[d-Arg1,d-Trp5,7,9,Leu11]Substance P Inhibits Bombesin-induced Mitogenic Signal Transduction Mediated by Both Gq and G12 in Swiss 3T3 Cells*

Substance P (SP) analogues including [d-Arg1,d-Trp5,7,9,Leu11]SP are broad spectrum neuropeptide antagonists and potential anticancer agents, but their mechanism of action is not fully understood. Here, we examined the mechanism of action of [d-Arg1,d-Trp5,7,9,Leu11]SP as an inhibitor of G protein-coupled receptor (GPCR)-mediated signal transduction and cellular DNA synthesis in Swiss 3T3 cells. Addition of [d-Arg1,d-Trp5,7,9,Leu11]SP, at 10 μm, caused a striking rightward shift in the dose-response curves of DNA synthesis induced by bombesin, bradykinin, or vasopressin and markedly inhibited the activation of p42mapk (ERK-2) and p44mapk (ERK-1) induced by these GPCR agonists. In addition, this SP analogue also prevented the protein kinase C-dependent activation of protein kinase D induced by these agonists. [d-Arg1,d-Trp5,7,9,Leu11]SP, at a concentration (10 μm) that inhibited these Gq-mediated events, also prevented GPCR agonist-induced responses mediated through the G proteins of the G12subfamily. These include bombesin-induced assembly of focal adhesions, formation of parallel arrays of actin stress fibers, increase in the tyrosine phosphorylation of focal adhesion kinase (FAK), p130Cas, and paxillin, and formation of a complex between FAK and Src. We conclude that [d-Arg1,d-Trp5,7,9,Leu11]SP acts as a mitogenic antagonist of neuropeptide GPCRs blocking signal transduction via both Gq and G12.

Neuropeptides stimulate DNA synthesis and cell proliferation in cultured cells and are implicated as growth factors in a variety of fundamental processes including development, inflammation, tissue regeneration, and neoplastic transformation (1,2). In particular, bombesin and its mammalian counterpart gastrin-releasing peptide (GRP) 1 bind to a G protein-coupled receptor (GPCR) (3,4) that promotes G␣ q -mediated activation of ␤ isoforms of phospholipase C (5-7) to produce two second messengers as follows: inositol 1,4,5-trisphosphate that mobilizes Ca 2ϩ from internal stores and diacylglycerol that activates PKC (8 -10). The bombesin/GRP GPCR also interacts with members of the G 12 family leading to rapid Rho-dependent assembly of focal adhesions, formation of actin stress fibers, and an increase in tyrosine phosphorylation of focal adhesion proteins including the non-receptor tyrosine kinase FAK and the adaptor proteins paxillin and p130 Cas (10 -17). Subsequently, bombesin induces striking activation of serine phosphorylation cascades including p42 mapk /p44 mapk (18 -21) leading to increased expression of immediate early response genes, stimulation of cell cycle events, and subsequent cell proliferation (10,(22)(23)(24)(25).
Neuropeptides and their receptors are implicated as autocrine growth factors for small cell lung carcinoma (SCLC), one of the most clinically aggressive human cancers (26). SCLC cells secrete and respond mitogenically to multiple neuropeptides including bombesin/GRP, bradykinin, cholecystokinin, galanin, gastrin, neurotensin, and vasopressin (26 -29). Neuropeptides have also been implicated as autocrine and/or paracrine growth factors for other common solid tumors including colon, breast, prostate, and pancreas (30 -34). Consequently, antagonists capable of blocking the biological effects of multiple neuropeptides (e.g. broad spectrum neuropeptide antagonists) could provide a novel approach to the treatment of SCLC and other human cancers in which multiple neuropeptides are involved as growth factors.
Substance P (SP) analogues including [D-Arg 1 ,D-Phe 5 ,D-Trp 7,9 ,Leu 11 ]SP and [Arg 6 ,D-Trp 7,9 ,MePhe 8 ]SP (6 -11) inhibit the activation of signal transduction pathways and the mitogenic action of a range of neuropeptides structurally unrelated to SP in mouse Swiss 3T3 cells (20,(35)(36)(37). These broad spectrum neuropeptide antagonists also inhibit the proliferation of SCLC cell lines in liquid culture, in soft agar, and as xenografts in nude mice (35, 36, 38 -41). As a result, [Arg 6 ,D-Trp 7,9 , MePhe 8 ]SP (6 -11) has entered into a phase I clinical trial. More recently, a more potent neuropeptide antagonist, [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP, has been identified that also inhibits signal transduction and cell proliferation (20,39). Since broad spectrum neuropeptide antagonists could provide a novel approach to the treatment of SCLC and other solid cancers, an understanding of their mechanism of action will be critical for their further development.
Several models have been proposed to explain the molecular mechanism(s) by which agonists promote GPCR activation (see Ref. 42 for review). The widely used conformational selection model envisages that GPCRs cycle spontaneously (i.e. in the absence of ligand) between different conformational states: the inactive (R) and the active (R*) state (42). Agonists are thought to stabilize the active conformation of the receptor thereby increasing the probability for receptor-mediated activation of G protein (42). Receptors capable of activating two different G proteins (e.g. the bombesin/GRP receptor that interacts with G q and G 12 ) have been proposed to exist in three conformational states as follows: an inactive state that is favored energetically in the absence of agonist (R), a state that activates G 12 (R* 1 ) and a state that activates G q (R* 2 ). In this context, Jarpe and collaborators (43) proposed recently that SP analogues act in a novel manner, stabilizing the active conformation of the bombesin/GRP receptor that interacts with G 12 and proportionally reducing the receptor population that interacts with G q . In the framework of this model, SP analogues would act as agonists for G 12 -mediated events and as antagonists for G q -mediated events. It was further suggested that the inhibitory effect of broad spectrum SP antagonists on neuropeptide-stimulated cell proliferation could result, at least in part, from the disruption of the coordinated signaling that normally emanates from GPCRs. In support of this model, Jarpe et al. (43) demonstrated that [D-Arg 1 ,D-Phe 5 ,D-Trp 7,9 ,Leu 11 ]SP, a less potent analogue of [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP induced actin reorganization and assembly of focal adhesions. Our previous results, however, lead us to conclude that [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP coordinately inhibits signal transduction pathways activated by bombesin and other neuropeptides, but focal adhesion assembly and actin remodeling were not evaluated (20,39).
The experiments presented here were designed to examine the mechanism of action of [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP as an inhibitor of neuropeptide-mediated signal transduction and cellular DNA synthesis. Specifically, we examined whether this synthetic peptide antagonist, at concentrations that selectively inhibit neuropeptide-induced DNA synthesis and ERK activation, acts as an agonist of G 12 -mediated events including assembly of focal adhesions, formation of stress fibers, and tyrosine phosphorylation of the focal adhesion proteins FAK, p130 Cas , and paxillin. On the basis of the results presented here, we conclude that [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP acts as a mitogenic antagonist of neuropeptide GPCRs blocking signal transduction via both G q and G 12 .

EXPERIMENTAL PROCEDURES
Cell Culture-Stock cultures of Swiss 3T3 cells were maintained in DMEM, supplemented with 10% fetal bovine serum in a humidified atmosphere containing 10% CO 2 and 90% air at 37°C. For experimental purposes, Swiss 3T3 cells were plated in 100-mm dishes at 6 ϫ 10 5 cells/dish or 35-mm dishes at 1 ϫ 10 5 cells/dish in DMEM containing 10% fetal bovine serum and used after 6 -8 days when the cells were confluent and quiescent.
Immunoprecipitation-Quiescent cultures of Swiss 3T3 cells (1-2 ϫ 10 6 cells) were washed twice with DMEM and incubated for 5 min at 37°C with or without [D-Arg 1 ,D-Trp 5,7,9 ,D-Leu 11 ]Substance P as indicated. Bombesin (1 nM) and other agonists were then added, and the cultures were incubated for a further 10 min at 37°C. The stimulation was terminated on ice by aspirating the medium and solubilizing the cells. For the immunoprecipitation of FAK and p130 Cas and paxillin the cells were lysed with 1 ml of ice-cold lysis buffer containing 50 mM HEPES, pH 7.4, 1% Triton X-100, 150 mM NaCl, 1.5 mM MgCl 2 , 1 mM EGTA, 1 mM sodium orthovanadate, 10 mM sodium pyrophosphate, 100 mM NaF, and 1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride. For the immunoprecipitation of Src-FAK complexes the above lysis buffer was supplemented with 1% sodium deoxycholate, 0.1% SDS, and 10% glycerol.
Western Blotting-After SDS-PAGE, proteins were transferred to Immobilon-P membranes. After transfer, membranes were blocked using 5% nonfat dried milk in phosphate-buffered saline, pH 7.2, and incubated for at least 2 h at 22°C with the desired antibodies diluted in phosphate-buffered saline, pH 7.2, containing 3% nonfat dried milk. Bound primary antibodies to immunoreactive bands were visualized by enhanced chemiluminescence (ECL) detection with horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibodies.
Assay of p42 mapk (ERK-2) and p44 mapk (ERK-1) Activation-Quiescent cultures of Swiss 3T3 cells grown on 35-mm dishes were washed twice with DMEM and incubated for 5 min at 37°C with or without [D-Arg 1 ,D-Trp 5,7,9 ,D-Leu 11 ]Substance P as indicated. The cultures were then treated with various agonists as indicated and incubated for a further 5 min at 37°C. The stimulation was terminated on ice by aspirating the medium and solubilizing the cells with 200 l of 2ϫ SDS-PAGE sample buffer. The samples were boiled for 10 min, resolved by 10% SDS-PAGE, and transferred to Immobilon-P membranes.
Activation of p42 mapk and p44 mapk occurs through phosphorylation of specific threonine and tyrosine residues (48) resulting in slower migrating forms in SDS-PAGE gels. These activated forms were monitored by using a specific anti-phospho-p44/p42 mapk -mAb (New England Biolabs) that recognizes only the activated forms phosphorylated on Thr-202 and Tyr-204.
Kinase Assay of PKD-The kinase activity of PKD was determined in an in vitro kinase assay by mixing 20 l of immunocomplexes with 10 l of a phosphorylation mixture containing (final concentration) 100 M [␥-32 P]ATP (specific activity 400 -600 cpm/pmol), 30 mM Tris-HCl, pH 7.4, 10 mM MgCl 2 , and 1 mM DTT. After 10 min of incubation at 30°C, the reaction was stopped by washing with 1 ml of kinase buffer and then adding 200 l equal volume of 2ϫ SDS-PAGE sample buffer (200 mM Tris-HCl, pH 6.8, 2 mM EDTA, 0.1 M Na 3 VO 4 , 6% SDS, 10% glycerol, and 4% 2-mercaptoethanol), followed by SDS-PAGE analysis. The gels were dried, and the 110-kDa radioactive band corresponding to autophosphorylated PKD (44) was visualized by autoradiography.
DNA Synthesis Measurements-Confluent and quiescent cultures of Swiss 3T3 cells were washed twice with DMEM and incubated with DMEM/Waymouth's medium (1:1, v/v) containing [ 3 H]thymidine (1 Ci/ml, 1 M) and various additions as described in the figure legends. After 40 h of incubation at 37°C, cultures were washed twice with PBS and incubated in 5% trichloroacetic acid at 4°C for 20 min to remove acid-soluble radioactivity, washed with ethanol, and solubilized in 1 ml of 2% Na 2 CO 3 and 0.1 M NaOH. The acid-insoluble radioactivity was determined by scintillation counting in 6 ml of Beckman Readysafe.
Immunostaining of Cells-Swiss 3T3 cells in 35-mm dishes were washed with serum-free DMEM and treated with agonists and/or inhibitors at 37°C as indicated. For staining of actin, cells were washed twice with PBS, fixed in 4% paraformaldehyde in PBS for 10 min at room temperature, permeabilized with 0.2% Triton X-100 in PBS for 10 min at room temperature, and blocked with 10% fetal bovine serum in PBS. The cells were then incubated with TRITC-conjugated phalloidin (0.25 g/ml) in PBS for 10 min at room temperature and washed five times with PBS. In order to visualize focal adhesions, Swiss 3T3 cells were fixed, permeabilized as described above, and incubated with antivinculin mAb (dilution 1:100) for 2 h at room temperature. Cells were subsequently washed three times with PBS and then incubated with FITC-labeled anti-mouse IgG as second antibody at a dilution of 1:100 for another 30 min at room temperature. Immunofluorescence was visualized in both cases using a Zeiss immunofluorescence microscope. Images were collected with a 63ϫ lens (Zeiss Plan Apochromat, NA1.4).

RESULTS
[D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP Selectively Inhibits Neuropeptide-induced DNA Synthesis-To examine whether [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP preferentially inhibits the mitogenic activity of neuropeptide-activated GPCRs, quiescent cultures of Swiss 3T3 cells, arrested in the G 0 phase of the cell cycle, were transferred to serum-free media supplemented with [ 3 H]thymidine and increasing concentrations of growth-promoting agonists either in the absence or in the presence of 10 M [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP. The cumulative incorporation of radiolabeled precursor into DNA was measured after 40 h of incubation.
Addition of [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP caused a striking rightward shift in the dose-response curves of DNA synthesis induced by bombesin, bradykinin, and vasopressin which act via distinct GPCRs (Fig. 1). The inhibitory effect was reversed at the higher concentrations of each peptide agonist (Fig. 1A), suggesting that [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP inhibits the mitogenic activity of neuropeptides in a competitive fashion.
In contrast, [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP did not interfere with the mitogenic effect induced via EGF or PDGF tyrosine kinase receptors or by pharmacological agents that directly activate signal transduction pathways bypassing receptors including the biologically active phorbol ester phorbol 12,13-dibutyrate (PDBu), a direct activator of the classic and novel isoforms of PKC (Fig. 1B), or the P. multocida toxin, a potent mitogen that activates G q (results not shown). Similarly, exposure to [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP did not affect DNA synthesis stimulated by PGF 2␣ which acts through a GPCR coupled to G q (49,50). Taken together, the results presented in Fig. 1 substantiate the notion that [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP selectively targets GPCRs for mitogenic neuropeptides.
[D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP Selectively Inhibits Neuropeptide-induced ERK Activation-In order to test further the selectivity of [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP as an inhibitor of neuropeptide-mediated mitogenic signals, we also determined the effect of this synthetic peptide on early activation of p42 mapk (ERK-2) and p44 mapk (ERK-1) induced by multiple agonists. To examine ERK activation, lysates of Swiss 3T3 cells incubated for 5 min in the absence or in the presence of 10 M [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP and subsequently challenged with multiple stimuli were analyzed by immunoblotting using a site-specific antibody that recognizes the dually phosphorylated (active) forms of ERK-1 and ERK-2.
[D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP Prevents G q -mediated PKD Activation-PKD/PKC is a serine/threonine protein kinase (51, 52) with structural, enzymological, and regulatory properties distinct from other members of the PKC family (53). PKD is rapidly activated by a variety of neuropeptide agonists including bombesin, bradykinin, and vasopressin that signal through heptahelical receptors coupled to G q (54 -56). Recent results indicate that G␣ q activation is sufficient to stimulate sustained PKD activation via PKC and show that the endogenous G␣ q mediates PKD activation in response to acute bombesin receptor stimulation (56). Here, we determined the effect of [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP on bombesin-stimulated PKD activation.
Confluent and quiescent cultures of Swiss 3T3 cells, treated with or without increasing concentrations of [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP, were stimulated with bombesin for 10 min and lysed, and PKD was immunoprecipitated with an antibody directed against the 15 carboxyl-terminal amino acids of this enzyme. The immunocomplexes were incubated with [␥-32 P]ATP and then analyzed by SDS-PAGE and autoradiography to examine the level of autophosphorylation. As illustrated by Fig. 3 and in agreement with previous results, stimulation of Swiss 3T3 cells with bombesin induced a striking increase in PKD activity that was maintained during cell disruption and immunoprecipitation. Shown in Fig. 3, for the first time, treatment of the cells with [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP blocked PKD activation induced by subsequent addition of bombesin, in a concentration-dependent fashion. Half-maximal inhibition was achieved at 7 M, and addition of [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP at 20 M inhibited bombesin-induced PKD activation by ϳ90%. This SP analogue also prevented PKD activation induced by stimulation with either bradykinin or vasopressin (Fig. 3B).

Assembly of Focal Adhesions, and Formation of Stress Fibers-
The preceding results indicate that [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP selectively targets neuropeptide-mediated signals. Recently, it has been proposed that antagonists of this class act in a novel manner, stabilizing the active conformation of GPCR that interacts with G 12 , thereby acting as an agonist for G 12 -mediated events but as an antagonist for G q -mediated events (43). In order to test this hypothesis, we determined the effect of [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP on cellular responses stimulated by bombesin receptor via the ␣ subunits of the G 12 subfamily. Activated G␣ 12 and G␣ 13 (which comprise the G 12 subfamily) are known to induce Rho activation (15,(57)(58)(59)(60) via recruitment and activation of a GDP/GTP exchange factor (61) and to promote Rho-dependent stress fiber formation and focal adhesion assembly in Swiss 3T3 cells (15,62). Consequently, if [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP acts as a selective G 12 agonist, it should induce focal adhesion assembly and stress fiber formation at concentrations that inhibit mitogenic signaling in Swiss 3T3 cells. To test this prediction, quiescent cultures of these cells were preincubated in the absence or in the presence of 10 M [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP, challenged with or without bombesin and then fixed. Focal adhesions were visualized by staining with anti-vinculin mAb (Fig. 4), and the organization of the actin cytoskeleton was revealed with TRITC-phalloidin (Fig. 5).
Quiescent cultures of Swiss 3T3 cells exhibited only very few focal adhesions. As expected, stimulation with 1 nM bombesin induced a dramatic increase in the assembly of well defined, pear-shaped focal adhesions (Fig. 4) and in the formation of parallel arrays of actin stress fibers (Fig. 5). In contrast, pretreatment of the cells with [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP at 10 M, a concentration that markedly inhibited neuropeptide-mediated G q signaling and DNA synthesis (Figs. 1-3), did not induce a significant increase in the formation of either focal adhesions (Fig. 4) or stress fibers (Fig. 5). The salient feature of these experiments is that pretreatment of the cells with 10 M [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP strikingly prevented the assembly of focal adhesions (Fig. 4) and the increase in actin stress fibers induced by bombesin (Fig. 5). The findings presented in Figs. 4 and 5 are inconsistent with the hypothesis that predicts that [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP induces G 12 -mediated events at concentrations that block G q -mediated signaling. 5,7,9 ,Leu 11 ]SP on Additional G 12 -mediated Events, Tyrosine Phosphorylation of FAK, p130 Cas , and Paxillin-G␣ 12 and G␣ 13 also mediate the increase in the tyrosine phosphorylation of FAK, paxillin, and p130 Cas through a Rho-dependent pathway (16). Consequently, if [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP acts as a selective G 12 agonist, this synthetic peptide should induce tyrosine phosphorylation of these focal adhesion proteins, at concentrations that prevent G q -mediated signaling. In order to test this prediction, lysates of Swiss 3T3 cells treated with or without 10 M [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP and challenged with or without bombesin were immunoprecipitated with anti-FAK antibody. The immune complexes were analyzed by SDS-PAGE followed by Western blotting with anti-Tyr(P) mAb. As shown in Fig. 6 (A  and B), addition of [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP at 10 M did not promote FAK tyrosine phosphorylation but, in contrast, dramatically inhibited (ϳ90%) the increase in the tyrosine phosphorylation of FAK induced by bombesin.

Inhibitory Effect of [D-Arg 1 ,D-Trp
Extensive evidence indicates that FAK translocation to nascent focal adhesions promotes its autophosphorylation as a result of clustering and/or conformational changes. Because the major site of FAK autophosphorylation, Tyr-397, is a potential high affinity binding site for the SH2 domain of Src, the phosphorylation of this site in response to GPCR activation facilitates the formation of a FAK/Src signaling complex (63). In order to test further whether [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP acts as an agonist for G 12 -mediated events, we examined the effect of this synthetic peptide on bombesin-induced tyrosine phosphorylation of FAK at Tyr-397 and on Src⅐FAK complex formation which depends on the tyrosine phosphorylation of Tyr-397. The results, shown in Fig. 6C, indicate that treatment with 10 M [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP neither induced a significant increase in the tyrosine phosphorylation of FAK at Tyr-397 nor promoted Src⅐FAK complex formation. In contrast, exposure to this SP analogue strikingly inhibited the increase in the tyrosine phosphorylation of FAK at Tyr-397 and the formation of a complex between Src and FAK induced by bombesin.
In order to substantiate the results obtained with FAK, we examined the effect of [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP on bombesininduced tyrosine phosphorylation of the adaptor proteins p130 Cas and paxillin. As illustrated by Fig. 7, addition of [D-Arg 1 , D-Trp 5,7,9 ,Leu 11 ]SP at 10 M did not induce tyrosine phosphorylation of either p130 Cas or paxillin. In contrast, this SP analogue markedly inhibited the increase in the tyrosine phosphorylation of p130 Cas and paxillin induced by bombesin. Taken together, our results support the conclusion that [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP (at 10 M) acts as an antagonist that coordinately prevents GPCR-induced events mediated by either G q or G 12 .

]SP Inhibits Focal Adhesion Assembly and Tyrosine Phosphorylation of FAK and Paxillin
Induced by Bombesin-The results presented here appear to contrast with those reported previously by Jarpe et al. (43) who demonstrated that [D-Arg 1 ,D-Phe 5 ,D-Trp 7,9 ,Leu 11 ]SP, a less potent analogue of [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP, induced actin reorganization and focal adhesion assembly. However, [D-Arg 1 ,D-Phe 5 ,D-Trp 7,9 ,Leu 11 ]SP promoted these responses at concentrations higher than those used in the present study, and the possibility that this SP analogue, at a lower concentration, could also inhibit G 12 -mediated events, as shown here with [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP, was not explored. In an effort to reconcile these discrepancies, we determined the effect of [D-Arg 1 ,D-Phe 5 ,D-Trp 7,9 ,Leu 11 ]SP on bombesin-induced focal adhesion assembly and tyrosine phosphorylation of FAK and paxillin.
[D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP Stimulates Morphological Responses and Tyrosine Phosphorylation of FAK and Paxillin at High Concentrations-We also examined the effect of [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP on focal adhesion assembly and tyrosine phosphorylation of FAK and paxillin at concentrations higher than 10 M. As shown in Fig. 9, exposure of Swiss 3T3 cells to 50 M [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP promoted the assembly of only few focal adhesions and caused actin cytoskeletal changes that were not equivalent to the parallel arrays of stress fibers induced by bombesin (results not shown). Furthermore, addition of [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP at 50 M induced a low level of tyrosine phosphorylation of FAK and paxillin as compared with the effect induced by bombesin in parallel cultures. Interestingly, exposure of the cells to [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP at 50 M markedly reduced the assembly of focal adhesions and the increase in tyrosine phosphorylation of focal adhesion proteins induced by bombesin. We conclude that [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP acts as an antagonist of neuropeptide GPCRs that signal via both G q and G 12 , but at higher concentrations, this synthetic peptide induces additional effects that lead to limited assembly of focal adhesions and to a low level of tyrosine phosphorylation of focal adhesion proteins. DISCUSSION SP analogues including [D-Arg 1 ,D-Phe 5 ,D-Trp 7,9 ,Leu 11 ]SP and [Arg 6 ,D-Trp 7,9 ,MePhe 8 ]SP (6 -11) are an interesting class of agents that inhibit the activation of signal transduction pathways and the mitogenic action of a range of neuropeptides structurally unrelated to SP. These synthetic peptides also inhibit the proliferation of human cancer cells both in vitro and in nude mice and have entered in phase I clinical trials. Consequently, the mechanism of action of these agents is attracting considerable interest.
Jarpe and collaborators (43) proposed recently that broad spectrum SP antagonists act in a novel manner, stabilizing the active conformation of GPCR that interacts with G 12 , thereby acting as an agonist for G 12 -mediated events but as an antagonist for G q -mediated events. It was further suggested that the inhibitory effect of these synthetic peptides on neuropeptidestimulated cell proliferation could result, at least in part, from the disruption of the coordinated signaling that normally emanates from the GPCRs. Our own results, however, lead us to conclude that [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP and other agents of this class coordinately inhibit signal transduction pathways activated by neuropeptides (20,39). In the present study we attempted to distinguish between these alternative mecha-nisms of action. Specifically, we examined whether [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP, used at a concentration that inhibits neuropeptide-induced DNA synthesis, ERK activation, and PKCdependent PKD activation, acts as a selective agonist of G 12mediated events.
It is well established that GPCR-induced increases in tyrosine phosphorylation of FAK, paxillin, and p130 Cas are downstream of Rho activation, stress fiber formation, and focal adhesion assembly (64 -70). Activated G␣ 12 and G␣ 13 are known to induce Rho activation (15,(57)(58)(59) via recruitment and activation of a GDP/GTP exchange factor (61) and to promote Rho-dependent stress fiber formation and focal adhesion assembly in Swiss 3T3 cells (15,62). Furthermore, activated G␣ 12 and G␣ 13 stimulate tyrosine phosphorylation of FAK, paxillin, and p130 Cas via Rho (16). Taken together, these findings indicate the existence of a signal transduction pathway whereby agonist occupation of GPCRs activates G␣ 12  way, we examined in the present study the effects of [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP on the assembly of focal adhesions, the formation of actin stress fibers, and the tyrosine phosphorylation of FAK, paxillin, and p130 Cas. Our results demonstrate that pretreatment of Swiss 3T3 cells with 10 M [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP, a concentration that markedly inhibited neuropeptide-mediated G q signaling and DNA synthesis, did not induce a significant increase in the formation of either focal adhesions or stress fibers. In contrast, pretreatment with [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP strikingly prevented the assembly of focal adhesions and the increase in actin stress fibers induced by bombesin. Furthermore, our results also demonstrate that treatment with [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP neither induced a significant increase in the overall tyrosine phosphorylation of FAK nor induced tyrosine phosphorylation of FAK at Tyr-397, the major autophosphorylation site in this enzyme. Accordingly, exposure to [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP did not promote Src⅐FAK complex formation. Similarly, [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP did not induce tyrosine phosphorylation of paxillin or p130 Cas . A salient feature of the results presented here is that treatment with [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP dramatically inhibited the increase in the tyrosine phosphorylation of these focal adhesion proteins induced by bombesin. Thus, [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP, at a concentration that inhibits G q signaling and mitogenesis by neuropeptides, also prevents G 12 -mediated events including focal adhesion assembly, stress fiber formation, and tyrosine phosphorylation of focal adhesion proteins.
Jarpe et al. (43) demonstrated that [D-Arg 1 ,D-Phe 5 ,D-Trp 7,9 , Leu 11 ]SP, a less potent analogue of [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP, induced actin reorganization and focal adhesion assembly at concentrations higher than that used in the present study. A possible explanation for the apparent discrepancy between our results obtained with [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP and those with [D-Arg 1 ,D-Phe 5 ,D-Trp 7,9 ,Leu 11 ]SP is that a single amino acid change at position 5 from D-Phe 5 to D-Trp 5 is responsible for the drastic change in the properties of these antagonists. However, Jarpe et al. (43) did not examine the possibility that [D-Arg 1 ,D-Phe 5 ,D-Trp 7,9 ,Leu 11 ]SP, at a lower concentration, could also inhibit G 12 -mediated events, as shown here with [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP. In the present study, we show that [D-Arg 1 ,D-Phe 5 ,D-Trp 7,9 ,Leu 11 ]SP (at 20 M) markedly inhibited bombesin-induced assembly of focal adhesions and tyrosine phosphorylation of FAK and paxillin. Furthermore, we could also demonstrate that exposure of Swiss 3T3 cells to 50 M [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP promoted cytoskeletal rearrangements, formation of some focal adhesions, and a low level of tyrosine phosphorylation of focal adhesion-associated proteins. These effects were much less prominent than those induced by bombesin in parallel cultures, and even at these concentrations, the SP analogue markedly reduced the assembly of focal adhesions and the tyrosine phosphorylation of focal adhesion proteins induced by bombesin.
[D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP, at high concentrations, may act as a low affinity partial agonist of an as yet unidentified GPCR expressed in Swiss 3T3 cells stabilizing an intermediate receptor conformation as suggested by the sequential binding model proposed by Gether and Kobilka (42). This sequential binding model takes into account structural and kinetic studies that are not readily accommodated by the conformational selection model. Alternatively, [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP could induce cellular effects via non-receptor pathways since SP analogues are known to insert into lipid membranes (71), and these charged and lypophilic peptides can directly modulate G protein activity (72).
In conclusion, [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP acts as a broad spectrum mitogenic antagonist of neuropeptide GPCRs blocking signal transduction via both G q and G 12 . However, at higher concentrations, this synthetic peptide induces additional effects that lead to a low level of focal adhesions and a low level of tyrosine phosphorylation of focal adhesion-associated proteins. These findings are not compatible with the hypothesis that [D-Arg 1 ,D-Trp 5,7,9 ,Leu 11 ]SP stabilizes a bombesin receptor conformation that interacts selectively with G 12 but indicate that this SP analogue inhibits bombesin signaling mediated by both G q and G 12 .