Bombesin activation of phospholipase C β 1 in rat acinar pancreatic cells involves the pertussis toxin-sensitive Gαi3 protein

REGULATORY

ELSEVIER

PEPTIDES Regulatory Peptides 62 (1996) 153 - 159

Bombesin activation of phospholipase C/31 in rat acinar pancreatic cells involves the pertussis toxin-sensitive G,~i3 protein Catherine Pigeon, Muriel Le Romancer, Christine Linard, Miguel J.M. Lewin, Florence Reyl-Desmars * INSERM Unit~ 10, lnstitut F~d~ratif de Recherches Cellules Epithdliales, H~pital Biehat-Claude Bernard, Paris Cedex, France Received 26 June 1995; revised 5 February 1996; accepted 5 February 1996

Abstract

Bombesin stimulation of inositol 1,4,5-trisphosphate (InsP3) formation in rat sonicated pancreatic acinar cells was inhibited by an antibody directed against the pertussis toxin (PTX)-sensitive GTP-binding Gai 3 protein but not by an anti-G,q_l, antibody. After solubilization and gel filtration, [125I-Tyr4]bombesin binding sites were recovered in a peak of protein of 67 ~ 90 kDa with a maximal enrichment corresponding to a molecular mass of 83-kDa. Results obtained from the non-hydrolysable GTP analog guanosine-5'-[ythio]triphosphate (GTPyS) binding, PTX-stimulated ADP-ribosylation and immunoblotting showed that the 83-kDa fraction contained the Gai 3 protein but not the G~q_ll protein. Furthermore, GTPyS increased the bombesin binding dissociation constant (K D) from 0.32 to 0.60 nM, while the anti-G,~3 antibody decreased the maximal binding capacity (Bmax) from 50 to 25 fmol/mg protein without affecting the K D. Mixing solubilized bombesin binding sites with a phospholipase C (PLC) preparation from rat pancreas reconstituted a bombesin-stimulated PLC activity which was markedly inhibited by the anti-G~i3 antibody but unaffected by the anti-G,~q_l~ antibody. In addition, this stimulation was inhibited by an anti-PLC/31 antibody. This result supports the involvement of the PLC/31 isoform in bombesin receptor activation. Keywords: Bombesin receptor; G protein; Solubilization; Phosphoinositide; Phospholipase C; Pancreas

1. Introduction

The amphibian tetradecapeptide bombesin and its mammalian homolog gastrin-releasing peptide (GRP) stimulate exocrine and endocrine pancreatic secretions [1,2]. They also exert a trophic effect on cell maturation and growth [3,4]. Receptor binding sites for bombesin and relative peptides have been kinetically characterized [5-7] and solubilized in pancreas as well as in other tissues [8-12]. Cloning studies show the existence of four receptor subtypes displaying different tissue distributions and physiological functions: the GRP-preferring receptor [13], the neuromedin B-preferring receptor [14] and bombesin receptor subtypes 3 and 4 [15-17]. Primary structures of all

* Corresponding author. INSERM U.10, H6pital Bichat-Claude Bernard, 170 Boulevard Ney, F-75877 Paris Cedex 18, France. Tel.: + 33 1.40.25.83.89; fax: +33 1.46.27.85.36. 0167-0115/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved PH S01 67-0 1 15(96)0001 8-3

these subtypes as deduced from corresponding cDNAs, display the seven-transmembrane-domains featuring the G protein-coupled receptor superfamily. However, the nature of the G protein(s) involved remains poorly documented. It is generally thought that bombesin stimulation of Ins P3 formation and intracellular Ca 2+ mobilization is insensitive to PTX [18-20]. However, our previous report [21] as well as two recent studies from other laboratories [22,23] demonstrated that PTX-sensitive G proteins could be associated with the bombesin receptor in rat pancreatic acinar cells and guinea pig lung. However a recent contradictory report suggested that, in the Swiss 3T3 cell line, both PTX-insensitive Gq and GI, proteins were activated by bombesin [24]. In the present study, we provide biochemical and immunological evidence to support that in rat pancreatic acinar cells, bombesin stimulation of at least the phospholipase C I31 isoform, is mediated through the PTX-sensitive G~i 3 protein activation.

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2. Materials and methods

2.4. GTPyS binding assay

2.1. Rat pancreatic acinar cells isolation

Fractions eluted from the Sephacryl $200 column were incubated, for 30 rain at 37°C, with 150000 cpm [35S]GTPTS (Amersham, France) and 10 /zM (total) or 10 mM (non-specific binding) unlabeled GTPTS. Bound [35S]GTPTS was then separated from the free radioactivity by filtration through HAWP nitrocellulose membrane (Millipore, France). Filters were washed three times with 10% trichloroacetic acid containing 10 mM sodium pyrophosphate, dissolved with acetone and the radioactivity counted.

Acinar cells were isolated from unstarved 200-300 g male Wistar rats (CERJ, France). Pancreases were dissociated by sequential incubations in the presence of 2 mM EDTA or collagenase (0.75 then 1.25 mg/ml, Sigma) according to a method originally described by Amsterdam and Jamieson [25]. After dispersion, isolated cells were resuspended in a modified oxygenated Krebs medium (Krebs buffer) equilibrated at pH 7.4 and containing: 0.5 mM NaH2PO 4, 1 mM Na2HPO 4, 20 mM NaHCO 3, 80 mM NaC1, 5 mM KC1, 1 mM CaCI 2, 1.5 mM MgC12, 50 mM Hepes NaOH, 11 mM glucose, 0.1% bovine serum albumin, added with 0.1% bacitracin and 0.1 m g / m l trypsin inhibitor to prevent proteolysis. 2.2. Solubilization and Sephacryl $200 chromatography

Isolated cells (108) were solubilized, for 30 min at 4°C, in 50 mM Hepes NaOH, pH 7.4, 0.1 m g / m l trypsin inhibitor, 0.01% bacitracin, and 1% Triton X-100 (Hepes buffer) as already described in our previous paper [8,26]. After centrifugation for 15 min at 12000 × g, the supernatant containing the solubilized proteins was applied to a Sephacryl $200 (Pharmacia Biotech., France) chromatography column equilibrated with Hepes buffer and fractions of 0.25 to 0.5 ml were collected. The column was calibrated using thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), lactate dehydrogenase (140 kDa), phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20.1 kDa) and lactalbumin (14.4 kDa) as standards. 2.3. Receptor binding assay

The incubation of 100000 cpm [125I-Tyr4]bombesin (2000 Ci/mmol, NEN, Du Pont de Nemours, France) with Sephacryl $200 eluted fractions was carried out for 30 min at 30°C in a total volume of 100 /zl in the presence (non-specific binding) or absence (total binding) of 1 /zM unlabeled Tyr4bombesin (Bachem, Switzerland). Binding was then stopped by the addition of 500/zl ice-cold Hepes buffer and filtration through Whatman G F / B glass fiber filters which were washed twice with 1 ml of the same Hepes buffer. In all fractions, the non-specific binding was estimated to about 20 ~ 25% of the total binding. When used, the GTPyS analog (Boehringer-Mannheim, Germany) and anti-Gaq_~l (NEN, Du Pont de Nemours, France, Cat. No. NEI-809), anti-G~il. 2 (NEI-801), anti-Gai 3 (NEI803), anti-Gao (NEI-804), or anti-Gas (NEI-805) antibodies directed against the C-terminus of the G protein a-subunits were preincubated with solubilized fractions, for 60 to 90 min, at 30°C.

2.5. ADP-ribosylation assay

Fractions eluted from the Sephacryl $200 column were incubated, for 30 min at 30°C, with 1 n g / m l of PTX (List Biologicals, USA) previously preactivated for 30 min at 37°C, in the presence of 10 mM thymidine, 2 mM EDTA, 1 mM ATP, 5 mM MgC12, 0.1 mM Gpp(NH)p, 50 /xM NAD, 1 mM dithiothreitol, 0.01% bacitracin, and 0.5 /xM [adenylate-32p]NAD (30 Ci/mmol, Amersham, France). Labeled ADP-ribosylated proteins were separated from free radioactivity by filtration as described above for the GTPyS binding assay. 2.6. InsPs assay

Isolated acinar pancreatic cells (107/ml) were incubated, for 2 h at 37°C, with 2.5 /zCi/ml myo-[2-3H]in ositol (17.5 Ci/mmol, Amersharn, France) in Krebs buffer, rinsed, and incubated for 15 rain at 37°C in the same buffer to improve the removal of free [3 H]inositol. Labeled cells were broken for 20 s at 12 /zm (20 kHz) using a sonicator MSE PG 100, power 150 W, and then incubated for 1 h at 37°C with 1 /xg/ml or 2 /xg/ml anti-Gaq_jl, anti-Ga,_ 2 , anti-Ga~ 3 , anti-G~o or anti-Gas antibodies described above or with an anti-IgG (Amersham, France) used as control, and then for 0 up to 10 s without or with 10 nM [Tyr4]bombesin or cholecystokinin (CCK, SigmaAldrich, France). The reaction was stopped at different times by the addition of 0.4 M perchloric acid. After centrifugation for 1 min in a microfuge, acid extracts were neutralized with half a volume of 0.72 N KOH and 0.6 M KCO 3 buffer. Then, supematants were applied to columns containing 1 ml of Dowex converted to formate form. Free inositol was eluted with 1 ml of 0.1 M formic acid. Sequential washes with 1 ml of 0.1 M formic acid containing 0.2 M, 0.4 M, 1 M, ammonium formate progressively eluted the InsP 1, InsP 2 and InsP 3 [21]. 2.7. PLC solubilization and reconstitution experiments

PLC was solubilized according to a modified method of Camps et al. [27]. Briefly, pancreases isolated from male Wistar rats were homogenized in a Krebs buffer containing 0.25 M sucrose, using a motordriven Potter-Elvehjem

C. Pigeon et al. / Regulatory Peptides 62 (1996) 153-159

Teflon-glass at 1800 rpm for 1 min. Membranes were prepared by two sequential centrifugations, one at 60 000 × g for 10 min and the second at 105000 × g for 1 h. They were resuspended in 20 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM dithiothreitol, 4 /xM GDP and frozen at -70°C. For solubilization, membranes were centrifuged three times at 105000 X g, for 10 min and homogenized with Krebs buffer containing 1% sodium cholate. After stirring for 60 min at 4°C, the insoluble material was removed by sequential centrifugations at 50000 × g, for 20 min, and then, at 120000 X g, for 1 h. The PLC activity was assayed, for 10 min at 30°C, in the presence (stimulated) or absence (basal) of 10 nM [Tyr4]bombesin, in a total volume of 100 /.tl containing 150 /zg crude solubilized PLC in 20 mM Tris-HC1, pH 7.5, 1.5 mM MgCI 2, 1 mM ATP, 3 inM benzamidine, 1 /xM leupeptin, 0.1 /xM phenylmethanesulfonyl fluoride, 2 /zg/ml soybean trypsin inhibitor, 1.25 nM EGTA, 25 /xl of each fraction issued from the Sephacryl $200 column and containing 6 mM MgCI2, l mM dithiothreitol, 20% glycerol, 80 mM Tris maleate, pH 7.3, 0.4 mM phosphatidylethanolamine, 0.06% sodium deoxycholate, 50 ~ M GTPTS, 15 mM EDTA, 0.1 /xM CaC12 and 0.4/xCi [3H]phosphatidylinositol 4,5-biphosphate (2-10 Ci/mmol, NEN, Du Pont de Nemours, France). The reaction was stopped by the addition of 0.4 M perchloric acid and Ins P~ isolated as described above. When used, 1 /xg/ml of anti-G,~ 3, antiG,q_l~, anti-PLC~, or 3 /xg/ml of antibody specifically directed against the PLC/3 1 (Upstate Biotechnology Inc, USA, Ref. 05-163) were preincubated for 1 h at 37°C before the assay, with the Sephacryl $200 eluted fractions containing the G~i 3 protein or the solubilized PLC, respectively.

155 A

42 32 E

.

22

m_.. 12 0

2

4 6 T i m e (s)

8

46

10

B

4, 36 2e

0

2

4 6 Time (s)

8

10

Fig. 1. Time course for lnsP3 formation in response to Tyr4bombesin (A) and cholecystokinin (B): effect of anti-G,i 3 an anti-G,~q4 j antibodies. Prelabeled acinar cells were incubated with 10 nM Tyr4bombesin or CCK for the indicated periods of time. Control sonicated cells ( • ) or 1-h pretreated sonicated cells with 1 /xg/ml ( • ) or 2 / z g / m l anti-G,i 3 ( © ) or 1 / x g / m l anti-G,~q_lt (El) antibodies. Values are means_+SEM of three experiments.

3. Results

3.1. Inhibition by the anti-G,i 3 antibody of bombesinstimulated Insp~ formation The addition of 10 nM [Tyr4]bombesin to the sonicated

2.8. Immunoblotting Solubilized fractions issued from the Sephacryl $200 column and containing both bombesin binding sites and G proteins were diluted in 60 mM Tris-HC1, pH 6.8, 3% sodium dodecyl sulfate (SDS), 10% glycerol and 5% /32mercaptoethanol. Samples were loaded onto a 10% SDSpolyacrylamide slab gel surmounted with a 5% stacking zone according to the procedure of Laemmli [28]. The electrophoresis was run overnight at a constant voltage (90 V). Then the gel was transferred onto a nitrocellulose paper for 3 h at room temperature at a constant voltage of 80 V. The nitrocellulose strips were incubated for 90 min at 37°C with PBS containing 10% non-fat dried milk and then incubated for 2 h with 1 /xg/ml of anti-G~i 3 , anti-G~q_l i or anti-/3 (NEN, Du Pont de Nemours, Cat. No. NEI-808) antibodies in the presence of 2% non-fat dried milk, and then for 2 h with the peroxidase-labeled anti-rabbit or anti-mouse IgG Fab fragment antibodies (Amersham, France). The antigen-antibody complex was revealed using the enhanced chemiluminescence procedure of Amersham.

myo-[2-3H]inositol-preincubated acinar cells resulted in a rapid and substantial stimulation of the basal Ins P3 production (from 12 to 42 fmol/106 cells) which peaked at 3 s and then slowly decreased (Fig. 1A). One-hour preincubation of the cells with 1 or 2 /zg/ml of anti-G,~3 antibody resulted in a decrease of about 46 _+ 4% and 65 + 6% of the bombesin maximal stimulation, respectively (Fig. 1A). However, under the same experimental conditions, no effect was observed with 1 /xg/ml of anti-G, q41 antibody (Fig. 1A), whereas it inhibited by 70 + 1% the InsP3 production stimulated by CCK (Fig. 1B). The specificity of this inhibition was also supported by the lack of effect of the anti-G,~i3 antibody on the CCK stimulation (Fig. 1B) as well as the absence of inhibition of the bombesin stimulation observed in the presence of anti-G~l_ 2, anti-G~o, anti-G~ or anti-IgG antibodies (not shown). The involvement of the Gc~i3 protein was also confirmed by the 70% decrease of the 3 s bombesin stimulation observed when the cells were pretreated during 4 h with 10 n g / m l of PTX (not shown) as already described in our previous paper [21].

C. Pigeon et al. / Regulatory Peptides 62 (1996) 153-159

156

0,08 20-1

94

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i

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0.5ml fractions Fig. 2. [125I-Tyr4]bombesin and [35S]GTPTS specific bindings or ADPribosylation to solubilized fractions 1 to 12. Cellular proteins were solubilized with 1% Triton X-100 and passed through a Sephacryl $200 column. Eluted fractions (0.5 ml) were then incubated for 30 min with [125-Tyr 4 bombesin in the absence (total binding) or presence (nonspecific binding) of unlabeled Tyr 4 bombesin or for 30 min with [35S]GTPyS or 60 min with [32p]NAD in the presence ((3) or absence ( O ) of 1 n g / m l PTX. Values are means + SEM of three experiments.

5

10 15 20 Bound (pM)

25

Fig. 4. Scatchard analysis of [1251-Tyr4]bombesin binding to the 83-kDa form: effect of GTP,/S, anti-G~j 3 or anti-G,~q_tl antibody. [1251Tyr4]bombesin was incubated for 30 min with the fraction No. 5 (83-kDa molecular form) with increased concentrations of [Tyr4]bombesin in the absence ( I ) or presence of 50 /xM GTPyS (D) or 1 /xg/ml anti-G,~13 antibody ( A ) or anti-G~q ]l antibody (©). Values are means+SEM of three experiments.

3.3. Interaction of the bombesin receptor with the G~i 3 protein 3.2. Coelution of the solubilized bombesin receptor with the Gi3 protein The Sephacryl $200 gel filtration of Triton X100solubilized cellular proteins, isolated [125I-Tyr4]bombesin binding sites in several fractions containing proteins of molecular masses from 90 to 67 kDa and with a maximal enrichment in fraction No. 5 corresponding to 83 kDa (Fig. 2). These bombesin binding sites were glycosylated, since their treatment with N-glycanase after cross-linking reduced the molecular mass from 83 to 45 kDa (not shown) as already described in the literature [29]. Furthermore, this fraction was enriched in [35S]GTPyS binding and PTX -sensitive ADP-ribosylation suggesting the presence of the G i proteins (Fig. 2). These results were consistent with the presence in fraction No. 5 of a 41-kDa antigenic band corresponding to the G~i 3 subunit and the absence of the PTX-insensitive G~q_]~ protein, the latter being present only in fraction No. 3 (Fig. 3). Western blot analysis revealed also the presence of the/3-subunit of 35 kDa (Fig. 3).

1 Gc~ i3 Gc~q-11

13

2

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4

5

-,='

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6

4--- 41 kDa

,=~

=,. =p

7

,~- 41 kDa

~=

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~

4-- 3 5 k D a

Fig. 3. Immunoblotting of the Sephacryl $200 fractions. Fractions of 0.25 ml eluted from Sephacryl $200 were electrophorated and immunoblotted using anti-Gc~ i3, anti-G~ qll or anti-/3 antibodies.

The Scatchard analysis of the bombesin binding obtained in fraction No. 5 indicated the presence of one single class of specific sites with a K o of 0.32 _ 0.03 nM and a Bma x of 50 q- 1.1 f m o l / m g proteins (Fig. 4). These values are close to those found for the native receptor ( K D = 0.41 + 0.02 nM and Bma x = 55 + 5 f m o l / m g protein, 5500 sites/cell, not shown), suggesting that detergent had no evident deleterious effect on the receptor structure. The addition of 50 /zM GTPyS increased the K D up to 0.60 + 0.06 nM without altering Bma x. In contrast, the preincubation with the anti-G~i 3 antibody reported above to inhibit the bombesin stimulation of Ins P3 formation in isolated sonicated acinar cells and to immunoreact with the solubilized 83-kDa proteins resulted in a 2-fold decrease in the Bma x (25.0 + 0.9 f m o l / m g protein) without affecting the K D (0.32 +__0.07 nM) (Fig. 4). No effect was observed with the anti-G~q_~l antibody (Fig. 4) or with the antiGenii_2, anti-G0~ and anti-G~s antibodies (not shown). 3.4. Reconstitution of the solubilized bombesin receptor and G~i 3 protein with PLC In an attempt to relate these findings to the bombesin stimulation of the Ins P3 formation in sonicated cells, we studied the effects of mixing the fractions with a PLC preparation. A basal phospholipasic activity was thereby reconstituted which distributed like [125i_Tyr 4]bombesin binding with a basal maximal activity observed in fraction No. 5 (83 kDa), corresponding to 0.5 pmol of released I n s P 3. In the presence of 10 nM [Tyr4]bombesin, this activity was stimulated up to 27-fold (13.5 pmol of re-

C. Pigeon et al. / Regulator), Peptides 62 (1996) 153-159 94

83

67 kDa

100 .~_ 80

~ 60 o ~_ 40200

0 1

2

3

4

5

6

7

O.5ml fractions

Fig. 5. PLC reconstitution with Sephacryl $200 fractions. Each eluted fraction issued from Sephacryl $200 was reconstituted with the crude solubilizedpancreatic PLC and the enzymeactivity was measuredin the presence of bombesinalone ( I ) or 1 /xg/ml anti-G,~13(O) or 1 /xg/ml anti-PLCT ([3) or 3 /.,g/ml anti-PLC/31 (0) antibodies. Values are means+ SEM of three experiments.

leased Ins P3) in fraction No. 5 (Fig. 5). Furthermore, this stimulation was 6 1 _ 2% inhibited when 3 /xg/ml of anti-PLC/31 antibody were preincubated with the PLC preparation, but apparently unaffected in the presence of the anti-PLCr antibody. In addition, it was 6 3 _ 6% decreased by the preincubation of Sephacryl $200 eluted fractions with 1 /zg/ml of anti-G~i 3 antibody, whereas the preincubation with the anti-G,q_ll antibody had no effect.

4. Discussion The present findings provide a molecular basis to our previous observation supporting that bombesin stimulation of the Ins P3 formation in rat acinar pancreatic cells is a PTX-sensitive process [21]. These results indicate that this stimulation is inhibited by a specific antibody directed against the PTX-sensitive Gai 3 regulatory subunit but not by other antibodies directed against G~il_2, G~o, G,s or by an anti-IgG antibody used under the same experimental conditions. It was also not affected by the anti-G~q_ll antibody which in contrast inhibits the stimulation by CCK known to be PTX-insensitive. We can speculate that the anti-G,i 3 antibody binding to the C-terminal region of the G protein could prevent the bombesin stimulation by impairing the coupling of the Gai 3 protein to the PLC. The sensitivity for PTX of the signal transduction activated by bombesin has been reported in a few studies [18-20]. More generally, the bombesin stimulation was reported to be PTX-insensitive and involved Gq and Gll proteins. In acinar pancreatic cells, a recent study demonstrated the interaction of activated bombesin receptors with Gil , Gi2 and Gi3 proteins [22]. However, no functional evidence for the involvement of these regulatory proteins in the bombesin activation of the signal transduction has been so far reported. In our experiments, we found no involvement of the Gil and G~2 proteins.

157

In the present study, pancreatic acinar cell bombesin receptors were solubilized in a functional form, still capable to bind bombesin with kinetic parameters close to our results found with native receptors and those previously published [23,30,31 ]. The apparent molecular mass that we found for the solubilized receptor is consistent with that (81 ~ 82 kDa) suggested from cross-linking [29,32,33], and solubilization studies [9-12]. It is consistent with a coelution of the bombesin receptor with the G protein heterocomplex of 80-85 kDa (the /3 and probably the 3/ subunit being present in this fraction) but not with an association between the two proteins (163-168 kDa). However, in the presence of bombesin, Scatchard analysis demonstrated the existence of only one population with high-affinity binding sites. The sensitivity of these sites to GTPyS suggests that in the presence of hormone, the whole receptors are associated with the G protein heterotrimeric complex in a high-affinity state. The decrease in K o observed upon addition of the non-hydrolysable GTP analog could be classically explained by the release of the G protein complex and the shift of the receptor from high to low affinity state. A similar 2-fold shift upon GTP addition was observed in membranes prepared from Swiss 3T3 and BALB/3T3 cells transfected with the GRP receptor cDNA [30]. The addition of the anti-G~i 3 antibody produced an apparent decrease in solubilized bombesin receptor number. This suggests that binding of the antibody to the G protein-receptor complex not only impaired the coupling of the receptor with the PLC but also produced an alteration of the receptor conformation preventing bombesin binding. Using reconstitution experiments, soluble bombesin receptors associated to the PTX-sensitive Gc~i3 proteins were found to stimulate the PLC activity, an enzyme known to catalyse the membrane phosphatidylinositide breakdown as the first step of the InsP3 production [34]. Little is known about the PTX-sensitive pathway of the PLC regulation. The subtypes G,~ and G,o could be involved but reconstitution experiments involving these purified subunits and PLC remained unsuccessful [35]. The purified Gc~0A, G~0B and recombinant G,0 A proteins were demonstrated to activate PLC isozymes in Xenopus oocytes [36]. In contrast, Boyer et al. found recently that recombinant G~q and G, j~ proteins expressed in Sf9 cells transfected using baculovirus could activate purified PLC/31 while Gc~il , G,i 2 and Gai 3 could be without effect [37]. In the same way, when reconstitution experiments with purified enzymes were used, members of the Gq family were found to couple bombesin receptors to the PLC 131, /33 and to a lesser extent, /32 [38]. In our study, a predominantly involvement of the isoform /31 was observed, since bombesin stimulation was decreased by 61% in the presence of the specific anti-PLC/31 antibody. We can suggest that the 49% of the stimulation that are insensitive to this antibody corresponded to the activation by bombesin receptors of other PLC/3 isozyme such as for example, the widely distributed PLC/3 3 [39].

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In conclusion, these findings provide biochemical and immunological evidence for a coupling of the PLC/31 with a G=i 3 regulatory protein in rat acinar pancreatic cells. However, the physiological effects triggered by this PTXsensitive transduction mechanism in the exocrine pancreas remain to be investigated. Furthermore, the additional involvement of /3y subunits or a PTX-insensitive G protein such as the small GTP-binding protein p21 ras in bombesin transduction pathways can be speculated [22].

[16]

[17]

[18]

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[1] Vaysse, N., Les peptides de la famille bomb6sine: de la physiologie aux applications th6rapeutiques potentielles, Gastroenterol. Clin. Biol., 12 (1988) 447-453. [2] Spindel, E.R., Giladi, E., Segerson T.P and Nagalla, S., Bombesinlike peptides: of ligands and receptors, Rec. Prog. Horm. Res., 48 (1993) 365-391. [3] Lhoste, E. F, Aprahamian, M., Balboni, G. and Damg6, C., Evidence for a direct trophic effect of bombesin on the mouse pancreas: in vivo and cell culture studies, Regul. Pept., 24 (1989) 45-54. [4] Lehy, T. and Puccio, F., Promoting effect of bombesin on the cell proliferation in the rat endocrine pancreas during the early postnatal period, Regul. Pept., 27 (1990) 87-96. [5] Scemama, J.L., Zahidi, A., Fourmy, D., Fagot-Revurat, P., Vaysse, N., Pradayrol, L. and Ribet, A., Interaction of [12SI]Tyr4 bombesin with specific receptors on normal human pancreatic membranes, Regul. Pept., 13 (1986) 125-132. [6] Logsdon, C.D., Zhang, J., Guthrie, J., Vigna, S. and Williams, J.A., Bombesin binding and biological effects on pancreatic acinar AR42J cells, Biochem. Biophys. Res. Commun., 144 (1987) 463-468. [7] Sinnett-Smith, J., Lehman, W. and Rozengurt, E., Bombesin receptor in membranes from Swiss 3T3 cells, Biochem. J., 265 (1990) 485-493. [8] Linard, C., Reyl-Desmars, F., Chen, W.W. and Lewin, M.J.M., Solubilization of pancreatic bombesin receptor(s), Ann. NY Acad. Sci., 547 (1988) 471-473. [9] Coffer, A., Fabregat, I., Sinnett-Smith, J. and Rozengurt, E., Solubilization of the bombesin receptor from Swiss 3T3 cell membranes. Functional association to a guanine nucleotide regulatory protein, FEBS Lett., 263 (1990) 80-84. [10] Naldini, L., Cirillo, D., Moody, T.W., Comoglio, P.M., Schlessinger, J. and Kris, R., Solubilization of the receptor for the neuropeptide gastrin-releasing peptide (bombesin) with functional ligand binding properties, Biochemistry, 29 (1990) 5 153-5160. [11] Feldman, R.L., Wu, J.M., Jenson, J.C. and Mann, E., Purification and characterization of the bombesin/gastrin-releasing peptide receptor from Swiss 3T3 cells, J. Biol. Chem., 265 (1990) 1736417372. [12] Staley, J., Coy, D.H., Jensen, R.T. and Moody, T.W., Solubilization and purification of bombesin/gastrin releasing peptide receptors from human cell lines, J. Mol. Neurosci., 29 (1993) 29-40. [13] Spindel, E.R., Giladi, E., Brehm, P., Goodman, R.H. and Segerson, T.P., Cloning and functional characterization of a cDNA encoding the murine fibroblast bombesin/GRP receptor, Mol. Endocrinol., 4 (1990) 1956-1963. [14] Wada, E., Way, J., Shapira, H., Kusano, K., Lebacq-Verheyden, A.M., Coy, D., Jensen, R. and Battey, J., cDNA cloning, characterization, and brain region-specific expression of a neuromedin-Bpreferring bombesin receptor, Neuron, 6, (1991) 421-430. [15] Fathi, Z., Corjay, M.H., Shapira, H., Wada, E., Benya, R., Jensen, R., Viallet, J., Sansville, E.A. and Battey, J.F., BRS-3: a novel

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