Bombesin and structurally related peptides including gastrin releasing peptide (GRP) are potent mitogens for Swiss 3T3 cells. The early cellular and molecular responses elicited by bombesin and structurally related peptides have been elucidated in detail. Further understanding of the molecular basis of the potent mitogenic response initiated by bombesin is required in order to elucidate the mechanism by which the occupied receptor communicates with effector molecules in the cell. Transmembrane signalling mechanisms involving either a tyrosine kinase or a guanine nucleotide-binding regulatory protein (G protein) have been proposed. Here we summarize our experimental evidence indicating that a G protein(s) is involved in the coupling of the bombesin receptor to the generation of intracellular signals related to mitogenesis. Evidence for the role of G proteins in bombesin signal transduction pathways has been obtained by assessing the effects of guanine nucleotide analogues on both receptor-mediated responses in permeabilized cells and ligand binding in membrane preparations. We found that [125I]GRP-receptor complexes were solubilized from Swiss 3T3 cell membranes by using the detergents taurodeoxycholate or deoxycholate. Addition of guanosine 5-[γ-thio]triphosphate (GTPγS) to ligand-receptor complexes isolated by gel filtration enhanced the rate of ligand dissociation in a concentration-dependent and nucleotide-specific manner. These results demonstrate the successful solubilization of [125I]GRP-receptor complexes from Swiss 3T3 cell membranes and provide evidence for the physical association between the ligand-receptor complex and a guanine nucleotide binding protein(s).

The cells of many tissues and organs in vivo are maintained in a non-proliferating state (G0/G1). However, such cells remain viable and can be induced to resume DNA synthesis and cell division when exposed to external stimuli such as hormones, antigens or growth factors. In this manner the growth of individual cells is regulated according to the requirements of the whole organism. The elucidation of the molecular mechanism by which these mitogens regulate growth and differentiation at the cellular level may prove crucial to understanding both the normal proliferative response and the unrestrained growth of cancer cells.

Many studies of growth factors have used cultured fibroblasts, such as murine 3T3 cells, as a model system. These cells cease to proliferate when they deplete the medium of its growth-promoting activity, and can be stimulated to re-initiate DNA synthesis and cell division either by replenishing the medium with fresh serum, or by the addition of purified growth factors or pharmacological agents in serum-free medium. Studies performed using such quiescent cells and defined combinations of growth factors have revealed the existence of potent and specific synergistically acting signal transduction pathways, initiated almost immediately following mitogen addition (Rozengurt, 1986, 1989).

It is increasingly recognized that small regulatory peptides act as molecular messengers in a complex network of information-processing by cells throughout the body. They may act on post-ganglionic receptors (neurotransmitters), nearby cells (paracine hormones) or distant target organs (endocrine hormones). The classical role of these peptides as fast-acting neurohumoral signallers has recently been challenged by the discovery that they also stimulate slow-acting mitogenesis (reviewed in Rozengurt, 1986; Zachary et al. 1987a; Woll and Rozengurt, 1989a). In particular, bombesin (Rozengurt and Sinnett-Smith, 1983), vasopressin (Rozengurt et al. 1979,1981), bradykinin (Woll and Rozengurt, 1988), vasoactive intestinal peptide (Zurier et al. 1988), endothelin (Takuwa et al. 1989) and vasoactive intestinal contractor (Fabregat and Rozengurt, 1990) can act as growth factors for cultured 3T3 cells. Collectively, these findings demonstrate that the mitogenic actions of regulatory peptides are mediated by multiple signalling pathways, and imply that the participation of regulatory peptides in the control of cell proliferation may be broader and more fundamental than previously thought.

The peptides of the bombesin family, including gastrin-releasing peptide (GRP), are of particular significance. These peptides are potent mitogens for Swiss 3T3 cells in the absence of other growth-promoting factors (Rozengurt and Sinnett-Smith, 1983) and may act as autocrine growth factors for small cell lung cancer (SCLC) (Woll and Rozengurt, 1989a). Indeed, the autocrine growth loop of bombesin-like peptides may be only a part of an extensive network of autocrine and paracine interactions involving a variety of neuropeptides in SCLC (Woll and Rozengurt, 1989b). Thus, murine Swiss 3T3 cells which express receptors for several mitogenic neuropeptides and are capable of multiple neuropeptide regulation, provide a model system relevant to SCLC. A detailed-understanding of the signal transduction pathways in this model system may identify novel targets for therapeutic intervention.

Signal transduction

The early cellular and molecular responses elicited by bombesin and structurally related peptides (1isted in Table 1) have been elucidated in detail. The cause-effect relationships and temporal organization of these early signals and molecular events have been reviewed elsewhere (Rozengurt, 1988, 1989; Rozengurt et al. 1988). They provide a paradigm for the study of other growth factors and mitogenic neuropeptides and illustrate the activation and interaction of a variety of signalling pathways.

Table 1.

Events in the action of bombesin in Swiss 3T3 cells

Events in the action of bombesin in Swiss 3T3 cells
Events in the action of bombesin in Swiss 3T3 cells

A central problem in understanding the molecular basis of the potent mitogenic response initiated by bombesin is to elucidate how the occupied receptor communicates with effector molecules in the cell. Transmembrane signalling mechanisms involving either a tyrosine kinase or a guanine nucleotide-binding regulatory protein (G protein) have been proposed to couple growth factor receptors to intracellular effectors (e.g. see Rozengurt, 1986). Bombesin stimulation of tyrosine phosphorylation has been reported (Cirillo et al. 1986; Gaudino et al. 1988) and the possibility that bombesin receptor signalling is associated with this kinase activity was raised. However, bombesin associated tyrosine kinase activity was not detected by another laboratory (Isacke et al. 1986) and the molecular weight of the tyrosine phosphorylated band does not coincide with that of the putative receptor identified by affinity cross-linking in intact cells or membrane preparations (see below).

It was suggested that a pertussis toxin-sensitive G protein could couple the bombesin receptor to the enzymes. which hydrolyze polyphosphoinositides (Letterio et al. 1986). However, the demonstration that pertussis toxin did not inhibit bombesin stimulation of inositol phosphate formation, Ca2+ mobilization or activation of protein kinase C in Swiss 3T3 cells (Zachary et al. 1987b), did not support this hypothesis. These experiments did not rule out, however, the possibility that another class of G protein, which was insensitive to pertussis toxin, might be involved in the pathway of polyphosphoinositide breakdown and activation of protein kinase C.

Evidence for the role of G proteins in signal transduction pathways can be obtained by assessing the effects of guanine nucleotide analogues on both receptor-mediated responses in permeabilized cells and ligand binding in membrane preparations. In what follows we summarize our experimental evidence indicating that a G protein(s) is involved in the coupling of the bombesin receptor to the generation of intracellular signals related to mitogenesis.

Protein phosphorylation in permeabilized cells

One of the most striking events induced by bombesin and structurally related peptides is a rapid increase in the phosphorylation of an acidic cellular protein of apparent Mr=80 000 (termed 80 K) which is a prominent substrate of protein kinase C in Swiss 3T3 cells (Rozengurt et al. 1983; Zachary et al. 1986; Isacke et al. 1986; Erusalimsky et al. 1988). The phosphorylation of this protein is also enhanced by biologically active phorbol esters (Rozengurt et al. 1983; Rodriguez-Pena and Rozengurt, 1985, 1986; Blackshear et al. 1985, 1986), diacylglycerols (Rozengurt et al. 1984; Issandou and Rozengurt, 1989) and other growth factors and neuropeptides (Rozengurt et al. 1983; Rodriguez-Pena and Rozengurt, 1985, 1986; Blackshear et al. 1985). Furthermore, the same 80 K protein is phosphorylated in cell-free systems either by activation of protein kinase C or by addition of the purified enzyme (Blackshear et al. 1986; Rodriguez-Pena and Rozengurt, 1986). A protein closely related to fibroblast 80 K has been purified from rat brain (Morris and Rozengurt, 1988) and partially sequenced (Erusalimsky et al. 1989). This protein, in turn, is related to a protein kinase C substrate purified from bovine brain (Albert et al. 1987) and recently cloned (Stumpo et al. 1989) but does not show significant homology to any other known protein. It is well established that the increase in the phosphorylation of 80 K provides a specific marker for protein kinase C activation.

Recent work from this laboratory has characterized the phosphorylation of 80 K in digitonin permeabilized Swiss 3T3 cells, and employed this technique to study the mechanism of bombesin-induced activation of protein kinase C (Erusalimsky et al. 1988; Erpsalimsky and Rozengurt, 1989). A salient feature of the results is that the GDP analogue GDPβS inhibited the stimulation of 80 K phosphorylation by bombesin in a selective manner. GDPβS is known to prevent the activation of G proteins by inhibiting the binding of GTP. The fact that GTP can reverse the inhibitory effect of GDP/1S is consistent with this notion. The findings indicate that guanine nucleotides modulate the transduction of the signal from the bombesin receptor and imply that a G protein links the bombesin receptor to the generation of an intracellular signal, which in turn activates protein kinase C in Swiss 3T3 cells. Further experimental work, using membrane or receptor preparations, was necessary to elucidate whether the putative G protein(s) is associated with the receptor and to clarify the role of tyrosine phosphorylation in the transduction of the bombesin signal.

Bombesin receptor in membranes from Swiss 3T3 cells

Binding measurements and chemical cross-linking experiments using [125I]GRP show that bombesin-like peptides interact with specific, high-affinity receptors located on the cell surface. [125I]GRP-binding is inhibited by various bombesin-like peptides in proportion to their ability to stimulate DNA synthesis, but not by structurally unrelated mitogens (Zachary and Rozengurt, 1985a,b). Two potent bombesin antagonists, [D-Arg1,D-Phe5,D-Trp79,Leu11]substance P and [Leu13-ψ (CH2NH)-Leu14] bombesin, inhibit both GRP-binding and bombesin/GRP-stimulated mitogenesis (Woll and Rozengurt, 1988; Woll et al. 1988). Thus, bombesin and related peptides interact with receptors that are distinct from those for other mitogens in Swiss 3T3 cells.

Recently, the properties of the mitogenic bombesin receptor have been examined in membrane preparations from Swiss 3T3 cells. It is noteworthy that addition of Mg2+ (5-10 mM) during the homogenization of these cells is crucial in order to stabilize the bombesin receptor in the resulting membrane preparation (Sinnett-Smith et al. 1990). [125I]GRP-binding to such membranes is specific, saturable and reversible. Scatchard analysis indicates the present of a single class of high-affinity binding sites. The Kd obtained from such analysis (2 × 10− 10M) is in excellent agreement with the equilibrium constant derived from rate constants (Rozengurt and Sinnett-Smith, 1990). These results are consistent with the existence of a homogeneous population of bombesin/GRP binding sites in membranes of Swiss 3T3 cells.

The physical properties of the bombesin/GRP receptor have been investigated using an affinity-labelling method. Analysis of extracts of cells which have been preincubated with [125I]GRP and then treated with disuccinimidyl cross-linking agents reveals the presence of a major band migrating with apparent Mr 75 000 – 85 000 (Zachary and Rozengurt, 1987a; Kris et al. 1987; Sinnett-Smith et al. 1988). Furthermore, [125I]GRP can be cross-linked to an Mr 75 000 – 85 000 glycoprotein of Swiss 3T3 membranes but not to membranes from cells lacking bombesin receptors (Sinnett-Smith et al. 1990). The radio-labelled Mr 75 000-85 000 protein binds to wheat germ lectin-sepharose columns and can be eluted with ALacetyl-D-glucosamine, suggesting that it is a glycoprotein (Sinnett-Smith et al. 1988; and unpublished results). In addition, treatment with endo- β -N- acetylglycosaminidase F reduces the apparent molecular weight of the affinity-labelled band from 75 000-85 000 to 43 000, indicating the presence of A-linked oligosaccharide groups (Kris et al. 1987; Sinnett-Smith et al. 1988). Thus, the bombesin/GRP receptor appears to be a glycoprotein of apparent Mr 75 000-85 000 with N-linked carbohydrate side-chains and a polypeptide core of Mr 43 000.

The availability of membrane preparations that retain specific bombesin receptors is useful in the identification of the signal-transduction mechanism(s) that couples the receptor for these neuropeptides with the generation of intracellular events. A decrease in ligand affinity for receptors produced by added guanine nucleotides is characteristic of a receptor-G protein interaction (Bourne et al. 1988). Sinnett-Smith et al. (1990) demonstrated that the non-hydrolyzable GTP analogue GTPγS caused a specific and concentration-dependent inhibition of [125I]GRP-binding and cross-linking to 3T3 cell membranes. The effect is due primarily to an increase in the equilibrium dissociation constant (Kd) rather than to a decrease in the number of receptors. A typical experiment is shown in Fig. 1A. This modulation of ligand affinity by guanine nucleotides provides further evidence that a G protein couples the mitogenic bombesin receptor with intracellular effector systems.

Fig. 1.

(A) GTPγS reduces the affinity of the bombesin receptor for [125I]GRP as determined by Scatchard analysis. Membranes in 100 μl of binding medium were incubated in the presence of various concentrations of [125I]GRP at 37°C either in the absence (○) or in the presence of GTPγS (10 μM), (•). Specific binding was determined after 10 min of incubation. Non-specific binding was measured by the addition of at least a 1000-fold excess of unlabelled bombesin, or 1 μM bombesin for concentrations of [125I]GRP below 1nM. Scatchard analysis of the data is shown: bound [125I]GRP is expressed as fmol25gg−1 of membrane protein; the free [125I]GRP concentration is expressed in μM. All other experimental details were as described by Sinnett-Smith et al. (1990). (B) Swiss 3T3 membranes show no detectable tyrosine phosphorylation by bombesin. Membranes (200 gg) were incubated in the absence (O) or presence of either bombesin (20 nM) (B) or PDGF (30 nM) (P) at 37°C for 7.5 min in 250gl of 50 mu Hepes, 5mM MgCl2, 0.5 mM MnCl2, 100 gM NaVO4, 20 μ M ZnCl2 (buffer A) containing [32P]ATP (15 μCi). The reaction was terminated by the addition of 250 ml of buffer A containing Triton X-100 (2%). The membranes were solubilized for 20 min at 4°C, and non-extractable material removed by centrifugation at 16 000 g for 10 min at 4 °C. The supernatant was then incubated with BSA-agarose (100 μl) for 1h at 4°C. Following centrifugation at 16 000 g for 10 s to remove the BSA-agarose, the supernatant was incubated with monoclonal antiphosphotyrosine antibody coupled to agarose at 4 °C for 2 h. Following 3 washes with buffer A, the phosphotyrosinyl proteins were eluted from the matrix with SDS-sample buffer (0.1 M Tris-HCl, pH 6.8, 10% (w/v) glycerol, 2% (w/v) SDS, 0.1M DTT) and analyzed by PAGE. The gel was then soaked in 1 M KOH at 55°C for 1h, dried and autoradiographed.

Fig. 1.

(A) GTPγS reduces the affinity of the bombesin receptor for [125I]GRP as determined by Scatchard analysis. Membranes in 100 μl of binding medium were incubated in the presence of various concentrations of [125I]GRP at 37°C either in the absence (○) or in the presence of GTPγS (10 μM), (•). Specific binding was determined after 10 min of incubation. Non-specific binding was measured by the addition of at least a 1000-fold excess of unlabelled bombesin, or 1 μM bombesin for concentrations of [125I]GRP below 1nM. Scatchard analysis of the data is shown: bound [125I]GRP is expressed as fmol25gg−1 of membrane protein; the free [125I]GRP concentration is expressed in μM. All other experimental details were as described by Sinnett-Smith et al. (1990). (B) Swiss 3T3 membranes show no detectable tyrosine phosphorylation by bombesin. Membranes (200 gg) were incubated in the absence (O) or presence of either bombesin (20 nM) (B) or PDGF (30 nM) (P) at 37°C for 7.5 min in 250gl of 50 mu Hepes, 5mM MgCl2, 0.5 mM MnCl2, 100 gM NaVO4, 20 μ M ZnCl2 (buffer A) containing [32P]ATP (15 μCi). The reaction was terminated by the addition of 250 ml of buffer A containing Triton X-100 (2%). The membranes were solubilized for 20 min at 4°C, and non-extractable material removed by centrifugation at 16 000 g for 10 min at 4 °C. The supernatant was then incubated with BSA-agarose (100 μl) for 1h at 4°C. Following centrifugation at 16 000 g for 10 s to remove the BSA-agarose, the supernatant was incubated with monoclonal antiphosphotyrosine antibody coupled to agarose at 4 °C for 2 h. Following 3 washes with buffer A, the phosphotyrosinyl proteins were eluted from the matrix with SDS-sample buffer (0.1 M Tris-HCl, pH 6.8, 10% (w/v) glycerol, 2% (w/v) SDS, 0.1M DTT) and analyzed by PAGE. The gel was then soaked in 1 M KOH at 55°C for 1h, dried and autoradiographed.

In view of the preceding results, it was important to determine whether bombesin stimulates tyrosine phosphorylation in membrane preparations in which the bombesin receptor remains functional, as judged by binding activity and modulation by guanine nucleotides. Membranes from Swiss 3T3 cells, prepared as described by Sinnett-Smith et al. (1990), were incubated with [y-32P]ATP in the absence or presence of bombesin and then immunoprecipitated with a monoclonal P-Tyr antibody. Platelet-derived growth factor (PDGF) was also tested for comparison. Fig. IB shows that bombesin failed to stimulate any discernible tyrosine phosphorylation, whereas PDGF, under identical conditions, caused a marked increase in the phosphorylation of its receptor. Thus, in contrast to the results of Cirillo et al. (1986), these results show that the bombesin receptor does not possess a closely associated tyrosine kinase activity. This conclusion is in line with the interpretation that the bombesin receptor signals via a G protein but does not rule out that the peptide could stimulate tyrosine phosphorylation in intact cells, in an indirect manner. This possibility warrants further experimental work.

Solubilization of the bombesin receptor from Swiss 3T3 cells membranes: coupling to a G protein

The molecular and regulatory characterization of plasma membrane receptors requires a procedure for their solubilization in a functional state. It is relevant that the binding of various hormonal peptides to their corresponding membrane receptors has been shown to stabilize the receptor molecules, as well as to induce tight association between the receptor and their respective G proteins (Dickey et al. 1987; Polakis et al. 1988; Marie et al. 1989). Consequently, attempts were made to solubilize bombesin/GRP receptors under conditions in which the ligand was pre-bound to the receptor prior to detergent extraction.

Coffer et al. (1990) found that [125I]GRP-receptor complexes were solubilized from Swiss 3T3 cell membranes by using the detergents taurodeoxycholate or deoxycholate. These detergents promoted ligand—receptor solubilization in a dose-dependent manner. In contrast, a variety of other detergents including Triton X-100, octylglycoside, CHAPS, digitonin, cholic acid and n-dodecyl-β-D maltoside, were much less effective. A typical experiment is shown in Fig. 2. Membrane preparations from Swiss 3T3 cells were incubated with [125I]GRP and then freed of unbound ligand by centrifugation. The membrane pellet was resuspended in a solution containing 0.5% deoxycholate and incubated for 30 min at 4°C. The solubilized material was separated from the non-extractable material by centrifugation for 60 min at 100 000 g and chromatographed on a Sephadex G-200 column at 4°C. Fig. 2A shows that a sharp peak of radioactivity was eluted with an apparent molecular weight of 190 000, while the remaining radioactivity co-eluted with free [125I]GRP. The peak of radioactivity eluting near the void volume of the G-200 column was abolished by adding an excess of unlabelled bombesin together with [125I]GRP during labelling of the membrane (Fig. 2). This experiment indicates that only a partial dissociation of the solubilized ligand—receptor complex occurred during the chromatographic separation and suggests that the ligand—receptor complex is physically associated to other proteins. This possibility was tested more stringently by using a higher detergent concentration (0.25% instead of 0.1%) during the chromatography. To circumvent the extensive dissociation of [125I]GRP that occurs at this detergent concentration, the ligand was cross-linked to the receptor prior to solubilization. Fig. 2 (inset) shows that even in the presence of 0.25% deoxycholate, the cross-linked ligand-receptor complex remains associated to other proteins.

Fig. 2.

Gel filtration profiles of solubilized [125I]GRP-receptor complexes. Swiss 3T3 membranes (0.9 mg) were incubated in 30 ma Hepes, 5mM MgCl2, 0.25 M sucrose, 10μgml−1 aprotonin, 1mgml−1 bacitracin (binding medium) for 10 min at 37°C with [125I]GRP (0.5 nM) in the absence (•) and presence (○) of 10μM bombesin. After centrifugation, the membranes were solubilized with 0.5% taurodeoxycholate in binding medium at a final protein concentration of 4 mg ml−1. The supernatant (200 pl) obtained was analyzed by gel filtration on a G-200 column (40 cm×0.9 cm) eluted with 30mM Hepes, 5ma MgCl2 and 0.1% taurodeoxycholate. Other experimental conditions were as described by Coffer et al. (1990). Vo, void volume of column. Inset: analysis of cross-linked complex by gel filtration. Membranes (100 μg) were chemically cross-linked to [125I]GRP (0.5 nM) using EGS as described previously (Sinnett-Smith et al. 1990). Following solubilization of the membranes with taurodeoxycholate (0.5%) as described above, the supernatant was analyzed on a G-200 column eluted with 0.25% taurodeoxycholate. Fraction of 0.4ml were collected at a flow rate of 8mlh−1 in both cases. The columns were calibrated using Blue Dextran (void volume of 12.8 ml), amylase (200×103Mr), bovine serum albumin (68xlO3Mr) and carbonic anhydrase (29×lO3Mr.).

Fig. 2.

Gel filtration profiles of solubilized [125I]GRP-receptor complexes. Swiss 3T3 membranes (0.9 mg) were incubated in 30 ma Hepes, 5mM MgCl2, 0.25 M sucrose, 10μgml−1 aprotonin, 1mgml−1 bacitracin (binding medium) for 10 min at 37°C with [125I]GRP (0.5 nM) in the absence (•) and presence (○) of 10μM bombesin. After centrifugation, the membranes were solubilized with 0.5% taurodeoxycholate in binding medium at a final protein concentration of 4 mg ml−1. The supernatant (200 pl) obtained was analyzed by gel filtration on a G-200 column (40 cm×0.9 cm) eluted with 30mM Hepes, 5ma MgCl2 and 0.1% taurodeoxycholate. Other experimental conditions were as described by Coffer et al. (1990). Vo, void volume of column. Inset: analysis of cross-linked complex by gel filtration. Membranes (100 μg) were chemically cross-linked to [125I]GRP (0.5 nM) using EGS as described previously (Sinnett-Smith et al. 1990). Following solubilization of the membranes with taurodeoxycholate (0.5%) as described above, the supernatant was analyzed on a G-200 column eluted with 0.25% taurodeoxycholate. Fraction of 0.4ml were collected at a flow rate of 8mlh−1 in both cases. The columns were calibrated using Blue Dextran (void volume of 12.8 ml), amylase (200×103Mr), bovine serum albumin (68xlO3Mr) and carbonic anhydrase (29×lO3Mr.).

Several hormone receptors known to be functionally coupled to G proteins remain physically associated with them after detergent solubilization. To determine whether the solubilized [125I]GRP-receptor complex is functionally coupled to a G protein(s), we tested whether the ligand-receptor complex isolated by gel filtration retains the ability to be regulated by guanine nucleotides. Swiss 3T3 membranes were incubated with [125I]GRP and then the detergent-solubilized extract was chromatographed on a G-100 column. The ligand-receptor complex eluting in the void volume was pooled and incubated in the absence or in the presence of increasing concentrations of GTPγS for 30 min at 37°C. Then, bound [125I]GRP was separated from dissociated ligand by chromatography (Coffer et al. 1990). As shown in Fig. 3, GTPγS caused a dose-dependent decrease in the level of [125I]GRP-receptor complex. In contrast, addition of GMP, ATP and ATPγS at 100 μ M did not have any detectable effect on the stability of the solubilized [125I]GRP-receptor complex. The results of Coffer et al. (1990) and those shown in Figs 2 and 3 demonstrate for the first time the successful solubilization of [125I]GRP-receptor complexes from Swiss 3T3 cell membranes, and provide functional evidence for a bombesin/GRP receptor-G protein interaction. The solubilization of the bombesin receptor in an active form may prove an important step for attempting its purification and reconstitution into phospholipid vesicles.

Fig. 3.

GTPγS promotes dissociation of [125I]GRP from [126I]GRP-receptor complexes in a concentration-dependent and specific manner. Swiss 3T3 membranes (2 × 1.0 mg) were labelled with [125I]GRP (0.5 nM) in binding medium at 37 °C for 10 min, solubilized with 0.5% deoxycholate and chromatographed on two identical Sephadex G-100 columns (20×0.9 cm) as described in Fig. 2. Fractions containing the solubilized [125I]GRP-receptor were then pooled. Samples (50-100 μ l) were incubated in either the absence or the presence of GTPγS at the indicated concentrations, or the specified nucleotides as indicated, all at 100 μm. Following incubation of the reaction mixture at 37 °C for 30 min, the dissociated, free [12SI]GRP was separated from the [125I]GRP-receptor complex by spun column chromatography as described by Coffer et al. (1990). The results are expressed as the percentage of [125I]GRP which remains bound to the receptor complex with respect to the control. The data represent the means±s.E.M.; n=6.

Fig. 3.

GTPγS promotes dissociation of [125I]GRP from [126I]GRP-receptor complexes in a concentration-dependent and specific manner. Swiss 3T3 membranes (2 × 1.0 mg) were labelled with [125I]GRP (0.5 nM) in binding medium at 37 °C for 10 min, solubilized with 0.5% deoxycholate and chromatographed on two identical Sephadex G-100 columns (20×0.9 cm) as described in Fig. 2. Fractions containing the solubilized [125I]GRP-receptor were then pooled. Samples (50-100 μ l) were incubated in either the absence or the presence of GTPγS at the indicated concentrations, or the specified nucleotides as indicated, all at 100 μm. Following incubation of the reaction mixture at 37 °C for 30 min, the dissociated, free [12SI]GRP was separated from the [125I]GRP-receptor complex by spun column chromatography as described by Coffer et al. (1990). The results are expressed as the percentage of [125I]GRP which remains bound to the receptor complex with respect to the control. The data represent the means±s.E.M.; n=6.

Bombesin initiates a complex cascade of signalling events that culminates in the stimulation of DNA synthesis and cell division (summarized in Table 1). PDGF stimulates a similar set of early signals (Rozengurt, 1986; Williams, 1989) and, like bombesin, promotes mitogenesis in the absence of other growth factors. These conclusions were recently substantiated using homodimers of the A chain of PDGF, which binds preferentially to one class of PDGF receptor rather than to all classes of receptors expressed in Swiss 3T3 cells (Mehmet et al. 1990). It is now well established that the tyrosine kinase activity of the PDGF receptor plays a central role in transduction of the mitogenic signal by PDGF. In contrast, the experiments summarized here provide strong evidence that the bombesin receptor is directly coupled to a G protein signal transduction pathway. Thus, it is likely that the bombesin receptor belongs to the superfamily of G protein-linked receptors which are glycoproteins of core Mr 40 000 – 50 000, thought to traverse the cytoplasmic membrane seven times (Lefkowitz and Caron, 1988). Other receptors for the regulatory peptides, substance P (Yokota et al. 1989; Hershey and Krause, 1990), substance K (Masu et al. 1987) and angiotensin (Jackson et al. 1988), belong to this superfamily. As yet, however, the elucidation of the structure of these receptors does not provide information concerning the class (i.e. Gs, G¡, Go etc.) or number of G proteins to which they are coupled.

The nature of the G protein(s) that couple the bombesin receptor to the activation of phospholipase C remains obscure. Indeed, none of the pertussis toxin-insensitive G proteins involved in regulating phospholipase C have been identified. The isolation and purification of other members of the G protein family (1ike Gs and Gi) has been greatly assisted by the use of bacterial toxins such as cholera toxin and pertussis toxin, which ADP-ribosylate their target proteins. A similar toxin directed to the G protein(s) that couples various neuropeptide receptors to phospholipase C has not been discovered.

Recently, Pasteurella multocida toxin (PMT) has been identified as an extremely potent mitogen for Swiss 3T3 cells and other cultured cells (Rozengurt et al. 1990). This toxin causes a dramatic increase in the formation of inositol phosphates (Rozengurt et al. 1990) and stimulates protein kinase C activity (Staddon et al. 1990). In contrast, PMT does not promote accumulation of cyclic AMP. While several possibilities remain open, it is intriguing to consider the possibility that PMT facilitates signal transduction by altering the properties of a G protein, such as that described here to be coupled to the bombesin receptor.

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