Solubilization and Molecular Characterization of Active Pancreastatin

0013-7227/97/$03.00/0 Endocrinology Copyright © 1997 by The Endocrine Society

Vol. 138, No. 4 Printed in U.S.A.

Solubilization and Molecular Characterization of Active Pancreastatin Receptors from Rat Liver Membranes* ´ NCHEZ-MARGALET VıĆTOR SA

AND

´ SANTOS-A ´ LVAREZ JOSE

Department of Medical Biochemistry and Molecular Biology, Investigation Unit of Virgen Macarena Hospital, Faculty of Medicine, University of Seville, Seville, Spain ABSTRACT Pancreastatin receptors were solubilized from rat liver membranes with the nonionic detergent Triton X-100. Binding of a iodinated analog of rat pancreastatin ([125I-Tyr0]pancreastatin) to the soluble fraction was time dependent, saturable, and reversible. Scatchard analysis of binding under equilibrium conditions indicated that the soluble extracts contained a single class of pancreastatin-binding sites, with a binding capacity of 14 fmol/mg protein and a Kd of 0.3 nM. As observed with membrane-bound receptors, binding of [125I]pancreastatin to soluble extracts was inhibited by guanine nucleotides with the following rank order of potency: guanyl-59-yl-imidodiphosphate . GTP . GDP . GMP, indicating that the soluble receptors are functionally linked to G proteins. Molecular analysis of the soluble pancreastatin receptor by covalent cross-linking to [125I]pancreastatin using disuccinimidyl suberate and further identification on SDS-

PAGE indicated a single band of 85,000 Mr. Gel filtration of soluble extracts on Sephacryl S-300 revealed two molecular components with binding abilities (Mr 80,000 and 170,000). The higher molecular mass component was more sensitive to guanine nucleotides, and covalent cross-linking of both components to [125I]pancreastatin and further SDS-PAGE analysis revealed again a single band of 85,000 Mr, suggesting an association of the receptor with a G protein. Moreover, direct evidence that a Gq was present in the same chromatographic fraction was obtained by specific immunodetection. The soluble receptor is a glycoprotein that can be specifically bound to the wheatgerm agglutinin lectin. We conclude that we solubilized active pancreastatin receptors from rat liver membranes, and these results support the conclusion that the liver pancreastatin receptor consists of a 80,000 Mr glycoprotein associated with G proteins. (Endocrinology 138: 1712–1718, 1997)

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Thereafter, we studied and characterized the PST-specific receptor in rat liver plasma membranes (33) as well as the specific signal transduction (34, 35). This receptor appears to be coupled to two different G proteins. A pertussis toxininsensitive G protein leads to the activation of phospholipase C and, therefore, mediates the glycogenolytic effect in the liver by increasing cytoplasmic free calcium and stimulating protein kinase C, as previously demonstrated for other receptors (36 – 40), whereas a pertussis toxin-sensitive G protein leads to the activation of guanylate cyclase (41). The role of cGMP in the action of PST is not known yet, although it seems to negatively regulate the activation of phospholipase C by PST and, therefore, function as a negative feedback for PST signaling (34, 41). The effect of PST in the liver glycogen metabolism both in vivo and in vitro suggests a role for PST as a counterregulatory peptide of insulin action. In fact, high PST levels have been found in insulin-resistant states (42– 44). To further characterize the molecular mechanism underlying PST action in the liver, we sought to solubilize the PST receptors from rat liver membranes in native conditions using the nondenaturing detergent Triton X-100. Here we report the successful solubilization of active PST receptors from rat liver membranes. Partial purification of the receptors by lectin adsorption chromatography has also been performed. Therefore, the glycoprotein nature of the receptor is confirmed (33). The binding properties of this soluble receptor were characterized, and its functionality was assessed by its interaction with G proteins. Gel filtration of the solubilized receptors and immunodetection provided evidence for the presence of a 80,000 Mr PST receptor and a 170,000 Mr complex containing the receptor coupled to G proteins.

ANCREASTATIN (PST), a 49-amino acid peptide isolated from porcine pancreas (1), arises from proteolytic cleavage of its precursor chromogranin A (CGA), a glycoprotein present in endocrine and neuronal cells (2, 3). In islets, PST appears to be localized to the insulin-containing b-cells, somatostatin-containing d-cells (4), and glucagoncontaining a-cells (5, 6). On the other hand, postsecretory processing of CGA also occurs (7, 8). Rat CGA A complementary DNA revealed the existence of a PST-like sequence homologous to porcine PST (9 –12). The role of PST as a regulatory enteropancreatic peptide has been established in the light of a variety of biological effects in a number of tissues that could be assigned to the carboxyl-terminal part of the molecule (see Ref. 13 for review). These effects are exerted on endocrine and exocrine pancreatic secretion (14 –19), gastric secretion (20, 21), PTH release (22), plasma catecholamine levels (23), and memory retention (24). Synthetic rat PST has also been shown to have biological activity in different tissues (25–27). However, it should be pointed out that no PST receptors have been found in these tissues to date, and therefore, the physiological role of PST in these systems remains to be solved. In rat liver, we have shown that PST has a calcium-dependent glycogenolytic effect (28 –31) and an inhibitory effect on the insulin stimulation of glycogen synthesis (32). Received September 19, 1996. Address all correspondence and requests for reprints to: Dr. Vıćtor Sańchez-Margalet, Departamento de Bioquı´mica Me´dica y Biologıá Molecular, Facultad de Medicina, Universidad de Sevilla. Av. Sańchez Pizjuan 4, 41009 Seville, Spain. E-mail: [email protected]. * This work was supported by the Fondo de Investigacioń Sanitaria (FIS 95/1411), Ministerio de Sanidad y Consumo (Spain).

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ACTIVE SOLUBLE LIVER PANCREASTATIN RECEPTORS Materials and Methods Materials Rat [125I-Tyr0]PST (1500 Ci/mmol) and rat PST were obtained from Peninsula Laboratories Europe (Merseyside, UK). Wheat-germ agglutinin (WGA) coupled to Sepharose 4B was obtained from Pharmacia Biotech (Uppsala, Sweden). Polyethylene Glycol 6000 was purchased from Merck (Darmstadt, Germany). Triton X-100, 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate (CHAPS), Nonidet P-40, bovine g-globulin, N-acetyl-b-glucosamine (NAG), bacitracin, leupeptin, N-p-tosyl-l-lysine chloromethyl ketone, phenylmethylsulfonylfluoride, pepstatin, aprotinin, dithiothreitol (DTT), BSA (fraction V), GTP, GDP, guanyl-59-yl-imidodiphosphate [GMP-P(NH)P], and other nucleotides were purchased from Sigma Chemical Co. (St. Louis, MO). The cross-linker disuccinimidyl suberate (DSS) was obtained from Pierce Chemical Co. (Rockford, IL). Electrophoretic chemicals and Mr standards were purchased from Novex (San Diego, Ca). Rabbit antisera against bcommon and aq (q,11) subunits of G proteins were obtained from DuPont New England Nuclear-DuPont de Nemours (Bad Homburg, Germany).

Preparation of rat liver membranes Male Wistar rats (150 –200 g) were used and were fed ad libitum. Rat liver membranes were prepared according to the method of Neville (45) up to step 11.

Membrane solubilization In optimal conditions, solubilization was carried out incubating membranes (14 mg/ml) in HEPES buffer (20 mm; pH 7.4) containing 1% (vol/vol) Triton X-100, NaCl (100 mm), KCl (1 mm), MgCl (2 mm), 10% (vol/vol) glycerol, bacitracin (200 mg/ml), phenylmethylsulfonylfluoride (0.1 mm), N-p-tosyl-l-lysine chloromethyl ketone (10 mg/ml), leupeptin (10 mg/ml), pepstatin (5 mg/ml), and aprotinin (10 mg/ml) for 1 h on ice. The membrane suspension was then centrifuged at 100,000 3 g (4 C). The supernatant was removed and used immediately or stored at 280 C. The yield of protein solubilization was estimated at 50%. Protein concentration was determined by the Bradford procedure (46) with a Bio-Rad kit (Richmond, CA), using BSA as standard. The ability of the solubilized material to bind [125I]PST was stable for 4 days at 4 C and for 3 months at 280 C.

Binding and cross-linking of [125I]PST to soluble extracts Binding to the solubilized receptors was performed with the radiolabeled rat PST analog [125I-Tyr0]PST (SA, 1500 Ci/mmol), which has been previously demonstrated to bind to specific receptors with high affinity in rat liver membranes (33). Soluble extracts were incubated with 5 3 10211 m [125I]PST. This is the same concentration of tracer previously employed for characterization of native membranes (33). The binding was performed at 25 C for 90 min in solubilization buffer (pH 7.4) containing 1% BSA. At the end of the incubation, soluble receptors were precipitated at 4 C by the addition of bovine g-globulins (0.4% final concentration) and polyethylene glycol (10% final concentration), pelleted by centrifugation at 4 C, and washed twice with cold binding buffer containing polyethylene glycol (10%). Specific binding was calculated as the difference between the amount of radioactivity bound in the absence (total binding) or presence (nonspecific binding) of an excess (1027 m) of rat PST. Specific binding represented about 2.5% of total radioactivity, and nonspecific binding represented about 35% of total binding. For cross-linking experiments, BSA was omitted and free [125I]PST and [125I]PST-receptor complexes were separated by gel filtration on a Sephadex G-25 column (11 3 0.9) from Pharmacia Biotech (Uppsala, Sweden), equilibrated, and eluted with solubilization buffer (without BSA); 0.5 ml of the void volume containing [125I]PST-receptors complexes was then incubated with 1 mm DSS (final concentration). After 20 min at 4 C, the cross-linking reaction was stopped by the addition of 10 ml ice-cold 1 m Tris, pH 6.8. The samples were denatured and separated by SDS-PAGE (8 –16%) (47).

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Gel filtration of solubilized materials The solubilized receptors were chromatographed on a Sephacryl S-300 high resolution prepacked column (60 3 1.6) from Pharmacia Biotech (Uppsala, Sweden), equilibrated, and eluted with HEPES buffer (pH 7.4) containing 0.1% Triton X-100, NaCl (100 mm), KCl (1 mm), MgCl (2 mm), glycerol (10%), and bacitracin (200 mg/ml). The column was run at 18 ml/h, and 1-ml fractions were collected and assayed for [125ITyr0]PST binding and cross-linking. The S-300 column was calibrated under conditions used for analyzing solubilized receptors, with marker proteins of known Stokes radii (Pharmacia Biotech). The elution volumes of the receptor and marker proteins were expressed in terms of Kav 5 (Ve 2 V0)/(Vt 2 V0), where Ve is the elution volume of the protein considered, V0 is the void volume, and Vt is the total liquid volume. To estimate the Stokes radius of PST receptors, Kav was plotted against the known Stockes radius of the marker proteins (48).

WGA affinity chromatography Solubilized receptors (5 mg protein) were incubated with agaroseWGA for 3 h at 4 C rotating. The mixture was then packed into a 5 3 1-cm chromatographic column. The column was then washed with 2 vol of the same buffer used for gel filtration. The receptor was eluted at 0.3 ml/min with the same buffer containing 0.3 m NAG. Fractions were assayed for binding activity as described above.

Immunodetection of b and aq subunits of GTP-binding proteins Soluble extracts and fractions from the S-300 column were denatured with Laemmli buffer and run on SDS-PAGE (8 –16%). Proteins were electrophoretically transferred onto nitrocellulose membranes. The membranes were first incubated with anti-bcommon or anti-aq, further incubated with second antibody conjugated with horseradish peroxidase, and developed by the Amersham enhanced chemiluminescence detection system (Arlington Heights, IL) (49).

Results Solubilization of rat liver PST receptor

We used 1% Triton X-100 for solubilizing liver membrane PST receptors, a commonly used detergent to solubilize native membrane proteins in an active state (50). The zwitterionic detergent CHAPS and the nonionic detergent Nonidet P-40 were previously tested and found to be inactive. Triton X-100 (1%) and a 14 mg/ml protein concentration were found to be the optimal conditions for solubilization of PST receptors (not shown), with a yield of 50% solubilized proteins of the total membrane proteins. Using these conditions, binding of [125I]PST to the incubated solubilized membranes was proportional to solubilized protein concentrations (Fig. 1). Characterization of binding of [125I]PST to solubilized rat liver membranes

[125I]PST specific binding to soluble receptors was time and temperature dependent. As shown in Fig. 2, the binding of [125I]PST to soluble receptors is a slowly reversible process at 25 C. At 30 C, the association-dissociation kinetics are faster (30 and 60 min, respectively); at 15 C, they are slower (120 and 180 min, respectively; not shown). Under optimal conditions, 90 min at 25 C, binding of PST was saturable (Fig. 3). Cold PST concentrations ranging from 10211-1028 m competitively inhibited the binding of [125I]PST to soluble receptors (Fig. 3). Other peptide hormones (vasopressin, glucagon, and insulin) were unable to inhibit the binding (data not

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ACTIVE SOLUBLE LIVER PANCREASTATIN RECEPTORS

FIG. 1. Dependency of protein concentration of soluble rat liver membranes on PST binding. Triton X-100-soluble extracts were incubated at the indicated concentrations to measure the specific binding of [125I]PST. Each point is the mean of three different experiments.

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FIG. 3. Inhibition of [125I]PST binding to soluble receptors by unlabeled PST. Soluble membranes were incubated with [125I]PST and increasing concentrations of unlabeled PST under standard conditions, as specified in Materials and Methods. Specific binding is expressed as the percentage of maximum binding measured in the presence of tracer alone. Each point is the mean of triplicate measurements from three different experiments. Inset, Scatchard analysis of the PST binding data.

FIG. 2. Time course of association and dissociation of specific [125I]PST to Triton X-100-soluble extracts. Association was performed at 25 C for 90 min. Thereafter, dissociation was measured after the addition of unlabeled PST (1027 M). Each point is the mean of triplicate determinations in a typical experiment. Two other experiments gave similar results.

shown). Scatchard analysis of the data (Fig. 3, inset) showed a straight line, indicating the presence of a single site with a dissociation constant (Kd) of 0.3 6 0.05 nm and a maximum concentration of binding sites at 14 fmol/mg protein. When soluble receptors were incubated with increasing concentrations of guanine nucleotides, binding of the tracer was reduced in a dose-dependent manner (Fig. 4), with the following rank order of potency: GMP-P(NH)P . GTP . GDP . GMP. Furthermore, guanine nucleotides have previously been shown to decrease the affinity of liver receptors to PST (33). These observations suggested that PST receptors and coupled G protein associations may survive the solubilization process. Molecular characterization of the solubilized liver PST receptor

To further analyze the PST receptor in rat liver, [125I]PST was covalently cross-linked to the soluble receptor with the homobifunctional hydroxysuccinimide ester, DSS. After the cross-linked materials were subjected to SDS-PAGE, autoradiography revealed a single band corresponding to the migration of a protein of 85,000 Mr (Fig. 5). The presence or

FIG. 4. Inhibition of binding of [125I]PST to soluble PST receptors by guanine nucleotides. Solubilized receptors were incubated with the tracer and various concentrations of GMP-P(NH)P (closed circles), GTP (open circles), GDP (closed triangles), or GMP (open triangles). Each point is the mean of triplicate determinations from three different experiments.

absence of 100 mm dithiothreitol did not affect the electrophoretic mobility of the 85,000 band. Assuming that one molecule of [125I]PST (Mr, 5,000) is bound per molecule of receptor, the average mol wt of the binding protein that is covalently linked to [125I]PST is 80,000. Figure 5 shows that the labeling of the 85,000 Mr component was inhibited by unlabeled PST. Moreover, the nonhydrolyzable nucleotide GMP-P(NH)P also reduced the labeling of the 85,000 Mr component (Fig. 5). To determine the mol wt of the soluble PST receptor, Triton X-100 extract was loaded onto a Sephacryl S-300 column and eluted with solubilization buffer with 0.1% Triton X-100. Aliquots of collected fractions were incubated with [125I]PST to determine the elution profile of binding activity (Fig. 6). The soluble receptor was eluted in two peaks, a major peak


FIG. 5. Autoradiogram of SDS-PAGE of [125I]PST covalently crosslinked to soluble PST receptors. Soluble receptors were incubated with [125I]PST in the absence or presence of unlabeled PST (1027 M) or GMP-P(NH)P (1024 M; Gpp). [125I]PST-receptor complexes were separated from free [125I]PST by gel filtration on a Sephadex G-25 column, then treated with 1 mM DSS, and analyzed by SDS-PAGE under nonreducing conditions (2 DTT) or under reducing conditions (1 DTT) in the presence of 0.1 M DTT. The addition of DTT diluted the sample, and as a consequence, 34% less protein was loaded in the gel.

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FIG. 7. Affinity cross-linking of [125I]PST to the gel filtration fractions containing the PST receptors. Fractions containing binding activity were incubated with [125I]PST and loaded onto a G-25 column, then treated with 1 mM DSS and subjected to SDS-PAGE. a, High Mr fraction; b, low Mr fraction; c, control (Triton X-100-soluble extracts).

FIG. 6. Gel filtration profile of soluble PST receptors. Triton X-100 extracts (7 mg) were loaded on a Sephacryl S-300 column (60 3 1.6). Specific binding of [125I]PST was measured in aliquots of each fraction as described in Materials and Methods, using native PST (1027 M) (closed circles) for nonspecific binding. The sensitivity to guanine nucleotides was assessed by performing binding experiments in aliquots of each fraction in the presence of GMP-P(NH)P (1024 M; open circles). The column was calibrated with proteins of known Stokes radii. The dashed line refers to the protein concentration.

corresponding to an apparent Mr of 80 kDa and a Stokes radius of 3.9 nm. In addition, we found a minor peak corresponding to a Mr of 170 kDa and a Stokes radius of 5 nm. The possible association with a G protein was assessed by inhibition of [125I]PST binding with GMP-P(NH)P in the fractions corresponding to the peaks. As shown in Fig. 6, the 170-kDa component was more sensitive to the nucleotide (same sensitivity observed for cold PST), whereas binding in the 80-kDa component was more sensitive to cold PST than to GMP-P(NH)P. This may suggest a possible association of the PST receptor with a putative G protein that could account for the 90 kDa that separate the two peaks. This explanation is supported by the cross-linking experiments of the chromatographic fractions with [125I]PST. Figure 7 shows the autoradiograms of the SDS-PAGE analysis of the cross-linking experiments from the two peaks described above. The radioactive band corresponding to the receptor-tracer complex showed the same migration pattern in the [125I]PST

FIG. 8. Immunodetection of the GTP-binding proteins in the gel filtration fractions. Triton X-100 extracts and fractions corresponding to the binding activity of the gel filtration chromatogram were resolved on SDS-PAGE and transferred to nitrocellulose membrane as described in Materials and Methods. Immunodetection of b-subunit (A) and aq-subunit (B) using specific antisera was carried out as described in Materials and Methods. Lane a refers to the fraction corresponding to the 170-kDa component. Lane b refers to the fraction corresponding to the 80-kDa component. Lane c refers to the control (total soluble extracts).

cross-linking of both chromatographic peaks, supporting the assumption that the 170-kDa peak consists of the soluble PST receptor component of 80,000 Mr associated with another protein. To further address this issue, we looked for the presence

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of G proteins in the chromatographed Triton X-100 extracts. As shown in Fig. 8A, a G protein was present in the fraction corresponding to the 170-kDa component, as assessed by anti-G protein bcommon-subunit immunoblotting. On the other hand, the fraction corresponding to 80 kDa contained much less G protein (Fig. 8A). Moreover, specific anti-asubunit antibodies revealed the presence of a Gq protein in the 170-kDa component (Fig. 8B), further suggesting the association of the 80-kDa receptor component with the 90-kDa component of the heterotrimeric Gq protein. The second band of about 60 kDa observed in the immunoblotting of the control soluble extracts (Fig. 8B, lane a) corresponds to a rat liver protein previously described to cross-react with this antibody (51). To test the glycoprotein nature of the soluble PST receptor, Triton X-100 extract was applied to wheat-germ lectinSepharose column, and glycoproteins were eluted from the column with 0.3 m NAG. Fractions from the column were assayed for [125I]PST binding. As shown in Fig. 9, a peak of [125I]PST binding protein was eluted with NAG. These data support the hypothesis of a functional PST receptor as a glycoprotein that can be purified by using lectins. Discussion

In this paper, we report solubilization of the PST receptor from rat liver membranes in an active and stable state using the nonionic detergent Triton X-100, which was more efficient than other detergents, such as CHAPS and Nonidet P-40 (not shown). We previously described the presence of PST receptors in rat liver membranes (33) and studied its function and characterization. Here, we found very similar characteristics in the soluble PST receptor. Thus, PST binds with a Kd of 0.3 nm, which is similar to the Kd (0.2 nm) for binding to membrane receptors (33). The binding capacity of the soluble receptor (14 fmol/mg protein) is close to that observed for membrane receptors (15 fmol/mg protein), suggesting that there is no selective solubilization compared to that for total proteins.

FIG. 9. Lectin affinity chromatography of soluble PST receptors. Five milligrams of solubilized receptors were loaded onto a 5-ml WGASepharose 6MB column that had been preequilibrated in HEPESglycerol buffer containing 0.1% Triton X-100. The column was washed, and glycoproteins were eluted from the column with 0.3 M NAG, as described in Materials and Methods. The fractions were then assayed for binding activity. A typical experiment is shown.

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The molecular characterization of soluble PST receptors by covalent cross-linking revealed a protein of 85,000 Mr. This is much higher than the Mr observed in native membranes. A possible explanation could be the dimerization of the receptor under soluble conditions, yielding a Mr about double that observed in membranes. However, as we had previously suggested (33), a proteolytic process may account for these differences. In this line, protein solubilization may protect against receptor degradation. Thus, a PST receptor of 80,000 Mr is within the range of any other known G protein-coupled receptor. Moreover, gel permeation chromatography of soluble PST receptors confirmed the mol wt and revealed a protein component with a Stokes radius of 3.9 nm. Another molecular form of 170,000 Mr and Stokes radius of 5 nm was found to have binding activity from the gel filtration chromatography fractions. However, cross-linking experiments in 170-kDa fractions and SDS-PAGE analysis revealed the same 85,000 component, suggesting the association of the receptor with a G protein. In fact, the binding of these fractions was very sensitive to GMP-P(NH)P, and the presence of a Gq protein detected by specific immunoblots was in line with this hypothesis. Besides, other peptide receptors, such as vasoactive intestinal peptide receptors, have been previously reported to cosolubilize with a G protein (48, 52). An alternative explanation could be the dimerization of the receptor, which would yield about the same size. However, both hypotheses remain speculative. Further purification and characterization of PST receptor will be required to determine the structures or subtypes of PST receptors. On the other hand, the small GMP-P(NH)P sensitivity of the 80-kDa peak may be explained by the coelution of receptor and G protein in the same fractions, taking into account their similar Mr. In fact, the presence of a Gq protein was assessed by specific immunoblotting in the same fraction, although significantly less than the amount found in the 170-kDa peak. In any case, the functional state of the soluble receptor was ascertained by the guanine nucleotide sensitivity of the binding of the tracer to solubilized PST receptors, as previously found in membrane-bound receptors, suggesting a functional coupling to G proteins (41). Moreover, the PST-dependent guanosine triphosphatase activity found in liver membranes (34) further supports this explanation. The role of calcium in PST signaling in the hepatocyte (30, 31), mediated by the stimulation of a phospholipase C (34), had raised the hypothesis of the possible involvement of a Gq in the signaling of PST receptor, although a pertussis toxinsensitive G protein seems to mediate the PST-stimulated guanylate cyclase activity (34). In fact, a dual signaling mechanism seems to mediate PST action in liver (41). cGMP negatively regulates the activation of phospholipase C by PST (34). Previous work also suggested that cGMP can alter hormone signaling by interfering with the production of inositol trisphosphate through the activation of cGMP-dependent protein kinase (53–55). Moreover, it has been shown that cGMP-dependent protein kinase may interfere with inositol trisphosphate formation and calcium mobilization by phosphorylation of a pertussis toxin-sensitive G protein (56, 57). There is also reported indirect evidence for the involvement of a pertussis toxin-sensitive G protein in the PST effect exerted in the insulin-secreting RIN m5F cell line and parietal


cells of gastric mucosa (21, 58), although no PST receptors have yet been described in these cells. As previously observed with other membrane-bound receptors, soluble PST receptor is a glycoprotein that interacts with WGA-Sepharose and is eluted with NAG. Therefore, substantial purification of the PST receptor may be achieved by this method. In conclusion, we have solubilized for the first time active PST receptors from rat liver membranes and partially characterize its function and molecular form. PST receptor seems to be a glycoprotein of 80,000 Mr functionally coupled to G proteins, one of which may be Gq. This successful solubilization of functional PST receptor and partial purification with lectins may lead to its ultimate purification and amino acid sequencing. Acknowledgments

22.

23. 24. 25. 26. 27. 28. 29.

We are grateful to Prof. R. Goberna for his continuous support. We also thank Concepcioń Munõz and Carmen Penã for their clerical work.

30.

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