©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Differential Modulation of Bombesin-stimulated Phospholipase C and Mitogen-activated Protein Kinase Activity by D-Arg, D-Phe, D-Trp,LeuSubstance P (*)

Fiona M. Mitchell (1)(§), Lynn E. Heasley (2) (3), Nan-Xin Qian (1), Jeffrey Zamarripa (2), Gary L. Johnson (1) (3)

From the (1) Division of Basic Sciences, National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado 80206 and the (2) Division of Renal Medicine and (3) Department of Pharmacology, University of Colorado Medical School, Denver, Colorado 80262

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Mitogenic stimulation of Swiss 3T3 fibroblasts with bombesin results in receptor-mediated activation of a complex array of effectors, including phospholipase C and mitogen-activated protein (MAP) kinase. Incubation of Swiss 3T3 fibroblasts with the 11-amino acid [ D-Arg, D-Phe, D-Trp,Leu]substance P peptide inhibited bombesin-stimulated cell proliferation and phospholipase C activation even at high bombesin concentrations. The peptide did not inhibit the activation of phospholipase C by a GTPase-deficient form of the G-like protein, G, indicating that the peptide does not inhibit phospholipase C and is acting at a point upstream of the activated form of the G protein subunit. The peptide inhibited MAP kinase activation at low bombesin concentrations, but unlike phospholipase C, this inhibition could be overcome with 30 n M bombesin. In control Swiss 3T3 cells, bombesin did not measurably activate Ras or Raf-1 above basal levels. Following incubation of the cells with the [ D-Arg, D-Phe, D-Trp,Leu]substance P peptide, 50 n M bombesin activated Raf-1 4-6-fold over basal levels. Platelet-derived growth factor-stimulated activities of PLC, Ras, Raf-1, and MAP kinase were unaltered after incubation of Swiss 3T3 cells with the [ D-Arg, D-Phe, D-Trp,Leu]substance P peptide, as was platelet-derived growth factor-stimulated growth of the Swiss 3T3 cells. Thus, the peptide behaves as an antagonist that differentially inhibited phospholipase C and MAP kinase signal transduction pathways. The growth arrest observed with the peptide indicates that the bombesin-stimulated activation of MAP kinase is not sufficient to support mitogenesis in Swiss 3T3 cells.


INTRODUCTION

Neuropeptides, such as bombesin, bind to specific, high affinity receptors that mediate a variety of biochemical responses fundamental to embryogenesis, tissue regeneration, and tumorigenesis (1) . The amphibian tetradecapeptide bombesin and structurally related mammalian peptides, including gastrin releasing peptide (GRP),() act as potent mitogens for quiescent murine Swiss 3T3 fibroblasts (2) . Activation of the bombesin receptor results in the stimulation of a variety of signaling enzymes including phospholipases C, A, and D, protein kinase C, and the mitogen-activated protein kinases (MAP kinases) (3, 4, 5, 6, 7) . The bombesin/GRP receptor was cloned from Swiss 3T3 cells and is a member of the G protein-coupled receptor superfamily with seven predicted hydrophobic transmembrane domains (8) . The cloned and expressed bombesin receptor couples to Gfamily members such as Gor G, activates phospholipase C (8) , and, as we demonstrate, also stimulates MAP kinase activity.

Neuropeptides, including bombesin, have been implicated as autocrine growth factors in the pathogenesis of some human small cell lung carcinomas (SCLC) (9) . Evidence for this in part involves a study using a monoclonal antibody directed against the carboxyl terminus of GRP, which has been shown to inhibit the in vitro growth of SCLC cell lines and tumor xenografts in nude mice (9) . Similarly, the 11-amino acid synthetic peptide, [ D-Arg, D-Phe, D-Trp,Leu]substance P, has been shown to act as a potent inhibitor of Swiss 3T3 and SCLC cell growth in vitro. Using Swiss 3T3 cells, the [ D-Arg, D-Phe, D-Trp,Leu]substance P peptide was demonstrated to diminish I-GRP binding to its receptor and inhibit GRP-stimulated calcium mobilization and DNA synthesis (10) .

We investigated the effect of the [ D-Arg, D-Phe, D-Trp,Leu]substance P peptide on the activation of the MAP kinase pathway under conditions in which it inhibits bombesin-stimulated PLC activation and growth arrests Swiss 3T3 cells. In this report, we demonstrate that the [ D-Arg, D-Phe, D-Trp,Leu]substance P peptide inhibits activation of the MAP kinase pathway at low concentrations of bombesin and that this can be overcome in a bombesin concentration-dependent manner. In contrast, PLC activation is inhibited even at high bombesin concentrations.


EXPERIMENTAL PROCEDURES

Materials

The [ D-Arg, D-Phe, D-Trp,Leu]substance P peptide was purchased from Bachem. myo-[2-H]Inositol and phosphorus-32 radionuclide were obtained from DuPont NEN. [-P]ATP was purchased from ICN Biomedicals, Inc. Pertussis toxin was obtained from List Biological Laboratories. The human bombesin/GRP receptor cDNA was from J. Battey, National Institutes of Health.

Cell Culture and Transfection

Swiss 3T3 fibroblasts were maintained in DMEM containing 5% newborn and 5% bovine calf serum, in humidified 7.5% CO, 92.5% air at 37 °C. All experiments were performed with cells between passages 1 and 10 from the original stocks. The cells were quiesced by replacing the growth medium with DMEM supplemented with 0.1% bovine serum albumin for 20-24 h. When the cells were assayed for kinase activity, this medium was replaced with DMEM, 0.1% bovine serum albumin, 15 m M Hepes (pH 7.4) for 5-10 min prior to agonist addition. The isolation of stable clones of Swiss 3T3 cells expressing the polypeptide has been described previously (11) . Stock HEK-293 cells were maintained in DMEM containing 10% bovine calf serum (HyClone) and were transfected transiently by the method of calcium phosphate precipitation (12, 13) .

Measurement of PtdIns-PLC Activity

Cells were seeded on 60-mm dishes and labeled with myo-[2-H]inositol (1 µCi/ml) for 24 h in DMEM/Ham's F-12 medium (1:1) containing 0.1% bovine serum albumin (DMEM/F-12/bovine serum albumin). The cells were then rinsed three times with DMEM, F-12, bovine serum albumin, 15 m M Hepes (pH 7.4) and incubated for 5 min with this medium supplemented with LiCl (20 m M). Agonists were added for an additional 20-min incubation at room temperature. The reactions were terminated with an equal volume of ice-cold methanol/HCl (100:1, v/v) and the cells scraped into an extraction mixture containing HO:methanol/HCl:chloroform (1:1:2, v/v). After vigorous mixing and centrifugation, the aqueous phase was passed over an AG 1-X8 (200-400 mesh) formate anion-exchange column (Bio-Rad). The total [H]inositol phosphates were eluted as described previously (14) and expressed as a percentage of the total [H]inositol incorporated into phospholipid.

Ras, Raf-1, and MAP Kinase Assays

The activation of these proteins was assayed as described previously with slight modifications (11, 15) . In the measurement of Ras activation, the elution buffer contained 0.1% SDS and the guanine nucleotides were resolved by thin layer chromatographic separation using 0.75 M KHPO(pH 3.4). In the experiments represented by Fig. 3 C, the MAP kinase activities were batch eluted from a DEAE-Sephacel column using a high salt concentration (0.5 M, NaCl). The eluate was then assayed in quadruplicate as described previously (15) .


Figure 3:A, bombesin-stimulated MAP kinase activity in Swiss 3T3 cells. Serum-starved Swiss 3T3 cells were stimulated with 50 n M bombesin for 2 min and assayed for MAP kinase activity as described under ``Experimental Procedures.'' The cells were treated with the [ D-Arg, D-Phe, D-Trp,Leu]substance P peptide exactly as described in Fig. 2. The results are from a single experiment representative of 12 independent experiments. B, PDGF-stimulated MAP kinase activity in Swiss 3T3 cells. Serum-starved Swiss 3T3 cells were stimulated with 10 ng/ml PDGF for 5 min in the absence or presence of the [ D-Arg, D-Phe, D-Trp,Leu]substance P peptide (50 µ M, 5-min preincubation) and assayed for MAP kinase activity as described above. Data points are from a single experiment. Three independent experiments gave similar results. C, dose-dependent activation of MAP kinase activity in Swiss 3T3 cells. Serum-starved Swiss 3T3 cells were stimulated with increasing concentrations of bombesin in the absence or presence of the substance P peptide (50 µ M, 5-min preincubation) and assayed for MAP kinase activity using the batch elution protocol as described under ``Experimental Procedures.'' Data points represent the mean ± S.D. of quadruplicate determinations from a single experiment representative of three independent experiments.



Quantification of Data

PhosphorImager analysis was used for a quantitative measure of the products of Raf-1 and Ras activation. Autoradiographs from single, representative experiments are shown, with data from several experiments presented as arbitrary phosphorimaging units.


RESULTS

Selective Inhibition of Cell Growth by the [ D-Arg, D-Phe, D-Trp,Leu]Substance P Peptide-Fig. 1 shows that the [ D-Arg, D-Phe, D-Trp,Leu]substance P peptide inhibited bombesin-stimulated Swiss 3T3 cell proliferation in a concentration-dependent manner. In contrast, the [ D-Arg, D-Phe, D-Trp,Leu]substance P peptide had little or no effect on PDGF-stimulated Swiss 3T3 cell proliferation. The results are similar to previously described cytostatic actions of this peptide with fibroblasts and small cell lung carcinoma lines (10) . The molecular action of the [ D-Arg, D-Phe, D-Trp,Leu]substance P peptide is poorly understood, except that it inhibits the ability of bombesin and other neuropeptides to stimulate calcium mobilization (10) . The [ D-Arg, D-Phe, D-Trp,Leu]Substance P Peptide Selectively Modulates Signal Transduction Pathways-We determined that incubation of Swiss 3T3 cells with [ D-Arg, D-Phe, D-Trp,Leu]substance P peptide inhibited calcium mobilization in response to bombesin stimulation but had little or no effect on the ability of PDGF to mobilize calcium (not shown). This finding indicated that the [ D-Arg, D-Phe, D-Trp,Leu]substance P peptide did not influence calcium stores or the ability of phospholipase C catalyzed release of inositol trisphosphate to mobilize intracellular calcium. Fig. 2 demonstrates that the [ D-Arg, D-Phe, D-Trp,Leu]substance P peptide inhibited the ability of 50 n M bombesin, a maximally effective concentration of bombesin in the absence of the inhibitory peptide, to stimulate inositol phosphate generation. The [ D-Arg, D-Phe, D-Trp,Leu]substance P peptide had no effect on PDGF-stimulated inositol phosphate generation.

The bombesin receptor has a predicted seven-transmembrane structure and couples minimally to Gmembers of the heterotrimeric G protein family (16, 17) . Gand subunits selectively activate phospholipase C (18) . Swiss 3T3 cells stably expressing a mutant GTPase-inhibited Gsubunit, a member of the Gsubfamily of G protein subunits (19) , have a constitutively activated phospholipase C (11) . The [ D-Arg, D-Phe, D-Trp,Leu]substance P peptide had no effect on the ability of the GTPase-inhibited Gto activate phospholipase C but inhibited bombesin stimulation of phospholipase C activity (Fig. 2). Thus, the [ D-Arg, D-Phe, D-Trp,Leu]substance P peptide inhibited receptor but not Gactivation of phospholipase C. The site of action of the [ D-Arg, D-Phe, D-Trp,Leu]substance P peptide is therefore upstream of G protein subunit interaction with phospholipase C.


Figure 2: Stimulation of PtdIns-PLC activity in Swiss 3T3 cells. Total inositol phosphate formation was measured in wild type or 16expressing Swiss 3T3 cells (denoted as G16QL). Serum-starved, myo-[H]inositol-labeled cells were challenged with 50 n M bombesin or 10 ng/ml PDGF for 20 min and assayed for PLC activity as described under ``Experimental Procedures.'' The cells were preincubated with the [ D-Arg, D-Phe, D-Trp,Leu]substance P peptide (50 µ M) for 5 min prior to agonist stimulation and the peptide was present for the duration of the stimulation. Data points represent the mean ± S.E. of between three and four independent experiments. In a single experiment each condition was measured in quadruplicate. Other clones of 16expressing Swiss 3T3 cells gave similar results to the clone represented in these experiments.



Bombesin and PDGF both activate MAP kinase activity in Swiss 3T3 cells (Fig. 3). The [ D-Arg, D-Phe, D-Trp,Leu]substance P peptide has no effect on PDGF stimulation of MAP kinase activity (Fig. 3 B). In contrast, incubation of Swiss 3T3 cells with a concentration of the [ D-Arg, D-Phe, D-Trp,Leu]substance P peptide (50 µ M) that totally inhibited PLC activation by 50 n M bombesin did not inhibit bombesin receptor stimulation of MAP kinase activity at this concentration of bombesin (Fig. 3, A and C). In fact, in many experiments the inhibitory peptide enhanced MAP kinase activity stimulated by 50 n M bombesin (Fig. 3 A). The [ D-Arg, D-Phe, D-Trp,Leu]substance P peptide had little or no effect on basal MAP kinase activity in the absence of bombesin. In Fig. 3 C we demonstrate that, in the presence of 50 µ M [ D-Arg, D-Phe, D-Trp,Leu]substance P peptide, bombesin activated MAP kinase in a concentration-dependent manner, although with a lowered affinity ( Kshift from 1.5 ± 0.5 n M to 20 ± 5 n M), consistent with the inhibitory peptide acting as a competitive antagonist. However, 50 n M bombesin could overcome the inhibition of MAP kinase, but not PLC activation, by the inhibitory peptide. These findings demonstrated that the [ D-Arg, D-Phe, D-Trp,Leu]substance P peptide influenced bombesin binding to its receptor, while differentially affecting PLC and MAP kinase regulation. Expression of the bombesin/GRP receptor in HEK-293 cells by transfection indicated that the cloned bombesin receptor mediates both phospholipase C and MAP kinase activation (Fig. 4). Thus, the [ D-Arg, D-Phe, D-Trp,Leu]substance P peptide differentially inhibits two signal transduction pathways regulated by the bombesin receptor. The [ D-Arg, D-Phe, D-Trp,Leu]substance P peptide must not simply competitively inhibit bombesin activation of its receptor but must have allosteric effects on the receptor. Regulation of the MAP Kinase Pathway in the Presence of the [ D-Arg, D-Phe, D-Trp,Leu]Substance P Peptide-MAP kinase is a serine-threonine protein kinase that is activated by phosphorylation on both tyrosine and threonine (20) . This reaction is catalyzed by a specific tyrosine/threonine-directed kinase, MEK (MAPK/ERK kinase) (21) . MEK is in turn phosphorylated and activated by the protein kinase Raf-1 (22) . Raf-1 interacts with RasGTP, and its activity is regulated in a Ras-dependent manner by growth factor receptor tyrosine kinases (23, 24) . Fig. 5 demonstrates that incubation of Swiss 3T3 cells with the [ D-Arg, D-Phe, D-Trp,Leu]substance P peptide alters the regulation of Raf-1 activation in response to bombesin but not PDGF. Treatment of Swiss 3T3 cells with PDGF or phorbol ester, but not bombesin, leads to a significant activation of Raf-1 (Fig. 5 A). PhosphorImager quantitation of MEK phosphorylation in Raf-1 immunoprecipitates from bombesin-stimulated cells of 15 different experiments was not statistically significant relative to basal Raf-1 activity (Fig. 5 B). In striking contrast, incubation of Swiss 3T3 cells with the [ D-Arg, D-Phe, D-Trp,Leu]substance P peptide resulted in a significant activation of Raf-1 activity in response to 50 n M bombesin relative to basal or peptide in the absence of bombesin. The peptide had little effect itself, on either MAP kinase or Raf-1 activity. The [ D-Arg, D-Phe, D-Trp,Leu]substance P peptide had no significant effect on PDGF receptor signaling or phorbol ester stimulation of Raf-1 activity (Fig. 5, A and B).


Figure 5:A, Raf-1 activity in Swiss 3T3 cells. Serum-starved Swiss 3T3 cells were stimulated with agonists in the absence or presence of the [ D-Arg, D-Phe, D-Trp,Leu]substance P peptide (50 µ M, 5-min preincubation) and assayed for Raf-1 activity as described under ``Experimental Procedures.'' Cells were stimulated with bombesin (50 n M, 2 min), TPA (200 n M, 5 min), or PDGF (10 ng/ml, 5 min). Purified recombinant wild type MEK was autophosphorylated and used as a standard (as indicated). An autoradiograph from a single experiment, representative of at least five experiments, is shown. B, PhosphorImager analysis of phosphorylated MEK-1 in Swiss 3T3 cells. Quantification of the [P]MEK protein is represented as -fold activation over basal in arbitrary units. The number of individual experiments quantified varied between 5 and 15, depending upon the condition. Bombesin in the absence or presence of peptide are conditions representative of 15 individual experiments and the increased activity observed in the presence of the peptide was significant ( p < 0.01 by Student's t test). C, PhosphorImager analysis of phosphorylated MEK-1 in control and pertussis toxin-treated Swiss 3T3 cells. Serum-starved cells were stimulated and assayed as described in A. When toxin-treated, cells were incubated for 18 h with pertussis toxin (100 ng/ml) prior to agonist stimulation. Quantification of the [P]MEK protein is represented as -fold activation over basal in arbitrary units. The number of individual experiments quantified varied between 3 and 5, depending upon the condition. The increased activity in the presence of the peptide was significant in both the control ( p < 0.01 by Student's t test) and the pertussis toxin-treated cells ( p < 0.05 by Student's t test). The apparent decrease in PDGF-stimulated Raf-1 activity upon pertussis toxin pretreatment of the Swiss 3T3 cells was not significant ( p < 0.10 by Student's t test).



The increased Raf-1 activation in response to bombesin in the presence of the [ D-Arg, D-Phe, D-Trp,Leu]substance P peptide was not due to the receptor coupling to Gor Gproteins. Pertussis toxin treatment of Swiss 3T3 cells, to uncouple Gand Gproteins from receptors, had no significant effect on the ability of the [ D-Arg, D-Phe, D-Trp,Leu]substance P peptide to enhance Raf-1 activation in response to bombesin (Fig. 5 C).

Even though the [ D-Arg, D-Phe, D-Trp,Leu]substance P peptide enhanced bombesin-stimulated Raf-1 activity, it had little or no effect on RasGTP loading (Fig. 6). The RasGTP loading assay (25) is non-catalytic and thus rather insensitive. Nonetheless, PDGF stimulation resulted in a 4.5-5-fold increase in RasGTP loading, whereas the response to bombesin was not significantly different from basal levels in the presence or absence of peptide (Fig. 6, A and B). Thus, the increased Raf-1 activity in response to bombesin in the presence of the [ D-Arg, D-Phe, D-Trp,Leu]substance P peptide is independent of a significant change in RasGTP loading.


Figure 6: A, Ras activation in Swiss 3T3 cells. [P]GTP and [P]GDP bound to immunoprecipitated Ras were measured as an index of the Ras activation state. Serum-starved, P-labeled Swiss 3T3 cells were stimulated with agonists in the absence or presence of the [ D-Arg, D-Phe, D-Trp,Leu]substance P peptide (50 µ M, 5-min preincubation) and assayed for Ras activity as described under ``Experimental Procedures.'' Cells were stimulated with bombesin (50 n M, 1 min) or PDGF (10 ng/ml, 2 min). Ras was immunoprecipitated and bound [P]GTP and [P]GDP resolved using polyethyleneimine-cellulose thin layer chromatography. An autoradiograph of a polyethyleneimine-cellulose TLC plate from a single experiment, representative of four experiments, is shown. B, PhosphorImager quantification of the percent ratio of GTP/(GTP + GDP). Quantification of the [P]GTP and [P]GDP is represented as the percentage of [P]GTP from the total P-labeled guanine nucleotide. The mean ± S.E. of four independent experiments is represented.




DISCUSSION

The [ D-Arg, D-Phe, D-Trp,Leu]substance P peptide inhibits bombesin but not PDGF-stimulated Swiss 3T3 cell growth and induces apoptosis of small cell lung carcinoma cells whose growth is dependent on neuropeptide autocrine or paracrine loops (26) . We have demonstrated that the [ D-Arg, D-Phe, D-Trp,Leu]substance P peptide inhibits phospholipase C and MAP kinase activation in response to bombesin, but that only the MAP kinase inhibition was overcome by high bombesin concentrations. The action of the [ D-Arg, D-Phe, D-Trp,Leu]substance P peptide is selective for bombesin receptor/G protein and not PDGF receptor tyrosine kinase regulated signal transduction pathways. The results clearly demonstrate that the [ D-Arg, D-Phe, D-Trp,Leu]substance P peptide differentially inhibits bombesin activation of receptor-mediated responses.

The enhanced Raf-1 activity in response to bombesin stimulation in the presence of the [ D-Arg, D-Phe, D-Trp,Leu]substance P peptide may be explained in part by the inhibition of phospholipase C activity. Stimulation of protein kinase C activity resulting from phospholipase C activation in Swiss 3T3 cells leads to enhanced cAMP synthesis and protein kinase A activation (11, 27) . Activation of protein kinase A in response to elevated cAMP levels has been shown to uncouple Raf-1 activation from RasGTP loading (28, 29, 30, 31, 32, 33) . In fact, protein kinase A activation in Swiss 3T3 cells inhibits the enhanced activation of Raf-1 in response to bombesin in the presence of the [ D-Arg, D-Phe, D-Trp,Leu]substance P peptide (data not shown). Thus, bombesin activation of Raf-1 is more pronounced in the presence of the [ D-Arg, D-Phe, D-Trp,Leu]substance P peptide. The robust RasGTP loading in response to PDGF is apparently sufficient to significantly overcome any protein kinase A-mediated uncoupling of Raf-1 activation that would occur in response to phospholipase C activation.

Saltiel and colleagues (7) have demonstrated that bombesin-stimulated, but not epidermal growth factor-stimulated, MAP kinase activation in Swiss 3T3 fibroblasts is inhibited by over 90% following TPA-mediated down-regulation of PKC. We have confirmed this observation with prolonged exposure to TPA, causing a 80-100% reduction in the bombesin-stimulated MAP kinase activity observed in both the absence and presence of the [ D-Arg, D-Phe, D-Trp,Leu]substance P peptide (data not shown). Prolonged exposure to TPA does not impair the ability of the bombesin receptor to activate PLC. PDGF-stimulated MAP kinase activity is also inhibited 50-60% after prolonged TPA treatment (data not shown). These observations could be interpreted to suggest that the activation of MAP kinase by bombesin, in the absence and presence of the [ D-Arg, D-Phe, D-Trp,Leu]substance P peptide, requires activation of a TPA-sensitive PKC isoform. This raises the possibility that in the presence of the [ D-Arg, D-Phe, D-Trp,Leu]substance P peptide an isoform of PKC is activated by a sn-1,2-diradylglycerol species independent from PLC activation. Bombesin regulation of a PLD-like activity, although currently poorly understood, is possible (5, 34) . The consequence of PLD and PLC stimulation of PKC isoforms must then have different regulatory functions. However, interpretation of the TPA down-regulation experiments should be qualified with the fact that TPA also activates the MAP kinase pathway. MAP kinase has been proposed to regulate activation of the Ras/Raf/MAP kinase pathway by feedback inhibition (see Ref. 35 and references therein). The potentially multiple effects observed with TPA make interpretation of the down-regulation experiments difficult and may explain in part the partial inhibition we observe for PDGF-stimulated MAP kinase activation.

The question arises how the [ D-Arg, D-Phe, D-Trp,Leu]substance P peptide selectively inhibits bombesin-stimulated phospholipase C but not MAP kinase activation at high bombesin concentrations (50 n M). Mukai et al. (36) used a novel truncated substance P-related peptide, p-Glu-Gln- D-Trp-Phe- D-Trp- D-Trp-Met-NH, which inhibited m2 muscarinic cholinergic receptor (m2 receptor) activation of Gand Gin reconstituted phospholipid vesicles. The inhibitory peptide interfered neither with ligand binding to the m2 receptor nor with binding of guanine nucleotides to Gand G. Their findings indicated that the inhibitory peptide bound to the G proteins and inhibited m2 receptor coupling to Gand G. The sequence of the [ D-Arg, D-Phe, D-Trp,Leu]substance P peptide is D-Arg-Pro-Lys-Pro- D-Phe-Gln- D-Trp-Phe- D-Trp-Leu-Leu-NH(10) . The two inhibitory peptides have the conserved 4-amino acid sequence Gln- D-Trp-Phe- D-Trp, although the flanking amino acids also appear critical for their pharmacological action (10, 36, 37) . Our findings are consistent with the [ D-Arg, D-Phe, D-Trp,Leu]substance P peptide uncoupling Gfrom the bombesin receptor. The site of action of the [ D-Arg, D-Phe, D-Trp,Leu]substance P peptide is presently unclear. It clearly reduces the affinity of the receptor for bombesin but selectively alters regulation of two effector pathways at high bombesin concentrations. This suggests that the [ D-Arg, D-Phe, D-Trp,Leu]substance P peptide most likely acts allosterically at a site distinct from the bombesin receptor ligand binding domain. This interpretation is consistent with the ability of the [ D-Arg, D-Phe, D-Trp,Leu]substance P peptide to have inhibitory effects on a number of neuropeptide receptors (10, 37) . The putative allosteric regulatory site could be either on the extracellular or cytoplasmic domain of the neuropeptide receptor. Studies to define the site of action of the [ D-Arg, D-Phe, D-Trp,Leu]substance P peptide are currently ongoing.

The bombesin receptor is, however, still functional and capable of signaling in the presence of the [ D-Arg, D-Phe, D-Trp,Leu]substance P peptide as demonstrated by the stimulation of the MAP kinase pathway at 50 n M bombesin. subunits of G proteins appear to play an important role in activation of the MAP kinase pathway (38, 39) . It is unclear if bombesin-stimulated MAP kinase activity in the presence of the [ D-Arg, D-Phe, D-Trp,Leu]substance P peptide allows subunits to be released from Gor if an additional G protein remains coupled to the bombesin receptor that is capable of regulating the MAP kinase pathway. If another G protein is coupled to the bombesin receptor it is not Gor G, because the bombesin response in the presence of the [ D-Arg, D-Phe, D-Trp,Leu]substance P peptide is not pertussis toxin-sensitive.

Woll and Rozengurt (37) demonstrated that the hexapeptide Arg- D-Trp-MePhe- D-Trp-Leu-Met-NHhad broad neuropeptide antagonist activity in Swiss 3T3 cells. This hexapeptide also inhibits the growth of small cell lung carcinoma cells in vitro (37) . In Swiss 3T3 cells this hexapeptide was a potent vasopressin antagonist and weak bombesin and bradykinin antagonist relative to the [ D-Arg, D-Phe, D-Trp,Leu]substance P peptide. The hexapeptide antagonist was, however, equipotent to the [ D-Arg, D-Phe, D-Trp,Leu]substance P peptide in arresting small cell lung carcinoma cell growth. From these observations the authors concluded that the actions of these peptides cannot simply be related to interruption or antagonism of bombesin-driven autocrine loops in small cell lung carcinoma (36) . Our findings demonstrate that the antagonist peptide can selectively inhibit the activation of one signal transduction effector but not another at high bombesin concentrations. We propose that it is the disruption of the coordinated regulation of multiple signaling pathways that contributes to the growth inhibitory properties of the [ D-Arg, D-Phe, D-Trp,Leu]substance P peptide. If this hypothesis is correct, it suggests that compounds that differentially regulate the different arms of signal transduction pathways regulated by G protein-coupled receptors will be potential anti-tumorigenic agents for small cell lung carcinoma and other carcinomas that proliferate in response to neuropeptide autocrine or paracrine loops.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants CA58187, DK37871, GM30324, and DK19928. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a travel fellowship from the Wellcome Trust. To whom correspondence should be addressed: Division of Basic Sciences, National Jewish Center for Immunology and Respiratory Medicine, 1400 Jackson St., Denver, CO 80206. Tel.: 303-398-1772; Fax: 303-398-1225.

The abbreviations used are: GRP, gastrin releasing peptide; SCLC, small cell lung carcinoma; PLC, phospholipase C; DMEM, Dulbecco's modified Eagle's medium; PtdIns, phosphatidylinositol; MEK, MAPK/ERK kinase; PDGF, platelet-derived growth factor; TPA, 12- O-tetradecanoylphorbol-13-acetate.


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