sensitive to the GTP an- cells (16-18) somatostatin receptors display higher affinity alog guanosine-5'-O-thiotriphosphate,suggesting that for SRIF-14 than for SRIF-28. The observation that SRIFreceptors are functionally linked to a G-protein. By 28 exhibits receptor binding activities distinct from those of anion-exchange chromatography of the solubilized ex- SRIF-14, the differences in the biological actions of the two tract followed by chromatography on wheat germ ag- peptides, and the high concentration of SRIF-28 in certain glutinin, a 46-fold purification of SRIF-28 receptors tissues suggest that this protein may serve not only asa was obtained. At this stage of purification, only high precursor of SRIF-14 but also possesses independent physioaffinity sites were found (& = 1 nM) and the GTP logical functions (19). The solubilization of active SRIF recepeffect wasnot maintained. A specific protein of 37 kDa tors has been reported in rat exocrine pancreas (20), brain was identified by sodium dodecyl sulfate-polyacryl- (21), and in a human gastric tumoral cell line HGT-1 (22). amide gel electrophoresis after photoaffinity labeling. While this article was inpreparation, the cloning of two We suggest that this protein is the putative SRIF-28 different somatostatin receptors, both with higher affinity for receptor or a subunit thereof. somatostatin-14 than somatostatin-28, has been reported (23). This confirms the existence of SRIF receptor subtypes. The hamster @ cell insulinoma is one of the richest sources of SRIF receptors (10) and also contains other regulatory Somatostatin-28(SRIF-28),' an N terminally extended, peptide receptors, related to the p cell function (24, 25). In bioactive form of somatostatin-14 (SRIF-14), was isolated the present work, we describe the solubilization, in a ligandfrom pig gut and hypothalamus (1,2) andsheep hypothalamus free form, of SRIF-28 preferringreceptors from p cell insulin(3), after the initial isolation of SRIF-14 from sheep hypotha- oma. Partial purification of the receptors by DEAE and WGAlamic extracts (4). Bothpeptides inhibit not only the release Sepharose chromatography has also been achieved. The bindof growth hormone but also that of insulin and glucagon (5). ing properties of this solubilized receptor were characterized The inhibitory actions of somatostatinare initiatedafter and itsfunctionality was supported by its interaction with Gproteins. *This workwas supported by Grants 1010 and 8902 from the Swedish Medical Research Council, by the Wenner-Gren Foundation, EXPERIMENTALPROCEDURES and by the InstitutNational de laSant6 et de la Recherche MBdicale. Materials-SRIF-28, [Leu', ~ - T r p " , Tyr25]somatostatin-28,and The costs of publication of this article were defrayed in part by the synthetic cholecystokinin octapeptide (CCK-8) were purchased from payment of page charges. This article must therefore behereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 Peninsula Laboratories (Belmont, CA). SRIF-14 was a gift by Dr. J. Diaz (Sanofi Recherche, Montpellier, France) and SMS 201-995 was solely to indicate this fact. 5 To whom correspondence should be addressed INSERM U.55, from Sandoz Pharmaceutical (Basel, Switzerland). [Leu', ~ - T r p " , Hijpital Saint-Antoine, 75571 Paris Cedex12, France. Tel.: 33-1- TyrZ5]SRIF-28was radioiodinated with carrier-free lZ5I-Na(referred to as '251[LWY]SRIF-28)and purified by high performance liquid 49284637; Fax: 33-1-43433234. The abbreviations used are: SRIF-28, somatostatin-28;SRIF-14, chromatography, according to the method previously described for somatostatin-14; SRIF, somatotropin release-inhibiting factor; SMS gastric inhibitory polypeptide (25),with some modifications (26). The 201-995, D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol; Gi, inhibitory specific activity of '251[LWY]SRIF-28was approximately 2000 Ci/ GTP-binding protein; CHAPS, 3-[(3-~holamidopropyl)dimethyl- mmol. Other products were from the followingsources: CHAPS, ammoniol-1-propanesulfonate; HEPES, 4-(2-hydroxyethyl)-l-piper- phenylmethylsulfonyl fluoride, pepstatin, leupeptin, and soybean azineethanesulfonic acid GTP-yS, guanosine-5'-O-thiotriphosphate; trypsin inhibitor, Boehringer (Mannheim, Germany); GTP-yS,bovine WGA, wheat germ agglutinin; ANB-NOS, N-5-azido-2-nitrobenzoy- serum albumin, and bacitracin, Sigma; lectin-coupled agarose, RBacloxysuccinimide;SDS-PAGE, sodium dodecylsulfate-polyacrylamide tifs IBF Biotechnics (Villeneuve la Garenne, France); protein standards for calibration of gel chromatography and SDS-PAGE, Bio-Rad; gel electrophoresis.

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Solubilized and Active Endocrine Pancreatic ANB-NOS, PierceChemical Co.; Sepharose 6Band DEAE-Sepharose CL-GB, Pharmacia LKB Biotechnology Inc. (Uppsala, Sweden). Solubilization of SRIF Receptors-Hamster pancreatic 0 cell membranes were prepared from serially transplantable tumors of Syrian hamster, as previously reported (25), but in the presence of 0.1 mM phenylmethylsulfonyl fluoride and 0.1% bacitracin. SRIF receptors were solubilized by treatment of the membranes with CHAPS. Membranes (2-4 mg protein/ml) were incubated, for 15 min a t 4 'C, in 25 mM HEPES buffer, pH 7.5, 0.3% (w/v) CHAPS, 35% (v/v) glycerol, and the following protease inhibitors: 0.1 mM phenylmethylsulfonyl fluoride, 1 p~ pepstatin, 1 p~ leupeptin, 0.1% (w/v) bacitracin and 0.2% (w/v)soybean trypsin inhibitor. The suspension was then centrifuged at 100,000 X g for 30 min a t 4 "C. The supernatant was removed and either used immediately or stored at -80 'C. Binding Experiments with CHAPS-solubilized Receptors-Binding of 1251[LWY]SRIF-28to solubilized receptors was evaluated by incubating the solubilized proteins (0.2-0.4 mg protein/ml) with 10 pM 1251[LWY]SRIF-28at 4 "C in the presence and absence of unlabeled SRIF-28. The final volume of incubation was 400 pl, and the assay buffer was 25 mM HEPES, pH7.5, 0.2% bovine serum albumin, 1 p M pepstatin, and 1 PM leupeptin. After incubation for 10 h, the separation of 1251[LWY]SRIF-28bound to solubilized receptors from free ligand was carried out by vacuum filtration through Whatman GF/B filters pretreated with 0.3% (v/v) polyethylenimine in H 2 0 (27). The filters were then washed twice with 4 ml of ice-cold 50 mM Tris-HCI, pH 7.5, and the radioactivity retained on filters was counted in a LKB 1282 y-counter. Nonspecific binding was measured in the presence of an excess (1 p ~ of) unlabeled SRIF-28 and subtracted from total binding to obtainspecific binding. Values of nonspecific binding were always lower than 10% of total binding. Gel Filtration of Solubilized Receptors-CHAPS-solubilized SRIF receptors (2 ml) were applied to a column of Sepharose 6B (1.5 X 83 cm), previously equilibratedwith 25 mM HEPES,pH 7.5, 0.1% CHAPS, and 20% glycerol at 4 "C. The column was eluted with the were same buffer at a flow rate of 11 ml/h. Fractions of2.5ml collected and aliquots (300 p l ) were assayed for binding activity as described above. DEAE-Sephnrose Chromatography of Solubilized Receptors-The CHAPS-solubilized extract of insulinoma membranes (665 ml) was applied to a DEAE-Sepharose CL-GB column (5 X 20 cm), previously equilibrated at 4 "C with 25 mM HEPES, pH 7.5, 40 mM NaCI, 20% glycerol, and 0.1% CHAPS, at a flow rate of 150 ml/h. After loading the sample, adsorbed proteins were eluted in the equilibrating buffer with a stepwise gradient of NaCI. Aliquots (100 p l ) from each fraction (12 ml) of the eluate were assayed for binding activity. Eluted fractions containing the highest specific activities (termed A and B)were pooled and stored at -80 "C. Lectin Affinity Chromatography-Two ml of wheat germ agglutinin (WGA)-agarose, in chromatographic tubes (1.1 X 10 cm), was equilibrated with 25 mM HEPES, pH 7.5,0.1% CHAPS, and 20% glycerol. The solubilized receptors(from the crude extract or the DEAE fraction A) were incubated with the gel for 5 h at 4 "C. Then the gel was washed with 12 ml of buffer before the elution of glycoproteins bound to WGA by 0.3 M N-acetylglucosamine (GlcNAc). Aliquots of 100 or 300 p1 from the collected fractions (2 ml) were assayed for binding activity as described above. Cross-linking ofIz5I[L WYISRIF-28 to Soluble Receptors-Crude CHAPS-solubilized extract (64 pg of protein) as well as aliquots of fractions containing receptoractivity that had been eluted from andB) or WGA-agarose or from DEAE-Sepharose(fractionsA chromatographed on Sepharose 6B were incubated with 1251[LWY] SRIF-28 (60 pM) for 10 h at 4 "C in the presence or absence of 1 p~ unlabeled SRIF-28. The final volume of incubation was 800 pl and the assay buffer was 25 mM HEPES, pH 7.5, to which a mixture of protease inhibitors had been added: 1 G M pepstatin, 1 p~ leupeptin, 0.1% bacitracin, and 0.2% soybean trypsin inhibitor. After the incubation, a freshly prepared solution of ANB-NOS (20 mM in dimethyl sulfoxide) was added to a final concentration of 0.1 mM. After 5 min stirring in the dark on ice, the samples were exposed to UV light for 10 min under a 275 watt mercury-vapor lamp at a distance of 16 cm. The reaction was stopped by addition of 50 mM Tris-HC1, 2 mM EDTA(final concentration),pH 7.5, as aquenching buffer. The samples were then concentrated using Centricon-10 microconcentrators (Amicon) and analyzed by SDS-PAGE. SDS-Polyacrylamide Gel Electrophoresis-The concentrated material was supplemented with sample buffer to a final concentration of 200 mM Tris-HCI, pH 6.8, 2%(w/v) SDS, 12% (v/v) glycerol, 0.001% (w/v) bromphenol blue, in either the presence or absence of

SRIF-28 Receptors

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3% (v/v) 2-mercaptoethanol. The samples were heated for 30 min at 60 "C before application to polyacrylamide slab gels for electrophoresis according to Laemmli (28), using a 5% acrylamide stacking gel and alineargradient resolving gel (10-15%). After staining with Coomassie Blue and destaining, gels were dried and exposed for 2-8 days at -80 "C with a Kodak X-Omat AR film and an enhancing screen. Protein Determination-Protein concentration was determined by bicinchoninic acid assay (29) using a BCA kit (Pierce Chemical Co.) with bovine serum albumin as standard. RESULTS

Conditions for Solubilization-Under our experimental conditions CHAPS was an appropriate detergentfor solubilizing SRIF receptors, whereas with Triton X-100 no measurable binding activity was detected in the solubilized extract. To determine the optimal CHAPS concentration for solubilization of active somatostatin receptors, membranes of fl cell insulinoma (2-4 mg protein/ml) were incubated at 4 "C with various detergentconcentrations. After centrifugation,the specific binding of 1251[LWY]SRIF-28was measured in the solubilized fraction.Binding activityrapidlyincreasedbetween 0.1% and 0.4% CHAPS, with a maximum at 0.3-0.4%, where approximately 50% of membrane protein was solubilized. A drastic decrease of SRIF binding activity was observed between 0.5 and 1% CHAPS, whereas the concentration of solubilized protein stillincreased. In order to optimize extraction conditions of activereceptors, we have incubated the membranes with 0.3% CHAPS for different times. The concentration of SRIF-binding sites in the extract reacheda maximum at 15 minand rapidlydecreased after 30 min. Addition of glycerol during solubilization resulted in a concentration-dependent increase of the binding activity in the solubilized extract witha &foldimprovement at 35% glycerol. CHAPS-glycerol-solubilized extracts frozen at -80 "C could be stored for several months without loss of binding activity. Therefore, routine solubilizations were performed for 15 min with 0.3% CHAPS and 35% glycerol. Binding Characteristics of 12'Z[L WYISRIF-28 to Solubilized Receptors-The 1251[LWY]SRIF-28binding of the solubilized extract was a time-dependent process. Fig. 1A shows that maximum binding at 4 "C was reached after 10 h of incubation. The binding was stable for a t least 27 h. As shown in Fig. 1B, the binding of '251[LWY]SRIF-28to solubilized receptors was reversible. When 1 PM unlabeled SRIF-28 or 100 PM of the non-hydrolyzable G T P analog GTPyS was added at equilibrium, about 40 and 35% of the bound radioactivity, respectively, was dissociated after 30 min of incubation at 30 "C. GTPyS, in combinationwith SRIF-28, accelerated the dissociation of 1251[LWY]SRIF-28 fromsolubilized receptors, reaching about 70% after 15 min of incubation. GTPyS inhibited specific 12sI[LWY]SRIF-28 bindingto solubilized receptors in a dose-dependent manner (Fig. 2). An inhibitory effect was observed already at 1 PM GTP+, and 100 ,LLMGTPyS (the highest concentration tested) caused a 70% decrease of specific 1251[LWY]SRIF-28binding. To further evaluate theeffect of solubilization on the specificity of SRIF-binding sites, the ability of SRIF-28, SMS 201-995, and SRIF-14for inhibiting 1251[LWY]SRIF-28binding to solubilized receptors was tested (Fig. 3). The concentrations of peptides required to produce half-maximal inhibition (1cb0)of 1251[LWY]SRIF-28binding tosolubilized receptors were inthe following order: SRIF-28 = 0.76 nM) < SMS 201-995 (ICs, = 9 nM) < SRIF-14 (ICE, = 32 nM). These results clearly indicated the presence of SRIF-28 preferring binding sites in the solubilized fl cell insulinoma. CCK-8, up to 1 HM, was unabletocompete with '251[LWY]SRIF-28. Scatchard analysis of the data, using the LIGAND program

Solubilized a n d Active Endocrine Pancreatic

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IO

0

SRIF-28Receptors

30

20

Time (hours) -11

B

20

.

,

20

.

,

.

40

,

60

.

.

-9

-8

-7

-6

-5

Peptide (log M)

40

04 0

-10

I

80

Time (min)

FIG. 1. Time course of association and dissociation of specific '261[LWY]SRIF-28 binding to the solubilized extract. Panel A , kinetics of association of '251[LWY]SRIF-28(10 PM) to solubilized membranes (0.2-0.4 mg protein/ml) were followedat 4 "C. Panel B, after a plateau value has been reached in the association kinetics described in panel A , dissociation of bound '251[LWY]SRIF28 was initiated at 30 "C by addition of 1 p~ unlabeled SRIF-28).( or 100 p~ GTPyS (0)or 1 p~ SRIF-28 + 100 p~ GTPyS ( 0 ) compared to control (A).Results are expressed as percentage of specific binding (in panels A and B ) . Each point represents(in panels A and B ) the mean of three experiments performed in duplicate.

FIG. 3. Competitive inhibition of binding of '2KI[LWY] SRIF-28 to solubilized receptors by somatostatin analogs. Solubilized receptors (150 pg of protein) were incubated at 4 "C for 10 h with 10 PM '251[LWY]SRIF-28and increasing concentrations of unlabeled SRIF-28 (O), SMS 201-995 (A) or SRIF-14 (m). Data are expressed as percentage of the specific binding measured in the absence of peptides. Each point representsthe mean of four separate experiments performed in duplicate. Inset, Scatchard analysis of data from competitive displacement by SRIF-28. The data are representative of four separate experiments.

gel filtration on Sepharose 6B at 4 "C. Aliquots of collected fractions were incubated with '251[LWY]SRIF-28to determine the elution profile of binding activity. Fig. 4 shows that the solubilized SRIF receptor complex was eluted as a single peak corresponding to an apparent molecular mass of550 kDa. DEAE-Sepharose Anion-Exchange Chromatography of the Solubilized SRIF-28 Receptor-In order to partiallypurify the SRIF-28 receptor, solubilized extract of insulinoma membranes was applied to DEAE-Sepharose and the eluted fractions were assayed for '251[LWY]SRIF-28binding activity. As shown in Fig. 5 (left), thesolubilized binding sites were found to distribute into two main peaks, referred to as fraction A and fraction B and eluted, respectively, at 40 and 150 mM NaC1. SRIF-28 strongly inhibited '251[LWY]SRIF-28binding to receptors infractionsA and B (Fig. 5,right). Binding parameters were: Kd = 0.12 f 0.03 nM for the fraction A and 0.55 f 0.16 nM for the fraction B, as calculated by Scatchard analyses (Table I). This indicates that both fractions contained high affinity receptors for SRIF-28. Table I1 showsthe results obtained after the partial purification of the crude extract on DEAE-Sepharose. The purification was 2.7-fold for the fraction A. The inhibition by GTP-yS of the specific '251[LWY]SRIF-28binding infractions A and B was less efficient in comparison to that in the crude extract (Fig. 2). -9 -8 -7 -6 -5 -4 At 100 PM GTP-yS, the inhibition represented 45 and 20% G T P 6 (1% M) decrease of specific binding in A and B, respectively. Lectin Affinity Chromatography-The solubilized SRIF-28 FIG. 2. GTPrS inhibition of specific 1261[LWY]SRIF-28 binding to solubilized receptors. The effect of varying concentra- receptor in cell insulinoma was found to be a glycoprotein tions of GTPyS on specific '251[LWY]SRIF-28binding to solubilized by its interaction with wheat germ lectin. Crude CHAPS receptors in the crude extract (O), and in the fractions A (0)and B extract was incubated with WGA-agarose and, after a wash, (0)eluted after DEAE chromatography (see Fig. 5) was tested. Values are expressed as percent of specific binding obtained in the absence 0.3 M GlcNAc was used to elute adsorbed glycosylated proof GTP-yS.An experiment representative of three separate studies is teins. Fig, 6 shows that a large part of the totalsoluble binding activity could be retained on WGA-agarose and specifically presented. eluted with 0.3 M GlcNAc. Scatchard analysis of the lectin (30), indicated the presence in the solubilized extract of two eluted binding activity demonstrated the presence of both classes of SRIF-28-bindingsites (Table I): a high affinity high and low affinity binding sites, as found in the crude binding site with a Kd of 0.32 f 0.04 nM and a B,,, of 0.94 f extract (Table I): &H = 0.55 f 0.12 nM and K ~ =L 6.4 f 2.3 0.17 pmol/mg of protein and a low affinity binding site with nM. The overall purification was 12.5-fold. Therefore, as a a Kd of 13.3 f 5 nM and a B,,, of 4.65 f 1.07 pmol/mg of next step in the purification, high affinity receptors in fraction A from DEAE-Sepharose were applied to WGA-Sepharose. protein. Gel Filtration of Solubilized SRIF Receptors-To estimate The receptors retained and eluted in this step maintained a the apparent molecular weight of the solubilized SRIF recep- high affinity binding for SRIF-28. According to Scatchard tor complex, the CHAPS-solubilized extract was subjected to analysis, the binding was consistent with the presence of a

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Active Endocrine

Pancreatic SRIF-28 Receptors

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TABLEI Binding parametersof solubilized receptors The parameters were determined from Scatchard plots of the equilibrium specific binding data. Values are the mean f S.E. of three senarate exueriments (fourfor the crude extract).

nM

pmollmg

nM

13.3 2 5 0.32 f 0.04 0.94 ? 0.17 Crude extract 6.4 f 2.3 0.55 f 0.12 11.8 f 2.7 WGA-Sepharose DEAE-Sepharose 0.12 f 0.03 2.5 f 0.4 Fraction A 0.55 f 0.16" 1.18 f 0.22 Fraction B 0.99 f 0.26* 43 f 7.3 WGA of DEAE fraction A Significantly differentfrom the values obtained in fractionA ( p < 0.01, n = 3). Significantly differentfrom the value obtained in fractionA before WGA ( p < 0.004, n = 3).

pmollw

4.65 f 1.07 52.3 f 20

purification, theG T P effect was not maintained. Affinity Labeling of Soluble SRIF Receptors-Crude solubilized insulinoma extract was covalently labeled with p 52 '251[LWY]SRIF-28 by theheterobifunctionalcross-linking f 5.0 0.0 0.1 0.2 0.3 0.4 0.5 0.6 reagentANB-NOS,andthen analyzed by SDS-PAGE. A Ka" radiolabeled band of 37 kDa was observed in the absence(Fig. 7, lune 1 ) but not in the presence (Fig. 7, lane 2 ) of 1 pM SRIF-28, indicating thespecificity of the labeling. The solubilized SRIF receptor in the -550 kDa complex observed by gel filtration chromatography (fraction19 in Fig. 0 10 20 30 40 50 60 4) was also cross-linked andanalyzed by SDS-PAGE. Again, Fraction number a specific 37-kDa band was photolabeled (data not shown). A further demonstration that the 37-kDa protein is the FIG. 4. Gel filtration of CHAPS-solubilized SRIF-28 receptors. Two ml of crude CHAPS-solubilizedextract was applied at 4 "C hormone-binding subunit of the putative SRIF receptor is two to a Sepharose 6B column (1.5 X 83 cm) equilibrated with HEPES provided by covalentlabeling of theproteinsinthe 25 mM, pH7.5, 0.1% CHAPS,and20%glycerol.Aliquots of the fractions (A and B) separated by DEAE chromatography. For eluted fractionswere assayed for specific binding of '251[LWY]SRIF- both fractions a major band corresponding to an apparent 28, as described under "Experimental Procedures." The column has molecular mass of 37 kDa was observed in SDS-PAGE analybeenpreviously calibrated withblue dextran (voidvolume = Vo), thyroglobulin (Mr, 669,000), ferritin (Mr, 440,000),catalase (Mr, sis (Fig. 7, lunes 3 and 5 ) ,specifically abolished by 1 p~ SRIF232,000) and aldolase (Mr, 158,000).Fractionvolume,2.5 ml; flow 28 (Fig. 7, lanes 4 and 6). rate, 11 ml/h. Inset, the logarithmic molecular weight of the marker Thematerialretainedandeluted from WGA by 0.3 M proteins (0)is plotted versus Kav.The apparent molecular weight of GlcNAc (fractions 7-8 in Fig. 6) has also been cross-linked the eluting bindingsite (0)was obtained from this plot. using the same procedure and analyzed by SDS-PAGE. A protein band of 37 kDa was photolabeled (Fig. 7, lane 7). The labeling of this band was specifically extinguished when 1 p~ unlabeled SRIF-28 was included during binding (Fig. 7, lune 8). DISCUSSION

0

20

40

60

80

IW

Fraruon number

FIG. 5. DEAE-Sepharose chromatography of CHAPS-sohbilized SRIF-28 receptors. Left, crude CHAPS-solubilizedextract (665 ml) was applied at 4 "C to a DEAE-Sepharose column (5 X 20 cm) equilibrated with25 mM HEPES, pH 7.5,40 mM NaC1,20% glycerol, and 0.1% CHAPS. Fraction volume, 12 ml; flow rate, 150 ml/h. Aliquots of the eluted fractionswere assayed for specific binding of '251[LWY]SRIF-28(O),as described under "Experimental Procedures." Absorbance at 280nmisshownby the broken line (- - -). Thestepwisegradient ofNaClis indicated by the dottedline (. . . .). Fractions used for further studies were named A and E , as indicated by the bars. Right, competitiveinhibition of '251[LWY] SRIF-28 binding to solubilized receptors in DEAE fractions A (0) and B (0)by unlabeled SRIF-28. Resultsare expressed as percentage of specific binding,determinedasdescribedunder"Experimental Procedures." Each point representsthe mean of four separate experiments performed in duplicate. single class of sites with a Kd of 0.99 k 0.26 nM and a B,,, of 43 f 7.3 pmol/mg of protein (Table I). These values correspond to a 46-fold purification (Table 11). A t this stage of

This paper reports the solubilization and partial purification of SRIF-28 preferring receptorsfrom the hamster endocrine pancreas in an active and unoccupied form. The highest specific bindingactivity could be recovered when 0.3% CHAPS and35% glycerol were used. The presence of glycerol was essential for obtaining high amounts of active solubilized receptors and for their stabilization. CHAPS has been efficiently employed for thesolubilization of somatostatin receptorsfromtherat exocrine pancreas(20)andbrain (21), although at higher concentrations (3 and 0.676, respectively) and usinga longer extraction time (60 min for the brain). Scatchard analysesof equilibrium competitive binding data indicated thepresence in thesolubilized extract of two classes of binding sites. The& for the high affinity site (0.32 f 0.04 nM) was similar to that reported for membrane-bound receptors (10). Solubilized p cell somatostatin receptors maintain the preference for SRIF-28 over SRIF-14 observed in membrane-bound receptors (9, 10). Also, the bindingof '251[LWY] SRIF-28 to solubilized receptorsis selective since CCK-8, which in pancreatic acinarcell membranes interacts with the somatostatin-14 receptor (31),did not interfere with SRIF-28

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TABLE I1 Purification of high affinity somatostatin-28receptors from hamster insulinoma The data in this table are from a single preparation, which is representative of four similar experiments. Total protein

Total binding activity

Specific activity

Purification

mRn

pmol

pmollmg

-fold

%

1

100 30 10

Crude extract 1012 1077 0.94 DEAE fraction A 300 120 2.5 DEAE WGA of fraction Ab 2.4 43 103 ’Protein was determined as described under “Experimental Procedures.” Values extrapolated from an experiment performed on a 3% aliquot of DEAE fraction A.

2.7 46

Yield

the different sensitivities of the two fractions to GTPyS. The low affinity sites, found in the crude extract, are no longer detected after DEAE-Sepharose chromatography. (The low affinity binding proteins may have been degraded during the procedure or alternatively may require >0.4 M NaCl for their elution.) Taking advantage of our finding of a glycoprotein nature of SRIF-28receptors,proteinsinfraction A were further purified by chromatography on WGA-agarose. The specific activity increased after DEAE chromatography to 2.5 pmol/ 0 2 4 6 X 1 0 1 2 Fraction number mg of protein and was further increased to 43 pmol/mg of FIG. 6. WGA affinity chromatography of solubilized SRIF- protein after lectin chromatography of the fraction A, with a 28 receptors. Three ml of the crude CHAPS-solubilized receptors recovery of 10% afterthese two purificationsteps. WGA was applied to WGA-agarose (2 ml). After washing the gel with 25 affinity chromatography has been useful in the purification mM HEPES, pH 7.5, 0.1% CHAPS, and 20% glycerol, the adsorbed of several receptors (33, 34). SRIF-28 receptors interacted proteins were eluted with 0.3 M GlcNAc in the same buffer as shown strongly withWGA-agarose, suggesting that thereceptor carby the arrow. Aliquots of the collected 2-ml fractions were assayed riesaglycosylatedmoiety containing N-acetylglucosamine for specific binding of ’251[LWY]SRIF-28,as described under “Exresidues and/or N-acetylneuraminic acid. Interestingly, soluperimental Procedures.” bilized SRIF receptorsfrom hamster endocrine pancreaswere eluted from the WGA gel by GlcNAc, whereas those from rat CRUDE DEAE WGA EXTRACT A B exocrine pancreas (20) could not be eluted by GlcNAc but -66 only by triacetylchitotriose, which possesses higher affinity -45 forWGA(35). This suggests thatthese two mammalian 37w receptors differ in the nature of their carbohydrate chains. Initially, SRIF-28 receptors were solubilized as a large complex, as determined by gel exclusion chromatography. GTPbinding proteinsmay be one componentof the SRIFreceptor complex because a potent ligand binding inhibitory and dissociating effect of GTPyS was found in p cell insulinomaSRIF-28 - + - + solubilized receptors. This GTP-binding unit might be the Gi 1 2 3 4 5 6 7 8 protein, since an inhibition of adenylate cyclase activity by FIG. 7. SDS-PAGE analysisof ’261[LWY]SRIF-28 cross- somatostatin has been found in pancreatic /3 cells (36). This linked to solubilized receptors. ’251[LWY]SRIF-28was crosslinked to crude CHAPS-solubilizedextract and to aliquots of receptor inhibitory effect was progressively lost duringpurification, in eluted from DEAE-Sepharose(fractionsA and B ) and WGA-agarose. parallel with the decreasein the affinity for the hormone Cross-linkingwas performed in the absence (lanes 1,3, 5, and 7) or noted after WGA chromatography of the fraction A (Table presence (lanes 2, 4, 6, and 8) of 1 p~ SRIF-28. The methods of I). A similar observation has been made during the purificacross-linking and autoradiography by SDS-PAGE are described under tion of SRIF receptors from the human gastric tumoral cell “Experimental Procedures.”The position to which protein markers line HGT-1, where the loss of the GTP effect was found to (kDa) had migrated is indicated on the right of the WGA autoradiograph, obtained after a separate cross-linking experiment.The ar- be accompanied by a decrease in the affinity(22). Covalent cross-linking of ‘2sI[LWY]SRIF-28 to crude solrows on the left indicate the molecular mass (kDa) of the specific labeled protein. ubilized receptors from p cell insulinoma revealed the presence of a specifically labeled band of an apparent molecular mass of 37 kDa, both in the absence or presence of 2-mercapreceptors.However, there was animportantshiftinthe suggesting that the receptor does affinity of the analog SMS 201-995, which bound efficiently toethanol (data not shown), to solubilized receptors, in contrast with its low affinity for not contain disulfide-linked subunits. The samespecific band the membrane-bound receptors (10). Changes ligand in affin- of 37 kDa was also identified when ‘251[LWY]SRIF-28 was ities between membrane-bound andsolubilized receptors have cross-linked to the receptor aftergel filtration, DEAE-Sepharose, or WGA-agarose. This provides evidence for a specific been previously reported (20, 32). After purification on DEAE-Sepharose, only high affinity 37-kDa binding protein in solubilized extracts from hamster sites were found distributed in two eluted fractions (A and /3 cell insulinoma, co-purifying with the high affinity binding B). The fractions A and B showed different Kd values, al- activity in the different chromatographic steps. Until now, molecularspeciesidentified, I). The thereisnoconsensusinthe though close to that found in the crude extract (Table reason for this is unclear, but one possible explanation could which have been found to range in size from 27 to 200 kDa be differences in interactions with G-proteins, indicated by (10,13, 15,37-42). Someof the observed discrepancies might

vr *

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9

+

Solubilized a n d Active Endocrine Pancreatic provide further support for the existence of different receptor subtypes distributed in different tissues (13, 43). Recently, the cloning of two dissimilar SRIF-14 receptors stresses the idea of a large SRIF receptor family (23). A difference can be noted between the mass of the solubilized putative SRIF receptor in our study (37 kDa) and the several higher molecular mass species (45, 132, and 196 kDa) previously reported in the samehamster fi cell insulinoma(10). This can be explained by the fact that, in the earlier report, during the cross-linking of membrane-bound receptors, other proteins may have become covalently attached to the hormone-binding receptor subunit. In the present experiment, such proteins are likely to have been separated from the receptor during solubilization. In conclusion, we describe the solubilization and a 46-fold purification of endocrine pancreatic high affinity SRIF-28 receptors. Moreover, we provide evidence for a specific SRIFbinding protein of 37 kDa and suggest that thisprotein is the putative SRIF-28 preferring receptor or a ligand-binding subunit thereof. Although the small size of this receptor is intriguing, it is not unique since the prolactin andthe growth hormone-releasing factor receptors are reported to have a mass of only 42 and 26 kDa respectively (44, 45). Moreover, the recently cloned somatostatin-14 receptorshave calculated molecular masses of roughly 41 and 43 kDa for their protein components (23). Acknowledgments-We thank Drs. E. Forgue-Lafitte, J. Mester, and G . Skoglund for helpful suggestions and A. Barakat for the preparation of membranes. REFERENCES 1. Pradayrol, L., Jornvall, H., Mutt, V., and Ribet, A. (1980) FEBSLett. 1 0 9 , 55-58 2. Schally, A,, Huang, W.-Y., Chang, R., Arimura, A,, Reddin T Millar R S. A. '77: Hunkapiller, M., and Hood, L. (1980) Pruc. Natl. Acad. &i. 4489-4493 3. Bohlen, P., Brazeau, P., Benoit, R., Ling, N., Esch, F., and Guillemin, R. (1980) Bwchem. Biuphys. Res. Cummun. 9 6 , 725-734 4. Brazeau, P., Vale, W., Burgus, R., Ling, N., Butcher, M., Rivier, J., and Guillemin, R. (1973) Science 1 7 9 , 77-79 5. Meyers, C., Murphy, W., Redding, T., Coy, D., and Schally, A. (1980) Pruc. Natl. Acad. Scr. U. S. A. 7 7 , 6171-6174 6. Lewin, M. J. M. (1986) Scand. J. Gastroenterol. 2 1 (Suppl. 119), 42-46 7. Amberdt, M., Patel, Y., and Orci, L. (1987) J . Clin. Invest. 80,1455-1458 8. Shapiro, S., Eto, S., Fleischer, N., and Baum, S. (1975) Enducrrnolugy 9 7 , 442-447 M., and Vale, W. (1982) 9. Reubi,J.-C.,Rivier,J.,Perrin,M.,Brown, Endocrinology 1 1 0 , 1049-1051 10. Cotroneo, P., Marie, J.-C., and Rosselin, G. (1988) Eur. J. Biuchem. 174, 219-224

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i.,