From the Program in Molecular Signal Transduction,
Division of Basic Sciences, National Jewish Medical Research Center,
Denver, Colorado 80206,
Cadus Pharmaceuticals, Tarrytown, New
York 10591, and the ** Department of Pharmacology, University of
Colorado Health Sciences Center, Denver, Colorado 80262
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ABSTRACT |
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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.
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INTRODUCTION |
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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
G12 and G
13 (18, 19). The regulation of
ERKs by neuropeptides is more complicated, suggesting a role for
PKC (20), G protein
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.
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EXPERIMENTAL PROCEDURES |
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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-
-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--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).
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RESULTS |
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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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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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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 -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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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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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 subunits that activate PLC
2 and
PLC
3 (35).
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DISCUSSION |
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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 Gq, for example, and R*2 can
activate G
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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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
Gq and G
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 G
12 but not G
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 G12 is 10-20-fold slower than that for other
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 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), 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
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.
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FOOTNOTES |
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* 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.
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REFERENCES |
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