Association of guinea pig lung bombesin receptors with pertussis toxin-sensitive guanine nucleotide binding proteins

ejp ELSEVIER

European Journal of Pharmacology Molecular Pharmacology Section 269 (1994) 87-93

molecular pharmacology

Association of guinea pig lung bombesin receptors with pertussis toxin-sensitive guanine nucleotide binding proteins Estelle Lach, Alexandre Trifilieff, Didier Scherrer, Jean-Pierre Gies * Laboratoire de Neuroimmunopharmacologie pulmonaire, INSERM CJF 91-05, Universitd Louis Pasteur - Strasbourg I, B.P. 24, 67401 I L L K I R C H Cedex, France Received 9 February 1994; revised MS received 20 May 1994; accepted 14 June 1994

Abstract

The possible interaction of bombesin receptors with guanine nucleotide binding protein in guinea pig lung was studied. The non-hydrolysable GTP analogue guanosine-5'-[y-thio]triphosphate (GTPyS) was shown to decrease [125I-Tyr4]bombesin binding in a concentration-dependent manner. The specificity of this effect was assessed by examining the effects of other guanine nucleotides on this binding at a concentration of 1 mM. GMP and GDP weakly inhibited [azsI-Tyr4]bombesin binding (2 and 19%, respectively), whereas GTP, guanosine-5'-[/3-thio]triphosphate (GDP/3S), and 5-guanylylimidodiphosphate (GppNHp) exhibited similar potencies, inducing 52%, 46%, and 43% inhibition of [125I-Tyr4]bombesin binding respectively. Saturation experiments performed in the absence and presence of 100 /zM GTPTS indicated the presence of a single population of receptors in both cases. However, the addition of GTPyS induced a marked decrease in the number of receptors (from 1.76 fmol/mg protein to 0.78 fmol/mg protein) without significantly altering the dissociation constant (Ka). These results provide evidence that bombesin receptors are coupled to a G-protein signal transduction pathway in guinea pig lung. We have further characterised this G-protein on the basis of its toxin sensitivity. Pretreatment of the lung membranes with either pertussis (10 /zg/ml) or cholera toxin (50 tzg/ml) was performed. Cholera toxin treatment did not affect the ability of GTPyS to inhibit [125I-Tyr4]bombesin binding to guinea pig lung membranes. However, pertussis toxin treatment induced a decrease in binding and resulted in the inability of GTPyS to inhibit [125I-Tyr4]bombesin binding in a concentration-dependent manner. These results suggest that bombesin receptors of the guinea pig lung interact with pertussis toxin-sensitive G-proteins.

Keywords: Bombesin receptor, lung; Guanine nucleotide sensitivity; G-protein; Pertussis toxin; Cholera toxin; (Guinea pig)

1. Introduction

The bombesin-like peptides are a family of peptides that regulate pulmonary epithelial, mesenchymal and neuroendocrine cell responses (Zachary et al., 1987b; Sunday et al., 1988). Some of the lung effects of bombesin-like peptides, which include the amphibian peptide bombesin and its mammalian counterparts, gastrin-releasing peptide (GRP) and neuromedins, are known. They are potent mitogens for Swiss 3T3 cells (Rozengurt and Sinnett-Smith, 1983), neuroendocrine ceils (Aguayo et al., 1990), human bronchial epithelial

* Corresponding author. Dr Jean-Pierre Gies, Laboratoire de Neuroimmunopharmacologie pulmonaire, INSERM CJF 91-05, Universit6 Louis Pasteur - Strasbourg I, B.P. 24, 67401 ILLKIRCH Cedex, France. Tel.: (33) 88.67.69.06; Fax: (33) 88.67.86.38. 0922-4106/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 9 2 2 - 4 1 0 6 ( 9 4 ) 0 0 1 0 0 - D

cells (Willey et al., 1984) and several small cell lung carcinoma cell lines (Weber et al., 1985; Cuttitta et al., 1985; Carney et al., 1987), and are also able to induce a potent bronchoconstrict0r effect in guinea pig lung both in vivo (Impicciatore and Bertaccini, 1973; Belvisi et al., 1991) and in vitro (Lach et al., 1993). To date, the only study undertaken in the lung describes the coupling of the bombesin receptor to its mitogenic effects which mainly involves G-protein as demonstrated in Swiss 3T3 cells (Fischer and Schonbrunn, 1988; Cattaneo and Vicentini, 1989) and small cell lung cancer (SCLC) cells (Sharoni et al., 1990). Contradictory results were obtained concerning the sensitivity to toxins of the G-proteins, previously described as being coupled to the bombesin receptors. In agreement with Letterio et al. (1986) who first reported the involvement of a pertussis toxin-sensitive G-protein in the bombesin-induced mitogenesis, similar G-pro-

88

E. Lach et ak / European Journal of Pharmacology - Molecular Pharmacology Section 269 (1994) 87-93

teins were also found to be implicated in the coupling to bombesin receptors in both Swiss 3T3 cells (Zachary et al., 1987a) and medullary thyroid carcinoma cells (Abe et al., 1992). Subsequent studies have, however, shown that pertussis toxin does not efficiently inhibit early events after bombesin stimulation, such as increase in Ca 2 + or inositol phosphates in Swiss 3T3 cells (Fischer and Schonbrunn, 1988; Zachary et al., 1987a) and pancreatic acini (Sekar et al., 1991). Evidence that bombesin-like peptides exert their effects by acting on different receptors has been provided in a variety of tissues (Von Schrenck et al., 1990). Recent molecular cloning has confirmed the existence of at least two different bombesin receptors, one GRP-preferring bombesin receptor (Spindel et al., 1990; Battey et al., 1991) and one neuromedin B-preferring receptor (Wada et al., 1991). Both receptors possess seven putative transmembrane spanning segments and belong to the guanine nucleotide-binding protein coupled receptor superfamily. Having described previously the presence of GRPpreferring bombesin receptors in guinea pig lung (Lach et al., 1991) and having demonstrated their involvement in bombesin-induced bronchoconstriction (Lach et al., 1993), we have further undertaken this study to determine whether the GRP-preferring bombesin receptor were coupled to a guanine nucleotide binding protein in guinea pig lung. This was achieved by studying the effects of guanine nucleotides on bombesin binding to lung membranes. In addition, we have examined the sensitivity to pertussis and cholera toxins of the G-proteins which are coupled to the GRP-preferring bombesin receptors in guinea pig lung.

2. Materials and methods

2.1. Membrane preparation Male albino Dunkin Hartley guinea pigs weighing 300-400 g (Elevage de Saint Antoine, Pleudaniel, France) were anaesthetised with 45 m g / k g sodium pentobarbitol injected intraperitoneally. The animals were decapitated, bled and the lungs were removed. Whole lungs were dissected free of connective and vascular tissues, rinsed, blotted dry, and then weighed. All membrane preparations were performed at 0-4°C as described previously (Lach et al., 1991). The protein concentrations were determined as described elsewhere (Gies et al., 1989).

2.2. Receptor binding assay [125I-Tyr4]bombesin binding was carried out in a total volume of 0.2 ml in binding medium containing;

50 mM TrisHC1, pH 6.8, 5 mM MgCl 2, 1 mM EGTA, 0.2% BSA, 0.1% bacitracin, 0.1 mM PMSF. The assays contained 100 pM [125I-Tyra]bombesin, various concentrations of unlabelled drugs and 180-200 tzg of membrane protein. Incubations were performed at 25°C for 70 min. The steady state for specific binding was verified under these conditions (Lach et al., 1991). Bound [125I-Tyr4]bombesin was harvested by rapid vacuum filtration through G F / C glass fiber filters (Whatman, Clifton, N J) pretreated with 0.2% aqueous polyethylenimine. Filters were rinsed three times with 4 ml of ice-cold buffer and radioactivity was measured directly using a y-counter with an efficiency of 80%. In all binding experiments no more than 10% of the total radioligand added was bound to the membranes. Specific [125I-Tyr4]bombesin binding was defined as the difference between total binding and binding in the presence of 1 mM of unlabelled bombesin. Non-specific binding did not exceed 50% of total [125I-Tyr4]bombesin binding.

2.3. ADP-ribosylation of membranes by toxins After membrane preparation, the last pellet was resuspended by vigorous vortexing in ribosylation buffer (50 mM TrisHCl, pH 7.4, 1 mM EDTA, 5 mM dithiothreitol, 10 mM thymidine, 1 mM ATP, 0.1 mM GTP, 10 mM nicotinamide, 0.016% sodium cholate, 0.2% BSA). ADP-ribosylation was assayed in a total volume of 400 p.l of ribosylation buffer in the presence of either 1 /zM NAD + for binding experiments, or 1 tzM NAD + and 10 /xCi [32p]NAD + for sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis. Cholera toxin or pertussis toxin was present at 50 g g / m l and 10 txg/ml respectively and had previously been pre-activated at 37°C for 30 min in 50 mM TrisHC1, pH 7.4 containing 25 mM dithiothreitol. ADP-ribosylation assays were carried out at 30°C for 1 hour. Membranes were pelleted, washed twice and resuspended in binding buffer for binding experiments. For SDS-polyacrylamide gel electrophoresis, the reaction was stopped by adding sodium dodecyl sulphate (2%) and 100 /zg/ml bovine serum albumin. Proteins were precipitated, treated, electrophoresed and autoradiographed as described elsewhere (Fischer et al., 1993).

2.4. Data analysis Experimental data for the saturation and inhibition studies were analysed using the non-linear regression analysis described by Munson and Rodbard (Ligand program; Elsevier-Biosoft, Cambridge, UK) (Munson and Rodbard, 1980). The precision of fit to a one- or two-site model was determined by an F-test ( P < 0.1) by comparing the residual sum of squares for fitting

89

E. Lach et al. / European Journal of Pharmacology - Molecular Pharmacology Section 269 (1994) 87-93

data to a one- or two-site model. Data were weighted with the reciprocal of the variance as described previously (Gies et al., 1989).

"~

2.5. Drugs

~,~ 60

[125I-Tyr4]bombesin (2200 Ci/mmol) and [32p] NAD + (30 Ci/mmol) were obtained from the Radiochemical Center (New England Nuclear Boston, MA). Unlabelled bombesin was purchased from Bachem (Bubendorf, Switzerland). GTP, GDP, GMP, bovine serum albumin, bacitracin, phenylmethylsulphonylfluoride and cholera toxin were from Sigma Chemical (St-Louis, MO). Pertussis toxin was obtained from List Biological (Campbell, CA). GppNHp, GDP/3S, GTPyS were purchased from Boehringer Mannheim Biochemicals (Indianapolis, IN). All other reagents were of the highest grade available.

~

3. Results

3.l. Interaction of guanine nucleotides with the bombesin receptor To determine whether the bombesin receptor is coupled to a G-protein, we first examined the effect of the non-hydrolysable GTP analogue, GTPyS, on the specific binding of [125I-Tyr4]bombesin to guinea pig lung membranes. Figure 1 shows that GTPyS inhibited [t25I-Tyr4]bombesin binding in a concentration-dependent manner, with half-maximal effect achieved at 12 /xM. The specificity of this effect was studied by

.~

100.

80

10-3M)

Guanine Nucleotides

Fig. 2. Inhibition of specific [~25I-Tyr4]bombesin binding by various guanine nucleotides analogues in guinea pig lung membranes. Effect of each compound, tested at 1 mM, is expressed as the percentage of radioactivity specifically bound in the absence of guanine nucleotides analogues (1100-1260 dpm/assay). Values are means from two independent experiments performed in triplicate on separate membrane preparations.

examining the effect of several guanine nucleotides on agonist binding to bombesin receptors in lung membranes. 1 mM GTPyS reduced the binding of [12sITyra]bombesin by greater than 60%, whereas 1 mM GMP and GDP were unable to displace [125ITyr4]bombesin binding. However, the specific binding of [125I-Tyr4]bombesin was inhibited by GTP, GDP/3S and GppNHp with approximately equal potencies, 52%, 46% and 43% inhibition respectively (Fig. 2). To define whether the GTPyS-induced inhibition of [125I-Tyra]bombesin binding was due to a decrease in the affinity or a decrease in the number of receptors, the specific binding of [125I-Tyr4]bombesin was measured as a function of increasing concentrations of the radiolabelled ligand in the absence and presence of 100 ~zM GTPTS (Fig. 3). 700

80-

•=-

[,-

40 20

e~

=

100

•

600

_m

~ , ~ 400

dO

R

u

O

L ' ~ 300

2o 0

100

9 IGTPTSI (-Log M) Fig. 1. Inhibition of [12Sl-TyrZ]bombesin binding by the non-hydrolysable GTP analogue, GTPyS, in guinea pig lung membranes. Membranes were incubated with 100 pM of [12SI-Tyr4]bombesin and various concentrations of GTPyS. Binding of [125I-Tyra]bombesin is expressed as the percentage of radioactivity specifically bound in the absence of GTPyS (1336+282 dpm/assay). Values are means_+ S.E.M. from three independent experiments performed in triplicate on separate membrane preparations.

0

i

0

100

i

200

i

300

i

400

1

500

600

[IZSl-Tyr4lbombesin, pM Fig. 3. Saturation experiments of [1251-Tyr4]bombesin in the absence ( l l ) and presence (D) of 100 p~M GTPyS. Non-specific binding was determined in the presence of 1 ~ M of unlabelled bombesin. Values are means from two independent experiments performed in triplicate on separate membrane preparations.

E. Lach et al. / European Journal of Pharmacology - Molecular Pharmacology Section 269 (1994) 87-93

90

Addition of G T P y S decreased specific binding of [lZSI-Tyr4]bombesin to lung membranes without modifying the proportion of non specific binding. Under both conditions, in the presence or absence of G T P y S , equilibrium binding data gave linear Scatchard plots which were best fit to an equation describing binding to a single population of saturable receptors. In the presence of GTP-/S, the receptors still appeared as one population but with a reduced number of binding sites for the ligand. Scatchard analysis of the binding data demonstrated that addition of G T P y S caused a marked decrease in B . . . . from 1.76 (1.60; 1.95) f m o l / m g protein to 0.78 (0.56; 0.98) f m o l / m g protein, but did not significantly change the K d, 116 pM (113; 120) compared with 91 pM (105; 77) (Fig. 4). This 56% reduction in Bmax accounts for the decreased binding observed in competition experiments.

3.2. Toxin sensiti~,ity of the G-protein coupled to the bombesin receptor We identified the nature of the G-proteins coupled to the bombesin receptors on the basis of their sensitivity to pertussis and cholera toxin. This was performed by measuring the effect of these toxins on bombesin receptor binding. Covalent modification of G-proteins by these toxins disrupts their normal interaction with receptors. One consequence of this perturbation is to reduce the ability of guanine nucleotides to inhibit agonist binding. Therefore, to determine whether the G-protein associated with the bombesin receptor was modified by either cholera or pertussis toxin, we examined the effect of these toxins on the capacity of G T P T S to modulate radioligand binding. Cholera toxin treatment did not significantly ( P = 0.87) alter the ability of G T P y S to 0.008

0.006

"

llO-

a~

I O0

-~

90

[.,-~

80

~

70

,~

60

=.

50

0

I 7

I 6

I 5

i 4

i 3

[ G T P y S ] (-Log M) Fig. 5. Inhibition of [12SI-Tyr4]bombesin binding by G T P y S in control ([]), cholera toxin-treated ( • ) , and pertussis toxin-treated ( • ) guinea pig lung membranes. Results are expressed as percentage of the initial specific binding (difference between total binding and non specific binding) in each condition (326 +_36 d p m / a s s a y ) . Values are m e a n s + s . e . m , from three independent experiments performed in triplicate on separate m e m b r a n e preparations.

inhibit [125I-Tyr4]bombesin binding to guinea pig lung membranes compared with control membranes (Fig. 5). On the other hand, pertussis toxin pretreatment of the membranes results in a 25% decrease in [~25ITyr4]bombesin binding and prevents G T P y S from modulating [125I-Tyr4]bombesin binding (Fig. 5). Both these results are in agreement with the coupling of the bombesin receptor via a pertussis-sensitive G-protein. A control experiment was performed to demonstrate that the absence of effect observed with cholera toxin is not a result of experimental conditions leading to the absence of cholera toxin-catalysed ADP-ribosylation. Thus, after pretreatment of the membranes with cholera toxin, in the presence of [32p-]NAD and unlabelled NAD, instead of unlabelled NAD alone, we observed the ADP-ribosylation of a protein of about 44-45 kDa which could correspond to G~ (Fig. 6). This ADP-ribosylation, not observed in the control experiment, demonstrates the effectiveness of the cholera toxin under our experimental conditions.

1.

4. Discussion

"~ 0.004

0.002

0

-~ 0.5 1 1.5 2 [12SI-Tyr4]bombesin bound (fmol/mg prot) Fig. 4. Representative Seatchard plots of [125I-Tyr4]bombesinbinding to guinea pig lung in the absence ( • ) and presence ([]) of 100 /xM GTPyS. Values are means from two independent experiments performed in triplicate on separate membrane preparations. 0

The major finding of the present study is the identification of a G-protein involved in the coupling of the lung GRP-preferring bombesin receptors. We show that binding of the radioligand agonist [~25I-Tyr4] bombesin is greatly inhibited by G T P and its stable analogues G T P y S , G p p N H p and GDP]3S, and that the specificity of this G T P effect can be assessed by the inability of G D P and G M P to decrease [tzsITyra]bombesin binding. The potency of G T P y S and GTP analogues to cause a 60% reduction in [125ITyra]bombesin binding is similar to the earlier findings

E. Lach et al. / European Journal of Pharmacology

kDa 66

45

36 29 24

20.1 Toxin

-t-

--

Fig. 6. Effect of cholera toxin pretreatment of guinea pig lung membranes on the ADP-ribosylation of proteins. Lung membranes were pretreated ( + ) or not ( - ) with cholera toxin (50 # g / m l ) and were incubated with 10 mCi [32p]NAD. Separation of the proteins was carried out by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The autoradiograph presented is of a gel showing radioactivity incorporated into ADP-ribosylated proteins and is representative of three independent experiments.

both with bombesin (Feldman et al., 1990; Coffer et al., 1990; Sinnett-Smith et al., 1990) and other peptides such as NPY (Walker and Miller, 1988). Guanine nucleotide inhibition of bombesin binding was also found in other cell types, namely GH4C1, HIT cell lines, (Fischer and Schonbrunn, 1988) and Swiss 3T3 fibroblasts (Sinnett-Smith et al., 1990). In saturation experiments, the binding sites in the presence or absence of GTPyS appear as a single population of sites. However, the addition of GTPyS to lung membranes reduces the apparent number of binding sites (Bmax) by approximately 56% without altering the radioligand affinity (Kd). The Bma x value of 1.78 fmol/mg protein (1.60; 1.95) for control membranes is in the same range as that found in our previous study (Lach et al., 1991). This decrease in receptor number in the presence of guanine nucleotides has also been observed with other peptide hormone receptors such as bradykinin (Etscheid and Villereal, 1989), substance P (Lee et al., 1983), neuropeptide Y (Walker and Miller, 1988; Unden and Bartfai, 1984) and somatostatin (Enjalbert et al., 1983) receptors. An unusual feature in the case of the lung GRPpreferring bombesin receptors is that the presence of

-

Molecular Pharmacology Section 269 (1994) 87-93

91

guanine nucleotides results in a decrease in receptors, rather than a decrease in affinity, as was originally observed for the glucagon receptor (Rodbell et al., 1971) and later also for bombesin receptors in GHaC ~ and HIT cells (Fischer and Schonbrunn, 1988) and Balb 3T3 fibroblasts (Benya et al., 1992). A possible explanation, which could account for the partial decrease in peptide binding in the presence of guanine nucleotides in guinea pig lung, would be the presence of two subpopulations of receptors, one of which is insensitive to guanine nucleotides and the other being responsive to guanine nucleotides resulting in such a large loss in affinity that these sites cannot be labelled in equilibrium binding experiments. However, in the case of bombesin receptors, there is no evidence of multiple binding sites in guinea pig lung (Lach et al., 1991, 1993). This possibility, however, cannot be ruled out considering that subtypes of GRP-preferring receptors can exhibit affinities in the same range (about 100 pM) which would appear as a single site in binding assays with [125I-Tyr4]bombesin. The discrimination between the two populations of sites would require analogues exhibiting a greater affinity for one GRPpreferring bombesin subtype compared with the other. None of the many bombesin binding assays in various tissues has revealed a heterogeneity of GRP-preferring receptors. Our results indicate that, in the absence of GTPyS, all the receptors are in a high affinity state (K d = 116 pM). One can speculate that the addition of GTPyS converts part of the whole pool of bombesin receptors to such a low affinity state that they become indistinguisable considering the high level of nonspecific binding for high concentrations of radioligand used and that a pool of 44% of bombesin receptors is insensitive to guanine nucleotides. Interestingly, the presence of a guanine nucleotide-resistant population of receptors was previously described for bombesin receptor in Swiss 3T3 cells (Coffer et al., 1990). However, Sekar et al. (1991) proposed another explanation, with the coupling of the bombesin receptor to different effector systems and perhaps also to different G-proteins. This was put forward on the basis of the ability of various bombesin agonists to activate a common bombesin receptor coupled to phosphoinositide, although their binding to this receptor was differentially regulated by guanine nucleotides. The nature of the G-proteins involved in the most widely studied bombesin transduction signal has not yet been clearly elucidated (Heslop et al., 1986; Takuwa et al., 1987; Zachary et al., 1986; Trepel et al., 1988). One way in which G-proteins can be distinguished is by determining whether they are either insensitive to, or inactivated by, pertussis toxin-mediated a n d / o r cholera toxin-mediated ADP-ribosylation of their a-subunits. It has been demonstrated that pertussis toxin catalyzes specifically the ADP-ribosylation of G i / G o regulatory

92

E. Lach et al. / European Journal of Pharmacology - Molecular Pharmacology Section 269 (1994) 87-93

proteins, whereas cholera toxin ADP-ribosylates specifically Gs-proteins (Graziano and Gilman, 1987). To determine whether the G-protein coupled to the bombesin receptor ressembled those which were substrates for cholera or pertussis toxin, we have examined the ability of G T P y S to modulate bombesin binding in toxin-treated lung membranes. Inhibition by guanine nucleotides of bombesin binding was not blocked by treatment with cholera toxin. In addition, after cholera toxin treatment of the m e m b r a n e s we observed the ADP-ribosylation of a protein of 44-45 kDa. This protein could be Gs (Kaziro et al., 1991). This result confirms that our experimental conditions allow the ADP-ribosylation of G~ and that the absence of an effect by cholera toxin does not result from an incorrect protocol. Concentrations higher than 10-5M G T P y S did not further reduce the [ 125I-Tyr 4]bombesin binding in control and cholera toxin-treated membranes. However, in normal m e m b r a n e s G T P y S was able to decrease [125I-Tyr4]bombesin binding at concentrations up to 10--3M. This discrepancy between normal and control conditions may be due to the ADP-ribosylation conditions, since the control m e m branes were treated in ADP-ribosylation conditions i.e. ADP-ribosylation buffer and incubation at 30°C during lh. In addition, pertussis toxin induces a decrease in bombesin binding. Moreover, the ability of G T P y S to decrease bombesin binding to these receptors is lost in pertussis toxin-treated membranes. These two results may suggest that a G-protein coupled to the bombesin receptor has the characteristics of G J G o regulatory proteins or at least is pertussis toxin-sensitive. The decrease in [~25I-Tyr4]bombesin binding induced by pertussis toxin treatment, was similar to that of G T P y S in untreated membranes, suggesting that GTP-binding protein are ADP-ribosylated by pertussis toxin and uncoupled from bombesin receptors. However, the magnitude of these decreases was not similar and this may be explained by incomplete ADP-ribosylation of GTP-binding proteins in our conditions. Such an observation was described for substance P receptors where, even after ADP-ribosylation of the membranes, addition of a high concentration of G T P cause a reduction of substance P binding (Morishima et al., 1989), Another possibility could be the participation of other GTP-binding proteins in addition to the pertussis toxin substrate. The observation that a G-protein linked to the lung GRP-preferring bombesin receptor is pertussis toxinsensitive is in agreement with other studies. Pertussis toxin was shown to block bombesin-stimulated D N A synthesis (Letterio et al., 1986) and mitogenesis in Swiss 3T3 cells (Zachary et al., 1987) and to inhibit the GRP-induced calcitonin secretion from medullary thyroid carcinoma cells (Abe et al., 1992). In addition, a

recent study demonstrates the direct involvement of Gi-proteins as well as p21 ra' proteins in bombesin receptor-mediated signal transduction in pancreatic acinar cells (Pr6frock et al., 1992). In summary, we have demonstrated for the first time that the GRP-preferring bombesin receptors of the lung are regulated by guanine nucleotides via a pertussis toxin-sensitive G-protein. We could nevertheless not rule out a possible additional coupling of the bombesin receptor to GTP-binding proteins insensitive to pertussis and cholera toxins. Direct evidence for the interaction of a single receptor with multiples classes of G proteins has been described for some other receptors (Milligan, 1993). The role of these proteins in the mechanism of bombesin action in the lung remains to be elucidated but the involvement of these G-proteins in the bombesin-induced bronchoconstrictor effect observed in the guinea pig lung (Lach et al., 1993) cannot be excluded, taking into account the fact that the most described bombesin transduction signal involves G-proteins coupled with phosphoinositide breakdown.

Acknowledgements This work was supported by the Ligue Nationale contre le Cancer (Comit6 du Haut-Rhin and F6d6ration Nationale des Centres de Lutte contre le Cancer) and the Institut National de la Sant6 et de la Recherche M6dicale (CJF 91-05). We acknowledge Y. Landry for useful discussions during the course of this work. We would also like to thank M. Mousli for his help and advice during part of this work.

References Abe, Y., A. Kanamori, Y. Yajima and T. Kameya, 1992, Increase in cytoplasmic Ca 2+ and stimulation of calcitonin secretion from human medullary thyroid carcinoma cells by the gastrin-releasing peptide, Biochem. Biophys. Res. Commun. 185, 833. Aguayo, S., T. Kino, J. Waldron, K. Sherritt, M. Kane and Y. Miller, 1990, Increased pulmonary neuroendocrine cells with bombesinlike immunoreactivity in adult patients with eosinophilic granuloma, J. Clin. Invest. 86, 838. Battey, J.M., J. Way, M.H. Coriay, H. Shapira, K. Kusano, R. Harkins, J.M. WU, T. Slattery, E. Mann and R.J. Feldman, 1991, Molecular cloning of the bombesin/GRP receptor from Swiss 3T3 cells, Proc. Natl. Acad. Sci. USA, 88, 395. Belvisi, M.G., C.D. Stretton and P.J. Barnes, 1991, Bombesin-induced bronchoconstriction in the guinea pig: mode of action, J. Pharmacol. Exp. Ther. 258, 36. Benya, R.V., E. Wada, J.F. Battey, Z, Fathi, L.H. Wang, S.A. Mantey, D.H. Coy and R.T. Jensen, 1992, Neuromedin B receptors retain functional expression when transfected into BALB 3T3 fibroblasts: analysis of binding, kinetics, stoichiometry, modulation by guanine nucleotide-binding proteins, and signal transduction and comparison with natively expressed receptors, Mol. Pharmacol. 42, 1058. Carney, D.N., F. Cuttitta, T.W. Moody. and J.D. Minna, 1987,

E. Lach et aL / European Journal of Pharmacology - Molecular Pharmacology Section 269 (1994) 87-93

Selective stimulation of small cell lung cancer clonal growth by bombesin and gastrin-releasing peptide, Cancer Res. 47, 821. Cattaneo, M.G. and L.M. Vicentini, 1989, Differential mechanisms of inositol phosphate generation at the receptors for bombesin and platelet-derived growth factor, Biochem. J. 262, 665. Coffer, A., I. Fabregat, J. Sinnett-Smith and E. Rozengurt, 1990, Solubilization of the bombesin receptor from Swiss 3T3 cell membranes. Functional association to a guanine nucleotide regulatory protein, FEBS Lett. 263, 80. Cuttitta, F., D.N. Carney, J. Mulshine, T.W. Moody, J. Fedorka, A. Fischler and J.D. Minna, 1985, Bombesin-like peptides can function as autocrine growth factors in human small cell lung cancer, Nature 316, 823. Enjalbert, A., R. Rasolonjanabary, E. Moyse, C. Kordon and J. Epelbaum, 1983, Guanine nucleotide sensitivity of [12Sl]-iodo ntyr somatostatin binding in rat adenohypophysis and cerebral cortex, Endocrinology 113, 822. Etscheid, B.G. and M.L. Villereal, 1989, Coupling of bradykinin receptors to phospholipase C in cultured fibroblast is mediated by a G-protein, J. Cell. Physiol. 140, 264. Feldman, R.I., J.M. Wu, J.C. Jenson and E. MANN, 1990, Purification and characterization of the bombesin/gastrin-releasing peptide receptor from Swiss 3T3 cells, J. Biol. Chem. 265, 17364. Fischer, J.B. and A. Schonbrunn, 1988, The bombesin receptor is coupled to a guanine nucleotide-binding protein which is insensitive to pertussis and cholera toxins, J. Biol. Chem. 263, 2808. Fischer, T., C. Bronner., Y. Landry and M. Mousli, 1993, The mechanism of inhibition of alkylamines on the mast-cell peptidergic pathway, Biochim. Biophys. Acta 1176, 305. Gies, J.-P., C. Bertrand, P. Vanderheyden, F. Waeldele, P. Dumont, G. Pauli and Y. Landry, 1989, Characterization of muscarinic receptors in human, guinea pig and rat lung, J. Pharmacol. Exp. Ther. 250, 309. Graziano, M.P. and A.G. Gilman, 1987, Guanine nucleotide-binding regulatory proteins: mediators of transmembrane signaling, Trends Pharmacol. Sci. 8, 478. Heslop, J.P., D.M. Blakeley, K.D. Brown, R.F. Irvine, R.F. and M.J. Berridge, 1986, Effects of bombesin and insulin on inositol (1,4,5)trisphosphate and inositol (1,3,4)trisphosphate formation in Swiss 3T3 cells, Cell 47, 703. Impicciatore, M. and G. Bertaccini, 1973 The bronchoconstrictor action of the tetradecapeptide bombesin in the guinea-pig, J. Pharm. Pharmacol. 25, 872. Kaziro, Y., H. Itoh., T. Kozasa., M. Nakafuku and T. Satoh, 1991, Structure and function of signal-transducing GTP-binding proteins, Annu. Rev. Biochem. 60, 349. Lach, E., E.-B. Haddad and J.-P. Gies, 1993, Contractile effect of bombesin on guinea pig lung in vitro: involvement of gastrin-releasing peptide-preferring receptors, Am. J. Physiol. 264, L80. Lach, E., A. Trifilieff, Y. Landry and J.-P. Gies, 1991, High-affinity receptors for bombesin-like peptides in normal guinea pig lung membranes, Life Sci. 48, 2571. Lee, C.M., J.A. Javitch. and S.H. Snyder, 1983, 3H-substance P binding to salivary gland membranes. Regulation by guanyl nucleotides and divalent cations, Mol. Pharmacol. 23, 563. Letterio, J.J., S.R. Coughlin and L.T. Williams, 1986, Pertussis toxin-sensitive pathway in the stimulation of c-myc expression and DNA synthesis by bombesin, Science 234, 1117. Milligan, G., 1993, Mechanisms of multifunctional signalling by G protein-linked receptors,Trends Pharmacol. Sci. 14, 239. Morishima, Y., Y. Nakata and T. Segawa, 1989, Comparison of the effects of ions and GTP on substance P binding to membranebound and solubilized specific sites, J. Neurochem. 53, 1428. Munson, J. and D. Rodbard, 1980, Ligand: a versatile computerized approach to characterization of ligand-binding systems, Anal. Biochem. 107, 220. Pr6frock, A., P. Zimmermann, P. and I. Schulz, 1992, Bombesin

93

receptors interact with G i and p21 ras proteins in plasma membranes from rat pancreatic acinar cells, Am. J. Physiol. 263, G240. Rodbell, M.H.M.J. Krans, S.L. Pohl and L. Birnbaumer, 1971, The glucagon-sensitive adenyl cyclase system in plasma membranes of rat liver. IV. Effects of guanyl nucleotides on binding of 1251glucagon, J. Biol. Chem. 246, 1872. Rozengurt, E. and J. Sinnett-Smith, 1983, Bombesin stimulation of DNA synthesis and cell division in culture of Swiss 3T3 cells, Proc. Natl. Acad. Sci. USA 80, 2936. Sekar, M.C., N. Uemura., D.H. Coy, B.I. Hirschowitz and E.J. Dickinson, 1991, Bombesin, neuromedin B and neuromedin C interact with a common rat pancreatic phosphoinositide-coupled receptor, but are differentially regulated by guanine nucleotides, Biochem. J. 280, 163. Sharoni, Y., J. Viallet, J.B. Trepel and E.A. Sausville, 1990, Effect of guanine and adenine nucleotides on bombesin-stimulated phospholipase C activity in membranes from Swiss 3T3 and small cell lung carcinoma cells, Cancer Res. 50, 5257. Sinnett-Smith, J., W. Lehmann. and E. Rozengurt, 1990, Bombesin receptor in membranes from Swiss 3T3 cells, Biochem. J. 265, 485. Spindel, E.R., E. Giladi, P. Brehm, R.H. Goodman and T.P. Segerson, 1990, Cloning and functional characterization of a complementary DNA encoding the murine fibroblast bombesin/gastrin releasing peptide receptor, Mol. Endocrinol. 4, 1956. Sunday, M.E., L.M. Kaplan, E. Motoyama, W.W. Chin and E.R., Spindel, 1988, Biology of disease. Gastrin-releasing peptide (mammalian bombesin) gene expression in health and disease, Lab. Invest. 59, 5. Takuwa, N., Y. Takuwa, W.E. Bollag and H. Rasmussen, 1987, The effects of bombesin on polyphosphoinositide and calcium metabolism in Swiss 3T3 cells, J. Biol. Chem. 262, 182. Trepel, J., J. Moyer, R. Heikkila and E. Sausville, 1988, Modulation of bombesin-induced phosphatidylinositol hydrolysis in a small cell lung-cancer cell line, Biochem. J. 255, 403. Unden, A. and T. Bartfai, 1984, Regulation of neuropeptide Y (NPY) binding by guanine nucleotides in the rat cerebral cortex, FEBS Lett. 177, 125. Von Schrenck, T., L.H. Wang, D.H. Coy, M.L. Villanueva, S. Mantey and R.T. Jensen, 1990, Potent bombesin receptor antagonists distinguish receptor subtypes, Am. J. Physiol. 259, GG468. Wada, E., J. Way, H. Shapira, K. Kusano, A.M. Lebacq-Verheyden, D. Coy, R. Jensen and J. Battey, 1991, cDNA cloning, characterization and brain region-specific expression of a neuromedin-Bpreferring bombesin receptor, Neuron, 6, 421. Walker, M.W. and R.J. Miller, 1988, 125I-neuropeptide Y and 12sIpeptide YY bind to multiple receptor sites in rat brain, Mol. Pharmacol. 34, 779. Weber, S., J.E. Zuckerman, D.O. Bostwick, K.G. Bensch, B.I. Sikic and T.A. Raffin, 1985, Gastrin releasing peptide is a selective mitogen for small cell lung carcinoma in vitro. J. Clin. Invest. 75, 306. Willey, J.C., J.F. Lechner and C.C. Harris, 1984, Bombesin and the C-terminal tetradecapeptide of gastrin-releasing peptide are growth factors for normal human bronchial epithelial cells, Exp. Cell Res. 153, 245. Zachary, I., J.W. Sinnett-Smith and E. Rozengurt, 1986, Early events elicited by bombesin and structurally related peptides in quiescent Swiss 3T3 cells. I. Activation of protein kinase C and inhibition of epidermal growth factor binding, J. Cell. Biol. 102, 2211. Zachary, I., J. Millar, E. Nanberg, T. Higgins and E. Rozengurt, 1987a, Inhibition of bombesin-induced mitogenesis by pertussis toxin: dissociation from phospholipase C pathway, Biochem. Biophys. Res. Commun. 146, 456. Zachary, I., P.J. Woll and E. Rozengurt, 198719, A role for neuropeptides in the control of cell proliferation, Dev. Biol. 124, 295.