From the
Mitogenic stimulation of Swiss 3T3 fibroblasts with bombesin
results in receptor-mediated activation of a complex array of
effectors, including phospholipase C
Neuropeptides, such as bombesin, bind to specific, high affinity
receptors that mediate a variety of biochemical responses fundamental
to embryogenesis, tissue regeneration, and tumorigenesis
(1) .
The amphibian tetradecapeptide bombesin and structurally related
mammalian peptides, including gastrin releasing peptide
(GRP),
Neuropeptides, including bombesin, have been implicated as
autocrine growth factors in the pathogenesis of some human small cell
lung carcinomas (SCLC)
(9) . Evidence for this in part involves
a study using a monoclonal antibody directed against the carboxyl
terminus of GRP, which has been shown to inhibit the in vitro growth of SCLC cell lines and tumor xenografts in nude mice
(9) . Similarly, the 11-amino acid synthetic peptide,
[
D-Arg
We investigated the effect of the
[
D-Arg
Selective Inhibition of Cell Growth by the
[
D-Arg
The bombesin receptor has a predicted
seven-transmembrane structure and couples minimally to G
Even though the
[
D-Arg
The
[
D-Arg
The enhanced Raf-1 activity in response
to bombesin stimulation in the presence of the
[
D-Arg
Saltiel and colleagues
(7) have demonstrated that bombesin-stimulated, but not
epidermal growth factor-stimulated, MAP kinase activation in Swiss 3T3
fibroblasts is inhibited by over 90% following TPA-mediated
down-regulation of PKC. We have confirmed this observation with
prolonged exposure to TPA, causing a 80-100% reduction in the
bombesin-stimulated MAP kinase activity observed in both the absence
and presence of the
[
D-Arg
The question arises how the
[
D-Arg
The bombesin receptor is, however,
still functional and capable of signaling in the presence of the
[
D-Arg
Woll and Rozengurt
(37) demonstrated that the hexapeptide
Arg-
D-Trp-MePhe-
D-Trp-Leu-Met-NH
and mitogen-activated protein
(MAP) kinase. Incubation of Swiss 3T3 fibroblasts with the 11-amino
acid
[
D-Arg
,
D-Phe
,
D-Trp
,Leu
]substance
P peptide inhibited bombesin-stimulated cell proliferation and
phospholipase C
activation even at high bombesin concentrations.
The peptide did not inhibit the activation of phospholipase C
by a
GTPase-deficient form of the G
-like protein,
G
, indicating that the peptide does not inhibit
phospholipase C
and is acting at a point upstream of the activated
form of the G protein
subunit. The peptide inhibited MAP kinase
activation at low bombesin concentrations, but unlike phospholipase
C
, this inhibition could be overcome with 30 n
M bombesin.
In control Swiss 3T3 cells, bombesin did not measurably activate Ras or
Raf-1 above basal levels. Following incubation of the cells with the
[
D-Arg
,
D-Phe
,
D-Trp
,Leu
]substance
P peptide, 50 n
M bombesin activated Raf-1 4-6-fold over
basal levels. Platelet-derived growth factor-stimulated activities of
PLC, Ras, Raf-1, and MAP kinase were unaltered after incubation of
Swiss 3T3 cells with the
[
D-Arg
,
D-Phe
,
D-Trp
,Leu
]substance
P peptide, as was platelet-derived growth factor-stimulated growth of
the Swiss 3T3 cells. Thus, the peptide behaves as an antagonist that
differentially inhibited phospholipase C
and MAP kinase signal
transduction pathways. The growth arrest observed with the peptide
indicates that the bombesin-stimulated activation of MAP kinase is not
sufficient to support mitogenesis in Swiss 3T3 cells.
(
)
act as potent mitogens for quiescent
murine Swiss 3T3 fibroblasts
(2) . Activation of the bombesin
receptor results in the stimulation of a variety of signaling enzymes
including phospholipases C, A
, and D, protein kinase C, and
the mitogen-activated protein kinases (MAP kinases)
(3, 4, 5, 6, 7) . The
bombesin/GRP receptor was cloned from Swiss 3T3 cells and is a member
of the G protein-coupled receptor superfamily with seven predicted
hydrophobic transmembrane domains
(8) . The cloned and expressed
bombesin receptor couples to G
family members such as
G
or G
, activates phospholipase C
(8) , and, as we demonstrate, also stimulates MAP kinase
activity.
,
D-Phe
,
D-Trp
,Leu
]substance
P, has been shown to act as a potent inhibitor of Swiss 3T3 and SCLC
cell growth in vitro. Using Swiss 3T3 cells, the
[
D-Arg
,
D-Phe
,
D-Trp
,Leu
]substance
P peptide was demonstrated to diminish
I-GRP binding to
its receptor and inhibit GRP-stimulated calcium mobilization and DNA
synthesis
(10) .
,
D-Phe
,
D-Trp
,Leu
]substance
P peptide on the activation of the MAP kinase pathway under conditions
in which it inhibits bombesin-stimulated PLC
activation and growth
arrests Swiss 3T3 cells. In this report, we demonstrate that the
[
D-Arg
,
D-Phe
,
D-Trp
,Leu
]substance
P peptide inhibits activation of the MAP kinase pathway at low
concentrations of bombesin and that this can be overcome in a bombesin
concentration-dependent manner. In contrast, PLC
activation is
inhibited even at high bombesin concentrations.
Materials
The
[
D-Arg,
D-Phe
,
D-Trp
,Leu
]substance
P peptide was purchased from Bachem.
myo-[2-
H]Inositol and phosphorus-32
radionuclide were obtained from DuPont NEN.
[
-
P]ATP was purchased from ICN Biomedicals,
Inc. Pertussis toxin was obtained from List Biological Laboratories.
The human bombesin/GRP receptor cDNA was from J. Battey, National
Institutes of Health.
Cell Culture and Transfection
Swiss 3T3
fibroblasts were maintained in DMEM containing 5% newborn and 5% bovine
calf serum, in humidified 7.5% CO, 92.5% air at 37 °C.
All experiments were performed with cells between passages 1 and 10
from the original stocks. The cells were quiesced by replacing the
growth medium with DMEM supplemented with 0.1% bovine serum albumin for
20-24 h. When the cells were assayed for kinase activity, this
medium was replaced with DMEM, 0.1% bovine serum albumin, 15 m
M Hepes (pH 7.4) for 5-10 min prior to agonist addition. The
isolation of stable clones of Swiss 3T3 cells expressing the
polypeptide has been described previously
(11) . Stock HEK-293 cells were maintained in DMEM containing
10% bovine calf serum (HyClone) and were transfected transiently by the
method of calcium phosphate precipitation
(12, 13) .
Measurement of PtdIns-PLC Activity
Cells were
seeded on 60-mm dishes and labeled with
myo-[2-H]inositol (1 µCi/ml) for 24
h in DMEM/Ham's F-12 medium (1:1) containing 0.1% bovine serum
albumin (DMEM/F-12/bovine serum albumin). The cells were then rinsed
three times with DMEM, F-12, bovine serum albumin, 15 m
M Hepes
(pH 7.4) and incubated for 5 min with this medium supplemented with
LiCl (20 m
M). Agonists were added for an additional 20-min
incubation at room temperature. The reactions were terminated with an
equal volume of ice-cold methanol/HCl (100:1, v/v) and the cells
scraped into an extraction mixture containing
H
O:methanol/HCl:chloroform (1:1:2, v/v). After vigorous
mixing and centrifugation, the aqueous phase was passed over an AG 1-X8
(200-400 mesh) formate anion-exchange column (Bio-Rad). The total
[
H]inositol phosphates were eluted as described
previously
(14) and expressed as a percentage of the total
[
H]inositol incorporated into phospholipid.
Ras, Raf-1, and MAP Kinase Assays
The activation
of these proteins was assayed as described previously with slight
modifications
(11, 15) . In the measurement of Ras
activation, the elution buffer contained 0.1% SDS and the guanine
nucleotides were resolved by thin layer chromatographic separation
using 0.75
M KHPO
(pH 3.4). In the
experiments represented by Fig. 3 C, the MAP kinase
activities were batch eluted from a DEAE-Sephacel column using a high
salt concentration (0.5
M, NaCl). The eluate was then assayed
in quadruplicate as described previously
(15) .
Figure 3:A, bombesin-stimulated MAP kinase
activity in Swiss 3T3 cells. Serum-starved Swiss 3T3 cells were
stimulated with 50 n
M bombesin for 2 min and assayed for MAP
kinase activity as described under ``Experimental
Procedures.'' The cells were treated with the
[
D-Arg,
D-Phe
,
D-Trp
,Leu
]substance
P peptide exactly as described in Fig. 2. The results are from a single
experiment representative of 12 independent experiments. B,
PDGF-stimulated MAP kinase activity in Swiss 3T3 cells. Serum-starved
Swiss 3T3 cells were stimulated with 10 ng/ml PDGF for 5 min in the
absence or presence of the
[
D-Arg
,
D-Phe
,
D-Trp
,Leu
]substance
P peptide (50 µ
M, 5-min preincubation) and assayed for MAP
kinase activity as described above. Data points are from a single
experiment. Three independent experiments gave similar results.
C, dose-dependent activation of MAP kinase activity in Swiss
3T3 cells. Serum-starved Swiss 3T3 cells were stimulated with
increasing concentrations of bombesin in the absence or presence of the
substance P peptide (50 µ
M, 5-min preincubation) and
assayed for MAP kinase activity using the batch elution protocol as
described under ``Experimental Procedures.'' Data points
represent the mean ± S.D. of quadruplicate determinations from
a single experiment representative of three independent
experiments.
Quantification of Data
PhosphorImager analysis was
used for a quantitative measure of the products of Raf-1 and Ras
activation. Autoradiographs from single, representative experiments are
shown, with data from several experiments presented as arbitrary
phosphorimaging units.
,
D-Phe
,
D-Trp
,Leu
]Substance
P Peptide-Fig. 1 shows that the
[
D-Arg
,
D-Phe
,
D-Trp
,Leu
]substance
P peptide inhibited bombesin-stimulated Swiss 3T3 cell proliferation in
a concentration-dependent manner. In contrast, the
[
D-Arg
,
D-Phe
,
D-Trp
,Leu
]substance
P peptide had little or no effect on PDGF-stimulated Swiss 3T3 cell
proliferation. The results are similar to previously described
cytostatic actions of this peptide with fibroblasts and small cell lung
carcinoma lines
(10) . The molecular action of the
[
D-Arg
,
D-Phe
,
D-Trp
,Leu
]substance
P peptide is poorly understood, except that it inhibits the ability of
bombesin and other neuropeptides to stimulate calcium mobilization
(10) . The
[
D-Arg
,
D-Phe
,
D-Trp
,Leu
]Substance
P Peptide Selectively Modulates Signal Transduction
Pathways-We determined that incubation of Swiss 3T3 cells
with
[
D-Arg
,
D-Phe
,
D-Trp
,Leu
]substance
P peptide inhibited calcium mobilization in response to bombesin
stimulation but had little or no effect on the ability of PDGF to
mobilize calcium (not shown). This finding indicated that the
[
D-Arg
,
D-Phe
,
D-Trp
,Leu
]substance
P peptide did not influence calcium stores or the ability of
phospholipase C
catalyzed release of inositol trisphosphate to
mobilize intracellular calcium. Fig. 2 demonstrates that the
[
D-Arg
,
D-Phe
,
D-Trp
,Leu
]substance
P peptide inhibited the ability of 50 n
M bombesin, a maximally
effective concentration of bombesin in the absence of the inhibitory
peptide, to stimulate inositol phosphate generation. The
[
D-Arg
,
D-Phe
,
D-Trp
,Leu
]substance
P peptide had no effect on PDGF-stimulated inositol phosphate
generation.
members of the heterotrimeric G protein family
(16, 17) . G
and
subunits
selectively activate phospholipase C
(18) . Swiss 3T3 cells
stably expressing a mutant GTPase-inhibited G
subunit, a member of the G
subfamily of G protein
subunits
(19) , have a constitutively activated
phospholipase C
(11) . The
[
D-Arg
,
D-Phe
,
D-Trp
,Leu
]substance
P peptide had no effect on the ability of the GTPase-inhibited
G
to activate phospholipase C
but inhibited
bombesin stimulation of phospholipase C
activity (Fig. 2).
Thus, the
[
D-Arg
,
D-Phe
,
D-Trp
,Leu
]substance
P peptide inhibited receptor but not G
activation of
phospholipase C
. The site of action of the
[
D-Arg
,
D-Phe
,
D-Trp
,Leu
]substance
P peptide is therefore upstream of G protein
subunit interaction
with phospholipase C
.
Figure 2:
Stimulation of PtdIns-PLC activity in
Swiss 3T3 cells. Total inositol phosphate formation was measured in
wild type or 16
expressing Swiss 3T3 cells
(denoted as G16QL). Serum-starved,
myo-[
H]inositol-labeled cells were
challenged with 50 n
M bombesin or 10 ng/ml PDGF for 20 min and
assayed for PLC activity as described under ``Experimental
Procedures.'' The cells were preincubated with the
[
D-Arg
,
D-Phe
,
D-Trp
,Leu
]substance
P peptide (50 µ
M) for 5 min prior to agonist stimulation
and the peptide was present for the duration of the stimulation. Data
points represent the mean ± S.E. of between three and four
independent experiments. In a single experiment each condition was
measured in quadruplicate. Other clones of
16
expressing Swiss 3T3 cells gave similar results to the clone
represented in these experiments.
Bombesin and PDGF both activate MAP
kinase activity in Swiss 3T3 cells (Fig. 3). The
[
D-Arg,
D-Phe
,
D-Trp
,Leu
]substance
P peptide has no effect on PDGF stimulation of MAP kinase activity
(Fig. 3 B). In contrast, incubation of Swiss 3T3 cells
with a concentration of the
[
D-Arg
,
D-Phe
,
D-Trp
,Leu
]substance
P peptide (50 µ
M) that totally inhibited PLC
activation by 50 n
M bombesin did not inhibit bombesin receptor
stimulation of MAP kinase activity at this concentration of bombesin
(Fig. 3, A and C). In fact, in many experiments
the inhibitory peptide enhanced MAP kinase activity stimulated by 50
n
M bombesin (Fig. 3 A). The
[
D-Arg
,
D-Phe
,
D-Trp
,Leu
]substance
P peptide had little or no effect on basal MAP kinase activity in the
absence of bombesin. In Fig. 3 C we demonstrate that, in
the presence of 50 µ
M [
D-Arg
,
D-Phe
,
D-Trp
,Leu
]substance
P peptide, bombesin activated MAP kinase in a concentration-dependent
manner, although with a lowered affinity ( K
shift from 1.5 ± 0.5 n
M to 20 ± 5
n
M), consistent with the inhibitory peptide acting as a
competitive antagonist. However, 50 n
M bombesin could overcome
the inhibition of MAP kinase, but not PLC
activation, by the
inhibitory peptide. These findings demonstrated that the
[
D-Arg
,
D-Phe
,
D-Trp
,Leu
]substance
P peptide influenced bombesin binding to its receptor, while
differentially affecting PLC
and MAP kinase regulation. Expression
of the bombesin/GRP receptor in HEK-293 cells by transfection indicated
that the cloned bombesin receptor mediates both phospholipase C
and MAP kinase activation (Fig. 4). Thus, the
[
D-Arg
,
D-Phe
,
D-Trp
,Leu
]substance
P peptide differentially inhibits two signal transduction pathways
regulated by the bombesin receptor. The
[
D-Arg
,
D-Phe
,
D-Trp
,Leu
]substance
P peptide must not simply competitively inhibit bombesin activation of
its receptor but must have allosteric effects on the receptor. Regulation of the MAP Kinase Pathway in the Presence of the
[
D-Arg
,
D-Phe
,
D-Trp
,Leu
]Substance
P Peptide-MAP kinase is a serine-threonine protein kinase
that is activated by phosphorylation on both tyrosine and threonine
(20) . This reaction is catalyzed by a specific
tyrosine/threonine-directed kinase, MEK (MAPK/ERK kinase)
(21) .
MEK is in turn phosphorylated and activated by the protein kinase Raf-1
(22) . Raf-1 interacts with Ras
GTP, and its activity is
regulated in a Ras-dependent manner by growth factor receptor tyrosine
kinases
(23, 24) . Fig. 5 demonstrates that incubation
of Swiss 3T3 cells with the
[
D-Arg
,
D-Phe
,
D-Trp
,Leu
]substance
P peptide alters the regulation of Raf-1 activation in response to
bombesin but not PDGF. Treatment of Swiss 3T3 cells with PDGF or
phorbol ester, but not bombesin, leads to a significant activation of
Raf-1 (Fig. 5 A). PhosphorImager quantitation of
MEK phosphorylation in Raf-1 immunoprecipitates from
bombesin-stimulated cells of 15 different experiments was not
statistically significant relative to basal Raf-1 activity (Fig.
5 B). In striking contrast, incubation of Swiss 3T3 cells with
the
[
D-Arg
,
D-Phe
,
D-Trp
,Leu
]substance
P peptide resulted in a significant activation of Raf-1 activity in
response to 50 n
M bombesin relative to basal or peptide in the
absence of bombesin. The peptide had little effect itself, on either
MAP kinase or Raf-1 activity. The
[
D-Arg
,
D-Phe
,
D-Trp
,Leu
]substance
P peptide had no significant effect on PDGF receptor signaling or
phorbol ester stimulation of Raf-1 activity (Fig. 5, A and B).
Figure 5:A, Raf-1 activity in Swiss 3T3
cells. Serum-starved Swiss 3T3 cells were stimulated with agonists in
the absence or presence of the
[
D-Arg,
D-Phe
,
D-Trp
,Leu
]substance
P peptide (50 µ
M, 5-min preincubation) and assayed for
Raf-1 activity as described under ``Experimental
Procedures.'' Cells were stimulated with bombesin (50 n
M,
2 min), TPA (200 n
M, 5 min), or PDGF (10 ng/ml, 5 min).
Purified recombinant wild type MEK was autophosphorylated and used as a
standard (as indicated). An autoradiograph from a single experiment,
representative of at least five experiments, is shown. B,
PhosphorImager analysis of phosphorylated MEK-1 in Swiss 3T3 cells.
Quantification of the [
P]MEK protein is
represented as -fold activation over basal in arbitrary units. The
number of individual experiments quantified varied between 5 and 15,
depending upon the condition. Bombesin in the absence or presence of
peptide are conditions representative of 15 individual experiments and
the increased activity observed in the presence of the peptide was
significant ( p < 0.01 by Student's t test).
C, PhosphorImager analysis of phosphorylated MEK-1 in control
and pertussis toxin-treated Swiss 3T3 cells. Serum-starved cells were
stimulated and assayed as described in A. When toxin-treated,
cells were incubated for 18 h with pertussis toxin (100 ng/ml) prior to
agonist stimulation. Quantification of the
[
P]MEK protein is represented as -fold
activation over basal in arbitrary units. The number of individual
experiments quantified varied between 3 and 5, depending upon the
condition. The increased activity in the presence of the peptide was
significant in both the control ( p < 0.01 by
Student's t test) and the pertussis toxin-treated cells
( p < 0.05 by Student's t test). The apparent
decrease in PDGF-stimulated Raf-1 activity upon pertussis toxin
pretreatment of the Swiss 3T3 cells was not significant ( p < 0.10 by Student's t test).
The increased Raf-1 activation in response to
bombesin in the presence of the
[
D-Arg,
D-Phe
,
D-Trp
,Leu
]substance
P peptide was not due to the receptor coupling to G
or
G
proteins. Pertussis toxin treatment of Swiss 3T3 cells,
to uncouple G
and G
proteins from receptors,
had no significant effect on the ability of the
[
D-Arg
,
D-Phe
,
D-Trp
,Leu
]substance
P peptide to enhance Raf-1 activation in response to bombesin (Fig.
5 C).
,
D-Phe
,
D-Trp
,Leu
]substance
P peptide enhanced bombesin-stimulated Raf-1 activity, it had little or
no effect on Ras
GTP loading (Fig. 6). The Ras
GTP loading
assay
(25) is non-catalytic and thus rather insensitive.
Nonetheless, PDGF stimulation resulted in a 4.5-5-fold increase
in Ras
GTP loading, whereas the response to bombesin was not
significantly different from basal levels in the presence or absence of
peptide (Fig. 6, A and B). Thus, the increased
Raf-1 activity in response to bombesin in the presence of the
[
D-Arg
,
D-Phe
,
D-Trp
,Leu
]substance
P peptide is independent of a significant change in Ras
GTP
loading.
Figure 6:
A, Ras activation in Swiss 3T3 cells.
[P]GTP and [
P]GDP bound
to immunoprecipitated Ras were measured as an index of the Ras
activation state. Serum-starved,
P
-labeled
Swiss 3T3 cells were stimulated with agonists in the absence or
presence of the
[
D-Arg
,
D-Phe
,
D-Trp
,Leu
]substance
P peptide (50 µ
M, 5-min preincubation) and assayed for Ras
activity as described under ``Experimental Procedures.''
Cells were stimulated with bombesin (50 n
M, 1 min) or PDGF (10
ng/ml, 2 min). Ras was immunoprecipitated and bound
[
P]GTP and [
P]GDP
resolved using polyethyleneimine-cellulose thin layer chromatography.
An autoradiograph of a polyethyleneimine-cellulose TLC plate from a
single experiment, representative of four experiments, is shown.
B, PhosphorImager quantification of the percent ratio of
GTP/(GTP + GDP). Quantification of the
[
P]GTP and [
P]GDP is
represented as the percentage of [
P]GTP from the
total
P-labeled guanine nucleotide. The mean ± S.E.
of four independent experiments is
represented.
,
D-Phe
,
D-Trp
,Leu
]substance
P peptide inhibits bombesin but not PDGF-stimulated Swiss 3T3 cell
growth and induces apoptosis of small cell lung carcinoma cells whose
growth is dependent on neuropeptide autocrine or paracrine loops
(26) . We have demonstrated that the
[
D-Arg
,
D-Phe
,
D-Trp
,Leu
]substance
P peptide inhibits phospholipase C
and MAP kinase activation in
response to bombesin, but that only the MAP kinase inhibition was
overcome by high bombesin concentrations. The action of the
[
D-Arg
,
D-Phe
,
D-Trp
,Leu
]substance
P peptide is selective for bombesin receptor/G protein and not PDGF
receptor tyrosine kinase regulated signal transduction pathways. The
results clearly demonstrate that the
[
D-Arg
,
D-Phe
,
D-Trp
,Leu
]substance
P peptide differentially inhibits bombesin activation of
receptor-mediated responses.
,
D-Phe
,
D-Trp
,Leu
]substance
P peptide may be explained in part by the inhibition of phospholipase
C
activity. Stimulation of protein kinase C activity resulting
from phospholipase C
activation in Swiss 3T3 cells leads to
enhanced cAMP synthesis and protein kinase A activation
(11, 27) . Activation of protein kinase A in response to
elevated cAMP levels has been shown to uncouple Raf-1 activation from
Ras
GTP loading
(28, 29, 30, 31, 32, 33) .
In fact, protein kinase A activation in Swiss 3T3 cells inhibits the
enhanced activation of Raf-1 in response to bombesin in the presence of
the
[
D-Arg
,
D-Phe
,
D-Trp
,Leu
]substance
P peptide (data not shown). Thus, bombesin activation of Raf-1 is more
pronounced in the presence of the
[
D-Arg
,
D-Phe
,
D-Trp
,Leu
]substance
P peptide. The robust Ras
GTP loading in response to PDGF is
apparently sufficient to significantly overcome any protein kinase
A-mediated uncoupling of Raf-1 activation that would occur in response
to phospholipase C
activation.
,
D-Phe
,
D-Trp
,Leu
]substance
P peptide (data not shown). Prolonged exposure to TPA does not impair
the ability of the bombesin receptor to activate PLC
.
PDGF-stimulated MAP kinase activity is also inhibited 50-60%
after prolonged TPA treatment (data not shown). These observations
could be interpreted to suggest that the activation of MAP kinase by
bombesin, in the absence and presence of the
[
D-Arg
,
D-Phe
,
D-Trp
,Leu
]substance
P peptide, requires activation of a TPA-sensitive PKC isoform. This
raises the possibility that in the presence of the
[
D-Arg
,
D-Phe
,
D-Trp
,Leu
]substance
P peptide an isoform of PKC is activated by a
sn-1,2-diradylglycerol species independent from PLC
activation. Bombesin regulation of a PLD-like activity, although
currently poorly understood, is possible
(5, 34) . The
consequence of PLD and PLC
stimulation of PKC isoforms must then
have different regulatory functions. However, interpretation of the TPA
down-regulation experiments should be qualified with the fact that TPA
also activates the MAP kinase pathway. MAP kinase has been proposed to
regulate activation of the Ras/Raf/MAP kinase pathway by feedback
inhibition (see Ref. 35 and references therein). The potentially
multiple effects observed with TPA make interpretation of the
down-regulation experiments difficult and may explain in part the
partial inhibition we observe for PDGF-stimulated MAP kinase
activation.
,
D-Phe
,
D-Trp
,Leu
]substance
P peptide selectively inhibits bombesin-stimulated phospholipase C
but not MAP kinase activation at high bombesin concentrations (50
n
M). Mukai et al. (36) used a novel truncated
substance P-related peptide,
p-Glu-Gln-
D-Trp-Phe-
D-Trp-
D-Trp-Met-NH
,
which inhibited m2 muscarinic cholinergic receptor (m2 receptor)
activation of G
and G
in reconstituted
phospholipid vesicles. The inhibitory peptide interfered neither with
ligand binding to the m2 receptor nor with binding of guanine
nucleotides to G
and G
. Their
findings indicated that the inhibitory peptide bound to the G proteins
and inhibited m2 receptor coupling to G
and G
.
The sequence of the
[
D-Arg
,
D-Phe
,
D-Trp
,Leu
]substance
P peptide is
D-Arg-Pro-Lys-Pro-
D-Phe-Gln-
D-Trp-Phe-
D-Trp-Leu-Leu-NH
(10) . The two inhibitory peptides have the conserved
4-amino acid sequence Gln-
D-Trp-Phe-
D-Trp, although
the flanking amino acids also appear critical for their pharmacological
action
(10, 36, 37) . Our findings are
consistent with the
[
D-Arg
,
D-Phe
,
D-Trp
,Leu
]substance
P peptide uncoupling G
from the bombesin receptor. The
site of action of the
[
D-Arg
,
D-Phe
,
D-Trp
,Leu
]substance
P peptide is presently unclear. It clearly reduces the affinity of the
receptor for bombesin but selectively alters regulation of two effector
pathways at high bombesin concentrations. This suggests that the
[
D-Arg
,
D-Phe
,
D-Trp
,Leu
]substance
P peptide most likely acts allosterically at a site distinct from the
bombesin receptor ligand binding domain. This interpretation is
consistent with the ability of the
[
D-Arg
,
D-Phe
,
D-Trp
,Leu
]substance
P peptide to have inhibitory effects on a number of neuropeptide
receptors
(10, 37) . The putative allosteric regulatory
site could be either on the extracellular or cytoplasmic domain of the
neuropeptide receptor. Studies to define the site of action of the
[
D-Arg
,
D-Phe
,
D-Trp
,Leu
]substance
P peptide are currently ongoing.
,
D-Phe
,
D-Trp
,Leu
]substance
P peptide as demonstrated by the stimulation of the MAP kinase pathway
at 50 n
M bombesin.
subunits of G proteins appear to
play an important role in activation of the MAP kinase pathway
(38, 39) . It is unclear if bombesin-stimulated MAP
kinase activity in the presence of the
[
D-Arg
,
D-Phe
,
D-Trp
,Leu
]substance
P peptide allows
subunits to be released from G
or if an additional G protein remains coupled to the bombesin
receptor that is capable of regulating the MAP kinase pathway. If
another G protein is coupled to the bombesin receptor it is not G
or G
, because the bombesin response in the presence
of the
[
D-Arg
,
D-Phe
,
D-Trp
,Leu
]substance
P peptide is not pertussis toxin-sensitive.
had
broad neuropeptide antagonist activity in Swiss 3T3 cells. This
hexapeptide also inhibits the growth of small cell lung carcinoma cells
in vitro (37) . In Swiss 3T3 cells this hexapeptide was
a potent vasopressin antagonist and weak bombesin and bradykinin
antagonist relative to the
[
D-Arg
,
D-Phe
,
D-Trp
,Leu
]substance
P peptide. The hexapeptide antagonist was, however, equipotent to the
[
D-Arg
,
D-Phe
,
D-Trp
,Leu
]substance
P peptide in arresting small cell lung carcinoma cell growth. From
these observations the authors concluded that the actions of these
peptides cannot simply be related to interruption or antagonism of
bombesin-driven autocrine loops in small cell lung carcinoma
(36) . Our findings demonstrate that the antagonist peptide can
selectively inhibit the activation of one signal transduction effector
but not another at high bombesin concentrations. We propose that it is
the disruption of the coordinated regulation of multiple signaling
pathways that contributes to the growth inhibitory properties of the
[
D-Arg
,
D-Phe
,
D-Trp
,Leu
]substance
P peptide. If this hypothesis is correct, it suggests that compounds
that differentially regulate the different arms of signal transduction
pathways regulated by G protein-coupled receptors will be potential
anti-tumorigenic agents for small cell lung carcinoma and other
carcinomas that proliferate in response to neuropeptide autocrine or
paracrine loops.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.