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J. Biol. Chem., Vol. 275, Issue 39, 30644-30652, September 29, 2000

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[D-Arg¹,D-Trp^5,7,9,Leu¹¹]Substance P Inhibits Bombesin-induced Mitogenic Signal Transduction Mediated by Both G_q and G₁₂ in Swiss 3T3 Cells^*

James Sinnett-Smith, Chintda Santiskulvong, Javier Duque, and Enrique Rozengurt^§

From the Department of Medicine, School of Medicine and Molecular Biology Institute, UCLA, Los Angeles, California 90095-1786

Received for publication, May 2, 2000, and in revised form, June 29, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Substance P (SP) analogues including [D-Arg¹,D-Trp^5,7,9,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-Trp^5,7,9,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-Trp^5,7,9,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 p42^mapk (ERK-2) and p44^mapk (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-Trp^5,7,9,Leu¹¹]SP, at a concentration (10 µM) that inhibited these G_q-mediated events, also prevented GPCR agonist-induced responses mediated through the G proteins of the G₁₂ subfamily. 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), p130^Cas, and paxillin, and formation of a complex between FAK and Src. We conclude that [D-Arg¹,D-Trp^5,7,9,Leu¹¹]SP acts as a mitogenic antagonist of neuropeptide GPCRs blocking signal transduction via both G_q and G₁₂.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 Ca²⁺ from internal stores and diacylglycerol that activates PKC (8-10). The bombesin/GRP GPCR also interacts with members of the G₁₂ 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, 22-25).

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-Arg¹,D-Phe⁵,D-Trp^7,9,Leu¹¹]SP and [Arg⁶,D-Trp^7,9,MePhe⁸]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, 35-37). 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, [Arg⁶,D-Trp^7,9,MePhe⁸]SP (6-11) has entered into a phase I clinical trial. More recently, a more potent neuropeptide antagonist, [D-Arg¹,D-Trp^5,7,9,Leu¹¹]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 G₁₂) 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 G₁₂ (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 G₁₂ and proportionally reducing the receptor population that interacts with G_q. In the framework of this model, SP analogues would act as agonists for G₁₂-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-Arg¹,D-Phe⁵,D-Trp^7,9,Leu¹¹]SP, a less potent analogue of [D-Arg¹,D-Trp^5,7,9,Leu¹¹]SP induced actin reorganization and assembly of focal adhesions. Our previous results, however, lead us to conclude that [D-Arg¹,D-Trp^5,7,9,Leu¹¹]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-Arg¹,D-Trp^5,7,9,Leu¹¹]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 G₁₂-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-Arg¹,D-Trp^5,7,9,Leu¹¹]SP acts as a mitogenic antagonist of neuropeptide GPCRs blocking signal transduction via both G_q and G₁₂.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Culture-- Stock cultures of Swiss 3T3 cells were maintained in DMEM, supplemented with 10% fetal bovine serum in a humidified atmosphere containing 10% CO₂ and 90% air at 37 °C. For experimental purposes, Swiss 3T3 cells were plated in 100-mm dishes at 6 × 10⁵ cells/dish or 35-mm dishes at 1 × 10⁵ 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 × 10⁶ cells) were washed twice with DMEM and incubated for 5 min at 37 °C with or without [D-Arg¹,D-Trp^5,7,9,D-Leu¹¹]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 MgCl₂, 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.

For the immunoprecipitation of PKD the cells were lysed with a lysis buffer containing 1% Triton X-100, 2 mM EDTA, 2 mM EGTA, 2 mM DTT, 1 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride in 50 mM Tris-HCl, pH 7.4 (44).

Lysates were clarified by centrifugation at 14,000 rpm for 10 min, and the pellets were discarded. After centrifugation, supernatants were transferred to fresh tubes, and proteins were immunoprecipitated at 4 °C for 4 h with protein A-agarose linked to the desired antibody, PA-1 antipeptide antiserum for PKD, polyclonal anti-FAK or anti-Src family (SRC-2) antibodies as described previously (12, 45-47), or anti-mouse IgG-agarose-linked mAb to p130^Cas or paxillin. Immunoprecipitates were washed three times with lysis buffer and extracted in 2× SDS-PAGE sample buffer (200 mM Tris-HCl, pH 6.8, 1 mM EDTA, 6% SDS, 2 mM EDTA, 4% 2-mercaptoethanol, 10% glycerol). The samples were boiled for 10 min and resolved by 8% SDS-PAGE.

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-Arg¹,D-Trp^5,7,9,D-Leu¹¹]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 µl of 2× 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 µl of immunocomplexes with 10 µl of a phosphorylation mixture containing (final concentration) 100 µM [-³²P]ATP (specific activity 400-600 cpm/pmol), 30 mM Tris-HCl, pH 7.4, 10 mM MgCl₂, 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 µl equal volume of 2× SDS-PAGE sample buffer (200 mM Tris-HCl, pH 6.8, 2 mM EDTA, 0.1 M Na₃VO₄, 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 [³H]thymidine (1 µCi/ml, 1 µM) 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% Na₂CO₃ 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 µg/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 anti-vinculin 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 63× lens (Zeiss Plan Apochromat, NA1.4).

Materials-- Bombesin, bradykinin, vasopressin, Pasteurella multocida toxin, anti-vinculin mAb, and TRITC-conjugated phalloidin were obtained from Sigma. ECL reagents were from Amersham Pharmacia Biotech. [D-Arg¹,D-Trp^5,7,9,Leu¹¹]SP and [D-Arg¹,D-Phe⁵,D-Trp^7,9,Leu¹¹]SP were obtained from Bachem. FAK polyclonal Ab C-20, Src polyclonal Ab, and Tyr(P) monoclonal Ab PY20 were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-p130^Cas and paxillin monoclonal Abs were from Transduction Laboratories (Lexington, KY). The phosphospecific antibody to tyrosine 397 of FAK was obtained from BIOSOURCE International (Camarillo, CA). The anti-phospho-p44/p42^mapk-mAb (E10) was from New England Biolabs (Beverly, MA). All other reagents used were of the purest grade available.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

[D-Arg¹,D-Trp^5,7,9,Leu¹¹]SP Selectively Inhibits Neuropeptide-induced DNA Synthesis-- To examine whether [D-Arg¹,D-Trp^5,7,9,Leu¹¹]SP preferentially inhibits the mitogenic activity of neuropeptide-activated GPCRs, quiescent cultures of Swiss 3T3 cells, arrested in the G₀ phase of the cell cycle, were transferred to serum-free media supplemented with [³H]thymidine and increasing concentrations of growth-promoting agonists either in the absence or in the presence of 10 µM [D-Arg¹,D-Trp^5,7,9,Leu¹¹]SP. The cumulative incorporation of radiolabeled precursor into DNA was measured after 40 h of incubation.

Addition of [D-Arg¹,D-Trp^5,7,9,Leu¹¹]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-Arg¹,D-Trp^5,7,9,Leu¹¹]SP inhibits the mitogenic activity of neuropeptides in a competitive fashion.

View larger version (23K): [in this window] [in a new window]   Fig. 1.   [D-Arg1,D-Trp5,7,9,Leu11]Substance P preferentially inhibits the mitogenic activity of neuropeptide-activated GPCRs. A, bombesin, bradykinin, and vasopressin dose-response relationships in the absence () and presence () of [D-Arg1,D-Trp5,7,9,Leu11]Substance P. Confluent and quiescent cultures of Swiss 3T3 cells were washed and incubated at 37 °C in 2 ml of DMEM/Waymouth's medium containing 1 µCi/ml [3H]thymidine and either increasing concentrations of bombesin (with 100 ng/ml insulin) or bradykinin (with 500 ng/ml insulin) or vasopressin (with 100 ng/ml insulin) either in the absence () or presence () of 10 µM [D-Arg1,D-Trp5,7,9,Leu11]Substance P. B, EGF, PDGF, PDBu, and PGF2 dose-response relationships in the absence () and presence () of [D-Arg1,D-Trp5,7,9,Leu11]Substance P. Confluent and quiescent cultures of Swiss 3T3 cells were washed and incubated at 37 °C in 2 ml of DMEM/Waymouth's medium containing containing 1 µCi/ml [3H]thymidine and increasing concentrations of EGF (with 100 ng/ml insulin), PDGF, PDBu (with 100 ng/ml insulin), or PGF2 (with 100 ng/ml insulin) either in the absence () or presence () of 10 µM [D-Arg1,D-Trp5,7,9,Leu11]Substance P. After 40 h, DNA synthesis was assessed by measuring the [3H]thymidine incorporated into acid-precipitable material. Results are expressed as a percentage of the incorporation induced by the highest concentration of agonists, and data are shown as the mean ± S.E. (n = 3).

In contrast, [D-Arg¹,D-Trp^5,7,9,Leu¹¹]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-Arg¹,D-Trp^5,7,9,Leu¹¹]SP did not affect DNA synthesis stimulated by PGF₂ which acts through a GPCR coupled to G_q (49, 50). Taken together, the results presented in Fig. 1 substantiate the notion that [D-Arg¹,D-Trp^5,7,9,Leu¹¹]SP selectively targets GPCRs for mitogenic neuropeptides.

[D-Arg¹,D-Trp^5,7,9,Leu¹¹]SP Selectively Inhibits Neuropeptide-induced ERK Activation-- In order to test further the selectivity of [D-Arg¹,D-Trp^5,7,9,Leu¹¹]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 µM [D-Arg¹,D-Trp^5,7,9,Leu¹¹]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.

As shown in Fig. 2, ERK activation induced by stimulation with 1 nM bombesin, 5 nM vasopressin, or 5 nM bradykinin was dramatically reduced by exposure of the cells to 10 µM [D-Arg¹,D-Trp^5,7,9,Leu¹¹]SP. In contrast, ERK activation induced by either PDGF (2.5 ng/ml) or EGF (2.5 ng/ml) was not affected by addition of [D-Arg¹,D-Trp^5,7,9,Leu¹¹]SP, at an identical concentration. Similarly, prior exposure to [D-Arg¹,D-Trp^5,7,9,Leu¹¹]SP did not affect ERK activation via direct stimulation of PKC with PDBu or through the G_q-coupled receptor for PGF₂.

View larger version (36K): [in this window] [in a new window]   Fig. 2.   [D-Arg1,D-Trp5,7,9,Leu11]Substance P preferentially inhibits the ERK activation of neuropeptide-activated GPCRs. A, inhibition of ERK activation induced by bombesin, bradykinin, and vasopressin by [D-Arg1,D-Trp5,7,9,Leu11]Substance P. Confluent and quiescent Swiss 3T3 cells were washed twice with DMEM and incubated for 5 min with 10 µM [D-Arg1,D-Trp5,7,9,Leu11]Substance P. Cells were subsequently challenged with bombesin (1 nM), bradykinin (5 nM), or vasopressin (5 nM) for 5 min. B-D, [D-Arg1,D-Trp5,7,9,Leu11]Substance P does not inhibit ERK activation induced by EGF, PDGF, PDBu, and PGF2. Confluent and quiescent Swiss 3T3 cells were washed twice with DMEM and incubated for 5 min with 10 µM [D-Arg1,D-Trp5,7,9,Leu11]Substance P. Cells were then challenged with EGF (2.5 ng/ml), PDGF (1 ng/ml), PDBu (at the indicated concentrations), or PGF2 (at the indicated concentrations) for 5 min. Cells were then lysed in 2× SDS-PAGE sample buffer and analyzed by SDS-PAGE and immunoblotting with phospho-ERK antibody as described under "Experimental Procedures." The results presented here are typical of three independent experiments.

[D-Arg¹,D-Trp^5,7,9,Leu¹¹]SP Prevents G_q-mediated PKD Activation-- PKD/PKCµ 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-Arg¹,D-Trp^5,7,9,Leu¹¹]SP on bombesin-stimulated PKD activation.

Confluent and quiescent cultures of Swiss 3T3 cells, treated with or without increasing concentrations of [D-Arg¹,D-Trp^5,7,9,Leu¹¹]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 [-³²P]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-Arg¹,D-Trp^5,7,9,Leu¹¹]SP blocked PKD activation induced by subsequent addition of bombesin, in a concentration-dependent fashion. Half-maximal inhibition was achieved at 7 µM, and addition of [D-Arg¹,D-Trp^5,7,9,Leu¹¹]SP at 20 µM inhibited bombesin-induced PKD activation by ~90%. This SP analogue also prevented PKD activation induced by stimulation with either bradykinin or vasopressin (Fig. 3B).

View larger version (33K): [in this window] [in a new window]   Fig. 3.   [D-Arg1,D-Trp5,7,9,Leu11]Substance P inhibits PKD activation by bombesin, bradykinin, and vasopressin. A, confluent and quiescent Swiss 3T3 cells were washed twice with DMEM and incubated for 5 min with increasing concentrations of [D-Arg1,D-Trp5,7,9,Leu11]Substance P (SP analogue), as indicated. The cells were then stimulated with 10 nM bombesin for 10 min at 37 °C and lysed. The lysates were immunoprecipitated with PA-1 antiserum and PKD activity in the immunocomplexes was determined by autophosphorylation as described under "Experimental Procedures." The autoradiogram shown is representative of two independent experiments. The results are expressed as a percentage of the maximal activation obtained with bombesin in the absence of [D-Arg1,D-Trp5,7,9,Leu11]Substance P. B, the experimental conditions were identical to those described in A except that the cells were stimulated with either 10 nM bradykinin (BRK) or 10 nM vasopressin (VP) instead of bombesin.

Effect of [D-Arg¹,D-Trp^5,7,9,Leu¹¹]SP on G₁₂-mediated Events, Assembly of Focal Adhesions, and Formation of Stress Fibers-- The preceding results indicate that [D-Arg¹,D-Trp^5,7,9,Leu¹¹]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 G₁₂, thereby acting as an agonist for G₁₂-mediated events but as an antagonist for G_q-mediated events (43). In order to test this hypothesis, we determined the effect of [D-Arg¹,D-Trp^5,7,9,Leu¹¹]SP on cellular responses stimulated by bombesin receptor via the  subunits of the G₁₂ subfamily.

Activated G₁₂ and G₁₃ (which comprise the G₁₂ subfamily) are known to induce Rho activation (15, 57-60) 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-Arg¹,D-Trp^5,7,9,Leu¹¹]SP acts as a selective G₁₂ 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 µM [D-Arg¹,D-Trp^5,7,9,Leu¹¹]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).

View larger version (78K): [in this window] [in a new window]   Fig. 4.   [D-Arg1,D-Trp5,7,9,Leu11]Substance P inhibits assembly of focal adhesions in Swiss 3T3 cells induced by bombesin. Confluent and quiescent Swiss 3T3 cells were washed and preincubated without () or with (SP analogue) 10 µM [D-Arg1,D-Trp5,7,9,Leu11]Substance P for 5 min at 37 °C and then challenged for a further 10 min either in the absence () or the presence (Bom) of 1 nM bombesin. Cells were then washed, fixed in 4% paraformaldehyde, and permeabilized with 0.2% Triton X-100. Focal adhesions were visualized by staining with vinculin mAb followed by FITC-labeled anti-mouse IgG and visualized using a Zeiss fluorescence microscope as described under "Experimental Procedures." Results are typical of three independent experiments.

View larger version (129K): [in this window] [in a new window]   Fig. 5.   [D-Arg1,D-Trp5,7,9,Leu11]Substance P inhibits the formation of actin stress fibers in cells induced by bombesin. Confluent and quiescent Swiss 3T3 cells were washed and preincubated without () or with (SP analogue) 10 µM [D-Arg1,D-Trp5,7,9,Leu11]Substance P for 5 min at 37 °C and then incubated for a further 10 min either in the absence() or the presence (Bom) of 1 nM bombesin. Cells were then washed, fixed in 4% paraformaldehyde, and permeabilized with 0.2% Triton X-100. Focal adhesions were visualized by staining with TRITC-conjugated phalloidin (0.25 µg/ml) for 10 min and visualized using a Zeiss fluorescence microscope as described under "Experimental Procedures." Results are typical of five independent experiments.

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-Arg¹,D-Trp^5,7,9,Leu¹¹]SP at 10 µM, 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 µM [D-Arg¹,D-Trp^5,7,9,Leu¹¹]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-Arg¹,D-Trp^5,7,9,Leu¹¹]SP induces G₁₂-mediated events at concentrations that block G_q-mediated signaling.

Inhibitory Effect of [D-Arg¹,D-Trp^5,7,9,Leu¹¹]SP on Additional G₁₂-mediated Events, Tyrosine Phosphorylation of FAK, p130^Cas, and Paxillin-- G₁₂ and G₁₃ also mediate the increase in the tyrosine phosphorylation of FAK, paxillin, and p130^Cas through a Rho-dependent pathway (16). Consequently, if [D-Arg¹,D-Trp^5,7,9,Leu¹¹]SP acts as a selective G₁₂ 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 µM [D-Arg¹,D-Trp^5,7,9,Leu¹¹]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-Arg¹,D-Trp^5,7,9,Leu¹¹]SP at 10 µM did not promote FAK tyrosine phosphorylation but, in contrast, dramatically inhibited (~90%) the increase in the tyrosine phosphorylation of FAK induced by bombesin.

View larger version (17K): [in this window] [in a new window]   Fig. 6.   [D-Arg1,D-Trp5,7,9,Leu11]Substance P inhibits tyrosine phosphorylation of FAK, phosphorylation of FAK at Tyr-397, and the formation of the Src·FAK complex induced by bombesin. A, [D-Arg1,D-Trp5,7,9,Leu11]Substance P inhibits tyrosine phosphorylation of FAK induced by bombesin. Confluent and quiescent cells were washed and incubated for 5 min at 37 °C without () or with (+) 10 µM [D-Arg1,D-Trp5,7,9,Leu11]Substance P (SP analogue). Cells were then stimulated with 1 nM bombesin (Bom) for 10 min at 37 °C and lysed. The lysates were immunoprecipitated with an anti-FAK polyclonal antibody, and the immunoprecipitates were analyzed by Western blotting using an anti-Tyr(P) mAb as described under "Experimental Procedures." Result shown are representative of four independent experiments. B, scanning densitometry. The results shown are the values (mean ± S.E.; n = 4) obtained by scanning densitometry, expressed as a percentage of the maximal FAK phosphorylation obtained with 1 nM bombesin in the absence of [D-Arg1,D-Trp5,7,9,Leu11]Substance P. All other experimental details are as described under "Experimental Procedures." C, [D-Arg1,D-Trp5,7,9,Leu11]Substance P inhibits phosphorylation of FAK at Tyr-397 and the formation of the Src·FAK complex induced by bombesin. Confluent and quiescent cells were washed and incubated for 5 min at 37 °C without () or with (+) 10 µM [D-Arg1,D-Trp5,7,9,Leu11]Substance P (SP analogue). Cells were then stimulated with 1 nM bombesin for 10 min at 37 °C and lysed. The lysates were immunoprecipitated with either anti-FAK or anti-Src polyclonal antibody as indicated, and the immunoprecipitates were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes. Phosphorylation of Tyr-397 in the FAK immunoprecipitates was detected by Western blotting using a phosphospecific polyclonal antibody (FAK 397). The formation of an Src·FAK complex in the Src immunoprecipitates (Src I.P.) was detected by Western blotting using an anti-FAK polyclonal antibody (FAK). All other details are as described under "Experimental Procedures."

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-Arg¹,D-Trp^5,7,9,Leu¹¹]SP acts as an agonist for G₁₂-mediated events, we examined the effect of this synthetic peptide on bombesin-induced tyrosine phosphorylation of FAK at Tyr-397 and on Src·FAK complex formation which depends on the tyrosine phosphorylation of Tyr-397. The results, shown in Fig. 6C, indicate that treatment with 10 µM [D-Arg¹,D-Trp^5,7,9,Leu¹¹]SP neither induced a significant increase in the tyrosine phosphorylation of FAK at Tyr-397 nor promoted Src·FAK 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-Arg¹,D-Trp^5,7,9,Leu¹¹]SP on bombesin-induced tyrosine phosphorylation of the adaptor proteins p130^Cas and paxillin. As illustrated by Fig. 7, addition of [D-Arg¹,D-Trp^5,7,9,Leu¹¹]SP at 10 µM 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-Arg¹,D-Trp^5,7,9,Leu¹¹]SP (at 10 µM) acts as an antagonist that coordinately prevents GPCR-induced events mediated by either G_q or G₁₂.

View larger version (16K): [in this window] [in a new window]   Fig. 7.   [D-Arg1,D-Trp5,7,9,Leu11]Substance P inhibits tyrosine phosphorylation of p130Cas and paxillin induced by bombesin. A, [D-Arg1,D-Trp5,7,9,Leu11]Substance P inhibits tyrosine phosphorylation of p130Cas induced by bombesin. Confluent and quiescent cells were washed and incubated for 5 min at 37 °C without () or with (+) 10 µM [D-Arg1,D-Trp5,7,9,Leu11]Substance P (SP analogue). Cells were then stimulated with 1 nM bombesin (Bom) for 10 min at 37 °C and lysed. The lysates were immunoprecipitated with an anti-p130Cas mAb, and the immunoprecipitates were analyzed by Western blotting using an anti-Tyr(P) mAb as described under "Experimental Procedures." Result shown are representative of three independent experiments. B, scanning densitometry. The results shown are the values (mean ± S.E. n = 3) obtained by scanning densitometry, expressed as a percentage of the maximal p130Cas phosphorylation obtained with 1 nM bombesin. All experimental details are as described under "Experimental Procedures." C, [D-Arg1,D-Trp5,7,9,Leu11]Substance P inhibits tyrosine phosphorylation of paxillin induced by bombesin. Confluent and quiescent cells were washed and incubated for 5 min at 37 °C without () or with (+) 10 µM [D-Arg1,D-Trp5,7,9,Leu11]Substance P (SP analogue). Cells were then stimulated with 1 nM bombesin for 10 min at 37 °C and lysed. The lysates were immunoprecipitated with an anti-paxillin mAb, and the immunoprecipitates were analyzed by Western blotting using an anti-Tyr(P) mAb as described under "Experimental Procedures." Results shown are representative of three independent experiments. D, scanning densitometry. The results shown are the values (mean ± S.E. n = 3) obtained by scanning densitometry, expressed as a percentage of the maximal paxillin phosphorylation obtained with 1 nM bombesin. All other experimental details are described under "Experimental Procedures."

[D-Arg¹,D-Phe⁵,D-Trp^5,7,9,Leu¹¹]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-Arg¹,D-Phe⁵,D-Trp^7,9,Leu¹¹]SP, a less potent analogue of [D-Arg¹,D-Trp^5,7,9,Leu¹¹]SP, induced actin reorganization and focal adhesion assembly. However, [D-Arg¹,D-Phe⁵,D-Trp^7,9,Leu¹¹]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 G₁₂-mediated events, as shown here with [D-Arg¹,D-Trp^5,7,9,Leu¹¹]SP, was not explored. In an effort to reconcile these discrepancies, we determined the effect of [D-Arg¹,D-Phe⁵,D-Trp^7,9,Leu¹¹]SP on bombesin-induced focal adhesion assembly and tyrosine phosphorylation of FAK and paxillin.

As shown in Fig. 8, treatment with 20 µM [D-Arg¹,D-Phe⁵,D-Trp^7,9,Leu¹¹]SP neither promoted focal adhesion assembly nor induced a significant increase in the tyrosine phosphorylation of FAK and paxillin. In contrast, exposure to this SP analogue markedly inhibited the increase in focal adhesion assembly and tyrosine phosphorylation of FAK and paxillin induced by bombesin stimulation (Fig. 8). Thus, [D-Arg¹,D-Phe⁵,D-Trp^7,9,Leu¹¹]SP, like [D-Arg¹,D-Trp^5,7,9,Leu¹¹]SP, inhibited focal adhesion assembly and tyrosine phosphorylation of FAK and paxillin.

View larger version (46K): [in this window] [in a new window]   Fig. 8.   [D-Arg1,D-Phe5,D-Trp7,9,Leu11]Substance P inhibits assembly of focal adhesions and tyrosine phosphorylation of FAK and paxillin induced by bombesin. A, confluent and quiescent Swiss 3T3 cells were washed and preincubated without () or with 20 µM [D-Arg1,D-Phe5,D-Trp7,9,Leu11]Substance P for 5 min at 37 °C and then challenged for a further 10 min either in the absence () or the presence of 1 nM bombesin (Bom). Cells were then washed, fixed in 4% paraformaldehyde, and permeabilized with 0.2% Triton X-100. Focal adhesions were visualized by staining with vinculin mAb followed by FITC-labeled anti-mouse IgG and visualized using a Zeiss fluorescence microscope as described under "Experimental Procedures." Results are typical of three independent experiments. B, [D-Arg1,D-Phe5,D-Trp7,9,Leu11]Substance P inhibits tyrosine phosphorylation of FAK and paxillin induced by bombesin. Confluent and quiescent cells were washed and incubated for 5 min at 37 °C without () or with (+) 20 µM [D-Arg1,D-Phe5,D-Trp7,9,Leu11]Substance P. Cells were then stimulated with 1 nM bombesin (Bom) for 10 min at 37 °C and lysed. The lysates were either immunoprecipitated with an anti-FAK mAb or anti-Tyr(P) mAb. The FAK immunoprecipitates were analyzed by Western blotting using an anti-Tyr(P) mAb, and the anti-Tyr(P) mAbs were analyzed by Western blotting using an anti-paxillin mAb as described under "Experimental Procedures." Result shown are representative of four independent experiments. C, scanning densitometry. The results shown are the values (mean ± S.E. n = 4) obtained by scanning densitometry, expressed as a percentage of the maximal FAK or paxillin phosphorylation obtained with 1 nM bombesin. All experimental details are as described under "Experimental Procedures."

[D-Arg¹,D-Trp^5,7,9,Leu¹¹]SP Stimulates Morphological Responses and Tyrosine Phosphorylation of FAK and Paxillin at High Concentrations-- We also examined the effect of [D-Arg¹,D-Trp^5,7,9,Leu¹¹]SP on focal adhesion assembly and tyrosine phosphorylation of FAK and paxillin at concentrations higher than 10 µM. As shown in Fig. 9, exposure of Swiss 3T3 cells to 50 µM [D-Arg¹,D-Trp^5,7,9,Leu¹¹]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-Arg¹,D-Trp^5,7,9,Leu¹¹]SP at 50 µM 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-Arg¹,D-Trp^5,7,9,Leu¹¹]SP at 50 µM markedly reduced the assembly of focal adhesions and the increase in tyrosine phosphorylation of focal adhesion proteins induced by bombesin. We conclude that [D-Arg¹,D-Trp^5,7,9,Leu¹¹]SP acts as an antagonist of neuropeptide GPCRs that signal via both G_q and G₁₂, 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.

View larger version (66K): [in this window] [in a new window]   Fig. 9.   Effect of high concentrations of [D-Arg1,D-Trp5,7,9,Leu11]Substance P on assembly of focal adhesions and tyrosine phosphorylation of focal adhesion proteins. A, effect of high concentrations of [D-Arg1,D-Trp5,7,9,Leu11]Substance P on assembly of focal adhesions in Swiss 3T3 cells challenged with or without bombesin. Confluent and quiescent Swiss 3T3 cells were washed and preincubated without () or with (SP analogue) 50 µM [D-Arg1,D-Trp5,7,9,Leu11]Substance P for 5 min at 37 °C. The cells were then incubated for a further 10 min either in the absence () or the presence of 1 nM bombesin (Bom). Cells were then washed, fixed in 4% paraformaldehyde, and permeabilized with 0.2% Triton X-100. Focal adhesions were visualized by staining with vinculin mAb followed by FITC-labeled anti-mouse IgG and visualized using a Zeiss fluorescence microscope as described under "Experimental Procedures." Results are typical of three independent experiments. B, effect of 50 µM [D-Arg1,D-Trp5,7,9,Leu11]Substance P on tyrosine phosphorylation of FAK and paxillin in Swiss 3T3 cells challenged with or without bombesin. Confluent and quiescent cells were washed and incubated for 5 min at 37 °C without () or with (+) 50 µM [D-Arg1,D-Trp5,7,9,Leu11]Substance P (SP analogue). Cells were then stimulated with 1 nM bombesin for 10 min at 37 °C and lysed. The lysates were immunoprecipitated with either anti-FAK polyclonal antibody or paxillin mAb, and the immunoprecipitates were analyzed by Western blotting using an anti-Tyr(P) mAb described under "Experimental Procedures." Result shown are representative of three independent experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

SP analogues including [D-Arg¹,D-Phe⁵,D-Trp^7,9,Leu¹¹]SP and [Arg⁶,D-Trp^7,9,MePhe⁸]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 G₁₂, thereby acting as an agonist for G₁₂-mediated events but as an antagonist for G_q-mediated events. It was further suggested that the inhibitory effect of these synthetic peptides on neuropeptide-stimulated 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-Arg¹,D-Trp^5,7,9,Leu¹¹]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 mechanisms of action. Specifically, we examined whether [D-Arg¹,D-Trp^5,7,9,Leu¹¹]SP, used at a concentration that inhibits neuropeptide-induced DNA synthesis, ERK activation, and PKC-dependent PKD activation, acts as a selective agonist of G₁₂-mediated 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 G₁₂ and G₁₃ are known to induce Rho activation (15, 57-59) 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 G₁₂ and G₁₃ 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 G₁₂ and/or G₁₃ leading to Rho-dependent formation of actin stress fibers and assembly of focal adhesions resulting in the recruitment of FAK to focal adhesions and in the tyrosine phosphorylation of FAK, paxillin, and p130^Cas. In order to elucidate whether [D-Arg¹,D-Trp^5,7,9,Leu¹¹]SP promotes the selective activation of this pathway, we examined in the present study the effects of [D-Arg¹,D-Trp^5,7,9,Leu¹¹]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 µM [D-Arg¹,D-Trp^5,7,9,Leu¹¹]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-Arg¹,D-Trp^5,7,9,Leu¹¹]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-Arg¹,D-Trp^5,7,9,Leu¹¹]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-Arg¹,D-Trp^5,7,9,Leu¹¹]SP did not promote Src·FAK complex formation. Similarly, [D-Arg¹,D-Trp^5,7,9,Leu¹¹]SP did not induce tyrosine phosphorylation of paxillin or p130^Cas. A salient feature of the results presented here is that treatment with [D-Arg¹,D-Trp^5,7,9,Leu¹¹]SP dramatically inhibited the increase in the tyrosine phosphorylation of these focal adhesion proteins induced by bombesin. Thus, [D-Arg¹,D-Trp^5,7,9,Leu¹¹]SP, at a concentration that inhibits G_q signaling and mitogenesis by neuropeptides, also prevents G₁₂-mediated events including focal adhesion assembly, stress fiber formation, and tyrosine phosphorylation of focal adhesion proteins.

Jarpe et al. (43) demonstrated that [D-Arg¹,D-Phe⁵,D-Trp^7,9,Leu¹¹]SP, a less potent analogue of [D-Arg¹,D-Trp^5,7,9,Leu¹¹]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-Arg¹,D-Trp^5,7,9,Leu¹¹]SP and those with [D-Arg¹,D-Phe⁵,D-Trp^7,9,Leu¹¹]SP is that a single amino acid change at position 5 from D-Phe⁵ to D-Trp⁵ is responsible for the drastic change in the properties of these antagonists. However, Jarpe et al. (43) did not examine the possibility that [D-Arg¹,D-Phe⁵,D-Trp^7,9,Leu¹¹]SP, at a lower concentration, could also inhibit G₁₂-mediated events, as shown here with [D-Arg¹,D-Trp^5,7,9,Leu¹¹]SP. In the present study, we show that [D-Arg¹,D-Phe⁵,D-Trp^7,9,Leu¹¹]SP (at 20 µM) 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 µM [D-Arg¹,D-Trp^5,7,9,Leu¹¹]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-Arg¹,D-Trp^5,7,9,Leu¹¹]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-Arg¹,D-Trp^5,7,9,Leu¹¹]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-Arg¹,D-Trp^5,7,9,Leu¹¹]SP acts as a broad spectrum mitogenic antagonist of neuropeptide GPCRs blocking signal transduction via both G_q and G₁₂. 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-Arg¹,D-Trp^5,7,9,Leu¹¹]SP stabilizes a bombesin receptor conformation that interacts selectively with G₁₂ but indicate that this SP analogue inhibits bombesin signaling mediated by both G_q and G₁₂.

	ACKNOWLEDGEMENTS

We thank Drs. Richard Waldron and Cliff Hurd for helpful comments and suggestions.

	FOOTNOTES

^* This work was supported by a grant from the Margaret E. Early Medical Research Trust.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Both authors contributed equally to this work.

^§ Ronald S. Hirshberg professor of Translational Pancreatic Cancer Research. To whom correspondence should be addressed: 900 Veteran Ave., Warren Hall Rm. 11-124, Dept. of Medicine, UCLA School of Medicine, Los Angeles, CA 90095-1786. Tel.: 310-794-6610; Fax: 310-267-2399; E-mail: erozengurt@mednet.ucla.edu.

Published, JBC Papers in Press, July 3, 2000, DOI 10.1074/jbc.M003702200

	ABBREVIATIONS

The abbreviations used are: GRP, gastrin-releasing peptide; DMEM, Dulbecco's modified Eagle's medium; EGF, epidermal growth factor; FAK, focal adhesion kinase; FITC, fluorescein isothiocyanate; GPCR, G protein-coupled receptor; mAb, monoclonal antibody; Ab, antibody; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PDBu, phorbol 12,13-dibutyrate; PDGF, platelet-derived growth factor; PKC, protein kinase C; SCLC, small cell lung carcinoma; mapk, mitogen-activated protein kinase; SP, Substance P; TRITC, tetramethylrhodamine B isothiocyanate; PKD, protein kinase D; PGF₂, prostaglandin F₂.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES