[D-Arg1,D-Phe5,D-Trp7,9,Leu11]Substance P Acts as a Biased Agonist toward Neuropeptide and Chemokine Receptors*

Matthew B. JarpeDagger §, Cindy KnallDagger , Fiona M. MitchellDagger , Anne Mette BuhlDagger , Emir Duzicpar , and Gary L. JohnsonDagger **

From the Dagger  Program in Molecular Signal Transduction, Division of Basic Sciences, National Jewish Medical Research Center, Denver, Colorado 80206, par  Cadus Pharmaceuticals, Tarrytown, New York 10591, and the ** Department of Pharmacology, University of Colorado Health Sciences Center, Denver, Colorado 80262

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Substance P derivatives are potential therapeutic compounds for the treatment of small cell lung cancer and can cause apoptosis in small cell lung cancer cells in culture. These peptides act as broad spectrum neuropeptide antagonists, blocking calcium mobilization induced by gastrin-releasing peptide, bradykinin, cholecystokinin, and other neuropeptides. We show that [D-Arg1,D-Phe5,D-Trp7,9,Leu11]substance P has unique agonist activities in addition to this described antagonist function. At doses that block calcium mobilization by neuropeptides, this peptide causes activation of c-Jun N-terminal kinase and cytoskeletal changes in Swiss 3T3 fibroblasts and stimulates migration and calcium flux in human neutrophils. Activation of c-Jun N-terminal kinase is dependent on the expression of the gastrin-releasing peptide receptor in rat 1A fibroblasts, demonstrating that the responses to the peptide are receptor-mediated. We hypothesize that [D-Arg1,D-Phe5,D-Trp7,9,Leu11]substance P acts as a biased agonist on neuropeptide and related receptors, activating certain guanine nucleotide-binding proteins through the receptor, but not others.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Neuropeptides are a structurally diverse group of hormones and neurotransmitters that bind to a related subfamily of G protein-coupled receptors (1) and function in neuron-to-neuron communication, as well as in signaling in the immune system and in tissue restructuring. Neuropeptides and their receptors are the principal driving force behind one of the most clinically aggressive cancers, small cell lung cancer (SCLC).1 SCLC tumors sustain their growth, in part, by maintaining neuropeptide autocrine and paracrine loops (2). These tumor cells in culture can secrete and respond mitogenically to multiple neuropeptides (3). For this reason, broad spectrum antagonists of neuropeptides have been examined for their ability to prevent growth of SCLC cells in vitro and in vivo. One such compound, [D-Arg1,D-Phe5,D-Trp7,9,Leu11]substance P (SPD-D) (4) can not only inhibit the action of substance P (5) but also inhibit binding and action of gastrin-releasing peptide (GRP), arginine vasopressin (6), and endothelin (7). To date, much of the research on the mechanism of action of SPDs has focused on their abilities to block ligand binding and Ca2+ flux, as well as on their cytostatic or cytotoxic effects on SCLC cells in culture (8).

The signal transduction pathways that mediate neuropeptide actions are rapidly becoming more clear. It is known that neuropeptides induce calcium mobilization by a pertussis toxin-insensitive mechanism, suggesting a role for Gq (9). Heterologous expression of receptors and G proteins in Sf9 cells have shown functional coupling between one neuropeptide receptor, NK-1, and Gz, Gq, and G13 but not Gs (10). In addition to the mobilization of Ca2+ from intracellular stores, neuropeptides can have diverse effects on cells in culture, including the induction of mitogenesis (11), the activation of both the extracellular signal-regulated kinase (ERK) (12) and the c-Jun N-terminal kinase (JNK) (13, 14) members of the mitogen-activated protein kinase family, and formation of actin structures such as filopodia (15) and stress fibers (16, 17). The activation of JNK and the reorganization of the actin cytoskeleton are most likely mediated by members of the G12 family of G proteins, such as Galpha 12 and Galpha 13 (18, 19). The regulation of ERKs by neuropeptides is more complicated, suggesting a role for PKC (20), G protein beta gamma subunits (12), and a pertussis toxin-sensitive mechanism possibly involving a Gi family member (21).

The effects of SPD-D and similar compounds on these signaling events downstream of neuropeptide receptors suggest a mechanism that is more complicated than that of a classical antagonist. The ERK response and the Ca2+ response through neuropeptide receptors are affected differently by SPD-D (22, 23). In Swiss 3T3 fibroblasts, SPD-D inhibits Ca2+ mobilization induced by bombesin (which acts on the human GRP receptor) with a maximal effect at 10 µM and an estimated IC50 of 2 µM. In contrast, SPD-D inhibits ERK-2 activation with a maximal effect at 50 µM and an estimated IC50 of 9 µM (22). In the presence of 3 nM bombesin, 10 µM SPD-D caused a nearly complete inhibition of the Ca2+ response and an approximately 50% inhibition of the ERK-2 response (22). In a similar manner, 50 µM SPD-D caused a 100% inhibition of the inositol 1,4,5-trisphosphate generation induced by 50 nM bombesin, but only a 20% inhibition of the total ERK response (23).

In an effort to uncover the mechanism behind these differences, we examined the effect of SPD-D on other signal transduction pathways downstream of neuropeptide receptors, such as the activation of JNK and rearrangement of the actin cytoskeleton. To our surprise, rather than acting to inhibit these responses caused by bombesin, SPD-D stimulated both JNK and cytoskeletal changes. These responses are shown to occur through the action of SPD-D on neuropeptide receptors, because rat 1A cells, which do not respond to SPD-D with a JNK response, gain the ability to respond when transfected with the GRP receptor. SPD-D can also act as an agonist toward chemoattractant receptors in human neutrophils. We show that SPD-D binds to both of the cloned interleukin-8 (IL-8) receptors, and the peptide can stimulate an increase in neutrophil migration and Ca2+ mobilization.

These results indicate that the mechanism of action of SPD-D is more complex than has previously been appreciated. Rather than acting as a classical antagonist, SPD-D is capable of initiating signals in multiple cell types in a receptor-dependent manner. Unlike a classical agonist, however, SPD-D does not induce receptor signaling to all available signal transduction pathways. SPD-D selectively activates JNK and actin reorganization in fibroblasts in the absence of Ca2+ mobilization. This is the first description of such a biased agonist. We expect that this novel mechanism of action will prove an important component of the cytotoxic activity such compounds have on SCLC. An understanding of these mechanisms is crucial to the development of this type of therapy for future clinical use.

    EXPERIMENTAL PROCEDURES
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Procedures
Results
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References

Materials-- [D-Arg1,D-Phe5,D-Trp7,9,Leu11]substance P, purchased from Bachem Bioscience, was stored in powder form at -20 °C and dissolved to 20 mM in degassed, sterile water and stored under argon for no more than 4 weeks. Rhodamine-conjugated phalloidin was purchased from Molecular Probes. Rabbit anti-beta -galactosidase antibody was purchased from Cappel. Recombinant human IL-8 was purchased from R&D Systems, recombinant C5a was from Sigma, and [Tyr4] bombesin was from Peninsula Laboratories. 125I-Labeled C5a, IL-8, and bombesin were purchased from NEN Life Science Products.

Cell Lines and Culture-- Swiss 3T3 fibroblasts were cultured in Dulbecco's modified Eagle's medium with 10% fetal calf serum (DME-10% FCS) and split every 3 days. For each experiment, Swiss 3T3 cells were split 1:10 and grown for 1 day in DME-10% FCS and then starved for 1 day in DME-0.1% calf serum prior to the experiment.

Rat 1A fibroblasts were grown in DME-10% calf serum. Cells transiently expressing the human gastrin-releasing peptide receptor (GRPR) were generated using LipofectAMINE (Life Technologies, Inc.). The cells were transfected with pCMV5 or pCMV5-GRPR and grown for 1 day in DME-10% FCS and then split 1:5 and grown for 1 more day in 10% FCS. The cells were then starved for 1 day in 0.1% calf serum prior to the experiment.

Human neutrophils were isolated from healthy donors and stored for no more than 6 h at 4 °C in Krebs-Ringer phosphate buffer containing 0.25% human serum albumin. Chemotaxis assays were performed essentially as described (24).

Jun Kinase-- Cells were treated as indicated and lysed in JNK lysis buffer, and JNK activity was determined as described previously (25). Bands were visualized by autoradiography and quantitated using a PhosphorImager (Molecular Dynamics).

Calcium Measurement in Rat 1A Cells-- Cells were transfected with the empty vector (pCMV5) or with pCMV5-GRPR using Pfx-3 transfection reagent (InVitrogen). After 1 day of recovery, the cells were placed with 0.1% calf serum into 12-well plates and starved for 1 day. The cells were then loaded with 1 µM Fluo-3 AM (Molecular Probes) for 30 min at room temperature in HEPES-buffered DME-0.1% calf serum. The cells were washed and intracellular calcium was determined by imaging on a laser confocal microscope. The images were processed using COMOS software (Bio-Rad). The brightness of each × 40 field of approximately 10 cells was determined and divided by the baseline value to give the relative Fluo-3 fluorescence.

Cytoskeletal Reorganization-- Swiss 3T3 cells were grown on glass coverslips for 1 day in DME-10% FCS and starved 1 day in DME-0.1% calf serum, followed by a 1-day starvation in DME-0.1% bovine serum albumin. Cells were treated for 10 min with control medium or medium containing the indicated concentration of SPD-D and fixed for 10 min in 3% paraformaldehyde, 3% sucrose, phosphate buffered saline. The cells were then permeablilized with 0.2% Triton X-100 in PBS for 10 min, nonspecific protein binding was blocked with a 30-min incubation in DME-10% FCS, and the actin cytoskeleton was stained with rhodamine-phalloidin at a 1:40 dilution. The slides were mounted in o-phenylendiamine mounting medium and visualized on a digital confocal microscope. The images were deconvolved using Slidebook software (Intelligent Imaging Innovations, Inc.)

Microinjection-- Cells were grown on glass coverslips and starved as described above. Microinjection was performed using an Eppendorf microinjector with an injection pressure of 600 hectopascals and an injection time of 0.3 s. A mixture of pCMV5-lacZ and either pCMV5 or pCMV5-N19Rho was microinjected into approximately 100 cells/coverslip. The cells were incubated for 2 h and then treated with or without SPD-D for 10 min, fixed, and stained as above, with the addition of anti-beta -galactosidase antibody (Cappel) and a fluorescein isothiocyanate-conjugated goat anti-rabbit secondary (Jackson Immunologicals).

Binding Assays-- HEK-293 cells stably expressing human C5a, CXCR1, or GRP receptors were grown in DME-10% FCS, 400 µg/ml hygromycin. Membranes were prepared as described (26) and diluted in binding buffer (50 mM HEPES, pH 7.4, 1 mM MgCl2, 2.5 mM CaCl2, 0.2% bovine serum albumin, 0.1 mM phenylmethylsulfonyl fluoride, 10 µg/ml pepstatin A, 10 µg/ml aprotinin) at a concentration of 0.05 mg/ml. Membranes were incubated with 125I-labeled C5a, IL-8, or bombesin (0.1 nM) at 25 °C for 1 h in the presence of unlabeled competing ligands in a 96-well microtiter plate. At the end of the incubation, the assay was terminated by the addition of ice-cold 50 mM HEPES, pH 7.4, followed by rapid filtration over glass fiber filters. The filters were dried and coated with scintillation fluid and counted on a TopCount scintillation counter (Packard).

    RESULTS
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Abstract
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Procedures
Results
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References

SPD-D Induces JNK in Fibroblasts-- Bombesin, a peptide agonist of the GRP receptor, was found to stimulate JNK activation in Swiss 3T3 fibroblasts (Fig. 1A) with a dose response that is similar to the stimulation of ERK activity (23) in these cells (data not shown). 50 µM SPD-D was used in previous studies to inhibit inositol 1,4,5-trisphosphate generation and ERK activation by bombesin (23). Rather than inhibiting bombesin-stimulated JNK activation, 50 µM SPD-D caused a similar activation of JNK (Fig. 1A), which reached a maximum at 5 min (Fig. 1B). This activity was sustained for 30 min and returned to baseline by 60 min (data not shown). The dose response curve for JNK activation is shown in Fig. 1C. The maximum activation was reached at 40 µM.


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Fig. 1.   SPD-D activates JNK in Swiss 3T3 fibroblasts. A, Swiss 3T3 fibroblasts were starved for 1 day in 0.1% calf serum and treated as indicated with 50 nM bombesin or 50 µM SPD-D for 10 min. Jun kinase activity was determined by precipitation with and phosphorylation of GST-c-Jun. B, time course of SPD-D-induced JNK activation. Swiss 3T3 cells were treated as above with 50 µM SPD-D for the indicated times. The amount of phosphorylation incorporated was quantitated by PhosphorImager analysis, and values are expressed as fold increase over control. An autoradiograph of one representative experiment is shown in the bottom panel. C, cells were treated with the indicated concentrations of SPD-D for 10 min, and Jun kinase activity was determined as above. An autoradiograph of one representative experiment is shown below. Error bars represent 1 S.D. of three (B) or two (C) samples.

The activation of JNK by SPD-D is surprising because thus far this peptide has been described as a competitive antagonist that acts by binding to the receptor and blocking binding of neuropeptides (4). The activation of JNK suggests that the peptide has agonist properties, not unlike similar compounds based on the structure of substance P (27, 28). However, this agonist activity is unique in that SPD-D does not mobilize Ca2+ in Swiss 3T3 cells (22). To test whether this unique agonist property was the result of SPD-D binding to neuropeptide receptors, we used a transient transfection assay in another cell line, rat 1A fibroblasts.

Untransfected rat 1A fibroblasts failed to respond to a dose of SPD-D as high as 50 µM. When these cells were transiently transfected with the human GRP receptor, the basal JNK activity increased (Fig. 2A). This increase in the basal JNK activity is most likely due to spontaneous activity in the expressed receptor, similar to that seen with the bradykinin receptor (29) and the calcitonin receptor (30), which activate adenylate cyclase in the absence of agonist when overexpressed. Cells transiently expressing the GRP receptor gained the ability to respond to SPD-D (Fig. 2A). SPD-D can therefore stimulate JNK activity through the GRP receptor.


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Fig. 2.   SPD-D induces JNK activation but not Ca2+ flux in rat 1A cells expressing the gastrin-releasing peptide receptor. A, rat 1A cells were transfected with empty vector (pCMV5) or vector containing the cDNA for the human GRPR. Cells were starved and treated with or without SPD-D, and Jun kinase was determined as described in the legend for Fig. 1. Error bars represent 1 S.D. of four samples. An autoradiograph of one representative experiment is shown in the bottom panel. B, rat 1A cells transfected with pCMV5 (top panel) or pCMV5-human GRPR (bottom panel) were treated with 50 nM bombesin (circles) or 50 µM SPD-D (squares). Calcium flux was measured by laser confocal microscopy using Fluo-3 as a calcium-sensitive dye. Curves are representative of two to four independent experiments. C, rat 1A fibroblasts transiently transfected with the GRP receptor were stimulated with 30 nM bombesin (top), 300 nM bombesin (middle) or 10% fetal calf serum (bottom) in the absence (filled squares) or presence (open squares) of 50 µM SPD-D. Calcium flux was measured as in B.

Rat 1A cells transfected with the empty vector did not respond to bombesin with an increase in intracellular calcium (Fig. 2B), but when these cells were transfected with the GRP receptor, 50 nM bombesin caused a rapid Ca2+ flux. SPD-D did not stimulate Ca2+ mobilization in rat 1A fibroblasts with or without the GRP receptor (Fig. 2B). This is in agreement with the inability of SPD-D to cause calcium flux in Swiss 3T3 or SCLC cells (4, 22). In these systems, SPD-D has been described as a competitive antagonist of bombesin and other neuropeptides. To confirm this competitive antagonism, we tested the ability of SPD-D to inhibit bombesin-stimulated calcium flux in receptor-expressing rat 1A cells (Fig. 2C). Whereas 50 µM SPD-D was able to inhibit calcium mobilization induced by 30 nM bombesin, 300 nM bombesin was able to overcome this inhibition. Neuropeptide-induced Ca2+ mobilization in fibroblasts is mediated by members of the Gq family of alpha  subunits (9), whereas it is likely that JNK activation is mediated by G12 family members (31). SPD-D therefore acts as a biased agonist, activating G12 and subsequent signaling components, without activating Gq and its downstream effectors.

An alternative explanation for the lack of Ca2+ mobilization by SPD-D is that SPD-D activates a receptor that initiates a signal transduction pathway that inhibits Ca2+ mobilization. To test this, we treated rat 1A cells with 10% fetal calf serum. Serum induced a rapid, sustained Ca2+ flux, which was not inhibited by SPD-D in untransfected cells (data not shown) or in cells expressing the GRP receptor (Fig. 2C). This and the fact that 300 nM bombesin can stimulate calcium mobilization in the presence of 50 µM SPD-D show that SPD-D inhibition of calcium mobilization is through competitive antagonism.

SPD-D Induces Actin Cytoskeletal Reorganization in Fibroblasts-- In addition to activating JNK, bombesin induces the formation of actin stress fibers in Swiss 3T3 cells (17). Other neuropeptides also cause reorganization of the actin cytoskeleton. For example, bradykinin causes the formation of peripheral actin microspikes and filopodia in Swiss 3T3 cells (15). Like JNK activation, this cytoskeletal reorganization is mediated by the G12 family of G proteins (19). To further test the idea that SPD-D can activate G12 in the absence of Gq activation, we visualized the actin cytoskeleton in cells treated with SPD-D. When 10 µM SPD-D was added to quiescent, starved Swiss 3T3 fibroblasts for 10 min, structures formed at the periphery of the cell outside the cortical actin ring (Fig. 3C, arrow). These structures resembled peripheral actin microspikes as they were described in Swiss 3T3 cells treated with bradykinin (15). Some cells showed the beginnings of stress fibers forming at the periphery (Fig. 3D, arrow).


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Fig. 3.   SPD-D causes the reorganization of the actin cytoskeleton in Swiss 3T3 fibroblasts. Swiss 3T3 cells were grown on glass coverslips and starved for 1 day in 0.1% calf serum, followed by 1 day of starvation in 0.1% bovine serum albumin. Cells were treated for 10 min with control medium (A and B), 10 µM SPD-D (C and D), 25 µM SPD-D (E and F), or 50 µM SPD-D (G and H). Cells were then fixed in paraformaldehyde. The actin cytoskeleton was stained with rhodamine-phalloidin and visualized on a digital confocal fluorescence microscope using a × 62 objective. Representative fields are shown for each concentration of SPD-D. Arrows indicate features discussed in the text.

At 25 µM SPD-D, structures resembling filopodia formed (Fig. 3E, arrow). Significant lamellar actin structures were found in most cells at this dose (Fig. 3F). These structures are two-dimensional, as opposed to membrane ruffles, in which the edge of the cell lifts off of the coverslip. At 50 µM SPD-D, adhesion plaques (Fig. 3G), as well as lamellar actin structures (Fig. 3H), formed. At concentrations up to 100 µM, the cells retained their ability to exclude trypan blue dye for up to 20 min (data not shown). This effect of SPD-D on the cytoskeleton again demonstrates that SPD-D acts as a biased agonist, stimulating G12 signal transduction pathways.

Previous work by this laboratory has mapped out the sequence of events by which G12 can induce cytoskeletal reorganization (19). This response is mediated by the low molecular weight GTP-binding protein Rho. Therefore, if Rho is inactivated by injection of C3 toxin into the cell or inhibited by the expression of a competitive inhibitory mutant of Rho, N19Rho-A, G12-induced stress fiber formation does not occur. To further show that SPD-D-induced responses, such as actin stress fiber formation, are mediated by G12, a plasmid encoding N19Rho-A was microinjected into Swiss 3T3 fibroblasts along with a second construct encoding beta -galactosidase. Injected cells, identified by staining for beta -galactosidase, could no longer form stress fibers in response to SPD-D (Fig. 4), whereas the neighboring cells that were not microinjected formed stress fibers. This laboratory has previously shown that microinjection of beta -galactosidase alone does not interfere with actin stress fiber formation (19). The effect of N19Rho-A on cytoskeletal reorganization provides further evidence that SPD-D acts on the cells through G12.


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Fig. 4.   SPD-D-induced cytoskeletal reorganization is blocked by the expression of N19Rho. Swiss 3T3 fibroblasts were grown on glass coverslips and starved as described in the legend for Fig. 3. Cells were injected with a mixture of plasmids encoding beta -galactosidase and N19Rho (pCMV5-lacZ and pCMV5-N19Rho), incubated for 2 h, and treated with SPD-D and fixed as described above. The expression of beta -galactosidase was determined by staining with a specific antibody to identify injected cells (A), and the actin cytoskeleton was visualized with rhodamine-phalloidin (B). A representative field is shown. In the field are three injected cells, which stained brightly with the anti-beta -galactosidase antibody. The injected cell in the upper right-hand corner is in the process of detaching from the coverslip. Two cells in the field did not stain with this antibody. Only the cells that were not injected show the formation of actin stress fibers.

SPD-D Binds to IL-8 and GRP Receptors-- SPD-D has been shown to bind to many neuropeptide receptors, acting as a broad spectrum antagonist (3). These receptors are related to one another by sequence homology in a large subfamily. Also members of this family are non-neuropeptide receptors such as those for alpha -thrombin, C5a, formylmethionylleucylphenylalanine, and IL-8 (32). In an effort to determine how broad the receptor specificity of SPD-D was, we tested binding of SPD-D to two of these receptors, as well as to the human GRP receptor. Receptor-transfected cells were used to make membrane fractions for direct binding assays. As shown in Fig. 5, SPD-D can compete for binding of IL-8 to the human CXCR1 with an IC50 of 12.8 µM. In agreement with the work of other groups (4), SPD-D inhibits binding of bombesin to the human GRP receptor with an IC50 of 5.8 µM. SPD-D does not inhibit binding of C5a to its receptor. Thus, the spectrum of receptors to which SPD-D binds is even broader than has been previously suspected. We know from the data presented above that SPD-D has agonist activity against the GRP receptor. We next tested whether SPD-D also has agonist activity against chemoattractant receptors. The IL-8 receptors are expressed in chemotactic cells of myeloid lineage and are involved in the chemotactic response (33). Neutrophils express not only the two IL-8 receptors but also several other chemoattractant receptors, including the substance P receptor NK-1 (34). We therefore considered it likely that SPD-D would have an affect on chemotaxis.


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Fig. 5.   SPD-D binds to the IL-8 receptor CXCR1 and the gastrin-releasing peptide receptor, but not to the C5a receptor. Membranes were prepared from HEK-293 cells stably expressing the the human C5a receptor (A), the human CXCR1 receptor (B), or the human gastrin-releasing peptide receptor (C). Binding of 125I-labeled C5a, IL-8, and bombesin was measured in the appropriate membranes in the presence of the indicated concentration of unlabeled ligand (filled circles) or SPD-D (open circles). IC50 values determined were 12.8 µM for SPD-D binding to the CXCR1 and 5.8 µM for SPD-D binding to the GRP receptor.

SPD-D Stimulates Migration of Human Neutrophils-- The chemotaxis assay is performed in a blind well Boyden chamber in which two sections are separated by a filter. The lower chamber is filled with medium containing a chemoattractant, such as IL-8, and the upper chamber is filled with medium containing human neutrophils. If the cells sense a gradient of chemoattractant, they will migrate through the filter toward the lower chamber (24). Neutrophils will also respond to most chemoattractants with an increase in random migration that is irrespective of the presence of a gradient. If both the upper and lower chambers contain chemoattractant, there will be an increase in the movement of the cells through the filter due to this increased nondirected motion, called chemokinesis. Thus, the chemotaxis assay, in which a gradient is present, actually measures chemotaxis and chemokinesis. The amount of migration measured in the chemokinesis assay, in which there is no gradient, must be subtracted from the chemotaxis value.

Human neutrophils responded to the presence of SPD-D with an increase in migration (Fig. 6), once again showing that it has agonist properties. This migration can entirely be accounted for by chemokinesis because chemotaxis was not significantly greater than chemokinesis. The dose response for chemokinesis (data not shown) was different from the dose response for SPD-D-induced JNK in fibroblasts (Fig. 1C). Neutrophils were able to respond to a much lower dose, as low as 1.5 µM (Fig. 6). This suggests either that the receptor that mediates this response in neutrophils has a higher affinity for SPD-D than either neuropeptide receptors or the IL-8 receptors or that the chemotaxis assay is much more sensitive than the competition binding analysis or the JNK activation. The identity of the receptor that mediates neutrophil chemotaxis in response to SPD-D is not known. Both the IL-8 receptors and the substance P receptor NK-1 are candidates.


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Fig. 6.   SPD-D induces chemokinesis in human neutrophils. Human neutrophils isolated from healthy donors were placed in the top section of a blind well Boyden chamber, separated from the lower chamber by a membrane filter with a pore size of 8 µm, and incubated for 2 h. Random cell movement was determined in the absence of any chemoattractant and was found to be minimal. Chemotaxis was determined by including peptide in the lower chamber only. Chemokinesis was determined by including peptide in both the upper and lower chambers. Migration through the filter was quantitated by counting the number of cells at a depth of 20 µm from the edge of the filter, and the number of cells in nine × 40 fields on three filters was determined. Error bars represent S.E.M.

SPD-D Induces Ca2+ Mobilization in Human Neutrophils-- IL-8 and substance P also cause a mobilization of Ca2+ in neutrophils. The mechanism for this Ca2+ mobilization is different from that elicited by neuropeptides in fibroblasts, in that it is sensitive to pertussis toxin treatment (35, 36). The pathway that leads to IL-8 and substance P-induced Ca2+ flux is the activation of a Gi family member, which releases beta gamma subunits that activate PLCbeta 2 and PLC beta 3 (35).

50 µM SPD-D was found to be capable of causing a release of Ca2+ from intracellular stores in human neutrophils (Fig. 7B). This Ca2+ flux is comparable to that caused by IL-8 (24). In contrast to the induction of chemokinesis, a low dose of SPD-D, 1.5 µM, was incapable of activating Ca2+ flux (Fig. 7A). This suggests either that the receptors involved in the induction of chemokinesis have a higher affinity for SPD-D than do the receptors involved in the Ca2+ response, or that the chemotaxis assay is much more sensitive than the Ca2+ mobilization assay. Either response could be mediated by one of the known receptors, either IL-8 receptors, NK-1, or another SPD-D receptor that is yet to be described.


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Fig. 7.   SPD-D induces a calcium mobilization in human neutrophils. Neutrophils were preloaded with Indo I AM, and Ca2+ release was monitored by flow cytometry. Cells were treated with 1.5 µM SPD-D (A) or 50 µM SPD-D (B). The ratio of the emission at 390 nm over the emission at 490 nm was determined and is presented in arbitrary units. Traces are representative of four independent experiments.

As mentioned above, the Ca2+ response to IL-8 and substance P is sensitive to pertussis toxin (36), suggesting the involvement of a Gi family member. The SPD-D-induced Ca2+ mobilization in neutrophils is also sensitive to pertussis toxin pretreatment (data not shown). These data therefore suggest that SPD-D can cause receptor association with Gi, as well as G12.

    DISCUSSION
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References

Compounds based on the mechanism of action of SPD-D may one day be used to treat small cell lung cancer. SPD-D causes apoptosis in SCLC cells in culture through an unknown mechanism. Part of this mechanism undoubtedly involves interruption of autocrine loops and prevention of calcium signaling. However, as we have shown in this report, SPD-D can act as an agonist against receptors that are expressed in SCLC tumors. This agonist activity introduces a signal, while the antagonist activity of the peptide serves to block the Ca2+ response. This asymetric signal transduction may cause a perturbation in cellular processes that leads to apoptosis.

The agonist activity of SPD-D is unique for two reasons. First, SPD-D binds to multiple receptors that recognize diverse ligands. By comparing the structures of other substance P derivatives and examining the biological activity of these compounds, we can speculate as to the mechanism of SPD-D function. Spantide I, for example, has a structure that is almost identical to SPD-D but is not cytotoxic to SCLC cells in culture (4) and has no ability to stimulate JNK or cytoskeletal rearrangement in Swiss 3T3 cells (data not shown). Spantide I contains a glutamine at position 5, whereas SPD-D contains a D-phenylalanine at this position. This suggests that residue 5 in SPD-D is critical in determining biological activity.

The second way in which SPD-D is unique as a ligand is that not all of the possible signals downstream of the receptors are initiated. This is difficult to demonstrate in Swiss 3T3 fibroblasts or in human neutrophils, which express multiple receptors that bind SPD-D. Because SPD-D can bind multiple known and uncharacterized receptors, it is only in a system in which one relevant receptor is expressed that biased agonism can be clearly shown. In rat 1A fibroblasts, we show that SPD-D induces a JNK activation that is dependent on the expression of the GRP receptor, but it does not induce a Ca2+ release when this receptor is present. Bombesin, on the other hand, stimulates both JNK and Ca2+ flux in fibroblasts. Work done in other laboratories supports these data. 10 µM SPD-D by itself does not cause Ca2+ flux in fibroblasts (22) and 50 µM SPD-D does not induce inositol 1,4,5-trisphosphate generation (23). However, SPD-D reliably induces JNK activation at a concentration as low as 10 µM. In contrast, a maximal dose of bombesin stimulates a rapid Ca2+ flux and JNK activation. This unique response is also seen in other cell systems. SPD-D has never been shown to flux Ca2+ in SCLC cells, but it is capable of blocking bombesin-induced Ca2+ flux (2), and it can activate a JNK response in these cells.2

SPD-D, therefore, does not fit the definition of either an agonist or an antagonist, so a new model and new terminology are needed to describe the action of this compound. A model of the way in which ligand and receptor interact has been proposed that differentiates between a competitive antagonist and an inverse agonist (37). The model assumes that a receptor can cycle between an inactive (R) and an active (R*) configuration spontaneously in the absence of ligand. This spontaneous activation is the likely mechanism behind increased basal activity of transfected receptors (Fig. 2 and Refs. 29 and 30). A true agonist acts to stabilize the active forms of the receptor, thus causing the receptor to remain in those states for longer and increasing the chance that the receptor will activate a G protein. This model is termed conformational selection (38).

A slight modification of this model can explain the activation of multiple G proteins (Fig. 8) In this model, when a receptor is capable of activating two different G proteins, three states of receptor activation exist: R, the unactivated receptor, and R*1 and R*2, the activated states. R is incapable of activating any G protein; R*1 can activate Galpha q, for example, and R*2 can activate Galpha 12. The receptor cycles between these three states, energetically favoring the inactive state. The existence of more than one active state of the receptor has been postulated based on a phenomenon called agonist trafficking (39). In systems in which a receptor is capable of coupling with multiple G proteins, signals that are downstream of one G protein are often more sensitive to low concentrations of agonist than are signals downstream of another G protein. For example, 0.1 nM calcitonin causes a significant activation of Gs through the C1a receptor, whereas it takes up to 10 nM calcitonin to activate Gq through this same receptor (30). There are two possible explanations for this difference. Either the Gs signal is more easily detected at low levels of receptor activation and thus at lower agonist concentration, or calcitonin can stabilize the form of the receptor that interacts with Gs with a greater affinity than it stabilizes the form that activates Gq. If the second case is true, there should be agonists that preferentially activate Gq. To date, no examples of such a reversal of agonist efficacy have been described for a native receptor.


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Fig. 8.   Biased agonist hypothesis for the activation of receptors by SPD-D. A receptor, R, is capable of spontaneously cycling between two active conformational states, R*1 and R*2, and one energetically favored inactive state, R. R*1 is the conformation that activates Galpha q, whereas R*2 is the state that activates Galpha 12. An agonist (A) binds to and stabilizes both activation states. An antagonist (AA) binds to all three states, competing with the agonist, and it may or may not stabilize the active states. If an antagonist stabilizes the active conformations of the receptor, it is termed a partial agonist. An inverse agonist (IA) binds to and further stabilizes the inactive state of the receptor. A biased agonist (BA) binds preferentially to one of the active states and stabilizes it, thus initiating one set of signal transduction events downstream of the receptor without activating others.

A competitive antagonist binds to the receptor in any state and competes for binding with the natural ligand. An antagonist may or may not stabilize different states of the receptor. The term "partial agonist" refers to a compound that binds to a receptor and competes with an agonist for binding but itself stabilizes the active states of the receptor to a lesser extent than does a full agonist.

An inverse agonist (29) favors the inactive state and therefore makes it less likely that the receptor will activate a G protein. Inverse agonists decrease the spontaneous activity of the receptor caused by cycling between active and inactive forms, therefore lowering the basal response seen without ligand.

We propose that SPD-D acts in a different manner. SPD-D strongly favors association with R*2 over R*1, resulting in a receptor configuration that activates G12 but not Gq. We call such an agonist a biased agonist. This model can be extended to include as many different activation states as there are potential G proteins coupling to the receptor. Our data suggest that SPD-D can cause receptors to couple to Gi and G12 but not to Gq. Therefore, SPD-D prefers the states of the receptor that can activate Gi and G12. Binding to the receptor prevents the binding of a full agonist, such as bombesin. Therefore, when the signals subsequent to Gq are measured, SPD-D appears to act as a competitive antagonist. Such a competitive antagonism can be overcome by an increased concentration of agonist. Thus, the inhibition of ERK activation (23), [3H] thymidine uptake (22), and calcium flux (Fig. 2C) by SPD-D can all be overcome by in increased concentration of bombesin.

There is a structural basis for the activation of one set of G proteins by a receptor in the absence of the activation of other G proteins. Mutations in the thyrotropin receptor uncouple the receptor from the G protein that mediates phospholipase C activation (most likely Gq) while maintaining coupling to Gs (40). Also, a deletion of part of the seventh transmembrane domain of the calcitonin receptor shows a similar change in G protein coupling, favoring Gs over Gq (30) There is also evidence that certain mutations in G protein-coupled receptors can lead to receptors that can more favorably achieve an active state in the absence of ligand (41).

This hypothesis can be tested by heterologous expression of G proteins and receptors (10). Whereas a classical agonist, such as bombesin, would stimulate the association of the GRP receptor with both Galpha q and Galpha 12 and a classical antagonist would stimulate association with neither G protein, SPD-D, acting as a biased agonist, would stimulate the association of the receptor with Galpha 12 but not Galpha q. If so, SPD-D would be the first compound described to activate receptor-G protein interaction selectively. The IL-8 receptors are coupled to members of the Gq class and to members of the Gi class (35). It would therefore be expected that SPD-D might cause the IL-8 receptor to couple to Gi.

In addition to biased agonism, there are two other possible mechanisms for the selective activation of signaling pathways by SPD-D. First, one of the receptors to which SPD-D binds may generate a signal that can suppress the Ca2+ response. Second, a difference in the kinetics of G protein activation may select for one downstream effector over another.

SPD-D has the unique property of associating with multiple G protein-coupled receptors. This peptide can displace bombesin, vasopressin, endothelin, IL-8, and other ligands from their respective receptors. This broad spectrum of interactions leaves open the possibility that the lack of Ca2+ signaling through neuropeptide receptors in fibroblasts and SCLC cells is due to a negative signal from an as yet uncharacterized receptor. However, a high dose of bombesin, or an unrelated agonist, such as fetal calf serum, can still stimulate Ca2+ mobilization in the presence of SPD-D. This shows that a second signal does not simply shut down calcium mobilization in SPD-D treated cells.

The kinetics of binding between ligand and receptor can vary greatly. It is possible that SPD-D has a much faster on/off rate than bombesin association with the GRP receptor. If this were true, the complex of receptor and SPD-D would be short lived. Only G proteins that are quickly activated would be stimulated by such a short lived complex. SPD-D would therefore favor the activation of one G protein over another without necessarily favoring one active conformation over another. However, the difference in GDP/GTP exchange rate between G12 family members and other G proteins would seem to suggest a bias in the other direction. The rate of dissociation of GDP from Galpha 12 is 10-20-fold slower than that for other alpha  subunits (42). This suggests that a longer association between ligand and receptor would be required to activate G12 family members than Gq family members. If SPD-D bound to receptor only briefly, it would therefore be more likely to activate Gq than G12.

If SPD-D does activate receptors in such a way as to cause association with only a subset of possible alpha  subunits, it would be the first agonist described to act in this manner. Such a biased agonist would become a valuable tool in the dissection of signal transduction pathways downstream of neuropeptide receptors. The concept can also be extended to the treatment of human diseases. Much work has been focused on the search for specific antagonists to receptors in an effort to achieve a clinical outcome without producing side effects. If it becomes possible to selectively affect signal transduction pathways downstream of those receptors, much more specific pharmacological agents could be generated. To pursue this possibility, peptides that are both receptor-specific and pathway-specific would have to be developed. SPD-D binds to several different receptors, which have activity in different physiological processes and may prove to be a biased agonist on some of these receptors and an agonist or antagonist on others.

The original goal of this research was to resolve the mechanism for the differential inhibition of neuropeptide-induced Ca2+ responses and ERK activation. In Swiss 3T3 fibroblasts, G proteins of all four major classes are present. Neuropeptide receptors have been shown to directly couple to three of these classes, Gq, Gi/o, and G12 (10). The activation of Ca2+ mobilization proceeds mainly through Gq (9) and JNK activation by neuropeptides may proceed through G12 (18).

The activation of ERKs by neuropeptides proceeds by a more complex mechanism, with potential roles for PKC (20), beta gamma subunits (12), and a pertussis toxin-sensitive signal (21). The latter two pathways leading to ERK activation may explain the residual activity in cells inhibited with SPD-D (23). In cells treated with a relatively high dose of bombesin (50 nM), SPD-D stimulates the activation of Raf-1 and ERK to a level beyond that stimulated by bombesin alone. This could be due to the release of beta gamma subunits from a G12 family member. Activation of Raf and ERK through a Gi pathway was eliminated as a possibility because this activation was not pertussis toxin-sensitive. SPD-D by itself does not stimulate ERK activity or Raf activity in these fibroblasts. This suggests that whatever signals SPD-D can generate, they are not sufficient to activate the ERK pathway, although they apparently are sufficient to activate the JNK pathway. The input of signals unique to bombesin, such as those mediated by Gq, are required for ERK activation. Thus, the biased agonist hypothesis explains many of the data that have been difficult to reconcile in previous reports, demonstrating the usefulness of this theory.

One of the most important biological systems in which neuropeptides play a role is small cell lung cancer. The substance P derivatives have already been identified as a potential therapy for SCLC, but the mechanism by which they act is unknown. Once we determine the manner in which SPDs selectively activate different signaling pathways, we can use them to determine which signals lead to the cytotoxic response in SCLC. Knowing the signals responsible for SCLC cytotoxicity will allow us to manipulate them to enhance the therapeutic effectiveness of compounds of this type.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants GM30324 and CA58187 (to G. L. J.), CA73037 (to M. B. J.), and HL09640 (to C. K.).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.

§ To whom correspondence should be addressed: Program in Molecular Signal Transduction, National Jewish Medical Research Center, 1400 Jackson St., Denver, CO 80206. Tel.: 303-398-1772; Fax: 303-398-1225; E-mail: jarpem{at}njc.org.

Recipient of a travel fellowship from the Wellcome Trust. Present address: CRC Center for Molecular and Cellular Biology, The Institute for Cancer Research, Chester Beatty Laboratories, 237 Fulham Rd., London SW3 6JB, United Kingdom.

1 The abbreviations used are: SCLC, small cell lung cancer; SPD-D, [D-Arg1,D-Phe5,D-Trp7,9,Leu11]substance P; GRP, gastrin-releasing peptide; GRPR, GRP receptor; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; IL, interleukin; DME, Dulbecco's modified Eagle's medium; FCS, fetal calf serum.

2 M. B. Jarpe and F. M. Mitchell, unpublished observations.

    REFERENCES
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Abstract
Introduction
Procedures
Results
Discussion
References

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