Bombesin (Bn) receptor subtype 3 (BRS-3) is an
orphan receptor that is a predicted member of the heptahelical
G-protein receptor family and so named because it shares a 50% amino
acid homology with receptors for the mammalian bombesin-like peptides
neuromedin B (NMB) and gastrin-releasing peptide. In a recent targeted
disruption study, in which BRS-3-deficient mice were generated, the
mice developed obesity, diabetes, and hypertension. To date, BRS-3's natural ligand remains unknown, its pharmacology unclear, and cellular
basis of action undetermined. Furthermore, there are few tissues or
cell lines found that express sufficient levels of BRS-3 protein for
study. To define the intracellular signaling properties of BRS-3, we
examined the ability of
[D-Phe6,
-Ala11,Phe13,Nle14]Bn-(6-14),
a newly discovered peptide with high affinity for BRS-3, and various Bn
receptor agonists and antagonists to alter cellular function in
hBRS-3-transfected BALB 3T3 cells and hBRS-3-transfected NCI-H1299
non-small cell lung cancer cells, which natively express very low
levels of hBRS-3. This ligand stimulated a 4-9-fold increase in
[3H]inositol phosphate formation in both cell lines under
conditions where it caused no stimulation in untransfected cells and
also stimulated an increase in [3H]IP1,
[3H]IP2, and 3H]IP3.
The elevation of [3H]IP was
concentration-dependent, with an EC50 of 20-35
nM in both cell lines.
[D-Phe6,
-Ala11,Phe13,Nle14]Bn-(6-14)
stimulated a 2-3-fold increase in [Ca2+]i, a
3-fold increase in tyrosine phosphorylation of p125FAK with
an EC50 of 0.2-0.7 nM, but failed to either
stimulate increases in cyclic AMP or inhibit forskolin-stimulated
increases. None of nine naturally occurring Bn peptides or three
synthetic Bn analogues reported to activate hBRS-3 did so with high
affinity. No high affinity Bn receptor antagonists had high affinity
for the hBRS-3 receptor, although two low affinity antagonists
for gastrin-releasing peptide and NMB receptors,
[D-Arg1,D-Trp7,9,Leu11]substance
P and
[D-Pro4,D-Trp7,9,10]substance
P-(4-11), inhibited hBRS-3 receptor activation. The NMB
receptor-specific antagonist
D-Nal,Cys,Tyr,D-Trp,Lys,Val, Cys,Nal-NH2 inhibited hBRS-3 receptor activation in a
competitive fashion (Ki = 0.5 µM).
Stimulation of p125FAK tyrosine phosphorylation by hBRS-3
activation was not inhibited by the protein kinase C inhibitor,
GF109203X, or thapsigargin, alone or in combination. These results show
that hBRS-3 receptor activation increases phospholipase C activity,
which causes generation of inositol phosphates and changes in
[Ca2+]i and is also coupled to tyrosine kinase
activation, but is not coupled to adenylate cyclase activation or
inhibition. hBRS-3 receptor activation results in tyrosine
phosphorylation of p125FAK, and it is not dependent on
activation of either limb of the phospholipase C cascade. Although the
natural ligand is not a known bombesin-related peptide, the
availability of
[D-Phe6,
-Ala11,Phe13,Nle14]Bn-(6-14),
which functions as a high affinity agonist in conjunction with
hBRS-3-transfected cell lines and the recognition of three classes of
receptor antagonists including one with affinity of 0.5 µM, should provide important tools to assist in the
identification of its natural ligand, the development of more potent
selective receptor antagonists and agonists, and further exploration of the signaling properties of the hBRS-3 receptor.
 |
INTRODUCTION |
The mammalian bombesin
(Bn)1-like peptides
gastrin-releasing peptide (GRP) and neuromedin B (NMB) contribute to
diverse biological functions in the central nervous system (1, 2) and
peripheral tissues (1, 2), which include thermoregulation (3), satiety (4), control of circadian rhythm (5), stimulation of pancreatic secretion (6), stimulation of gastrointestinal hormone release (7-9),
and macrophage activation (10). These peptides also have important
developmental effects (11, 12) and potent growth effects (13-15),
causing proliferation of normal cells (13, 14, 16, 17) and various
tumor cell lines (15, 16, 18-20). To date, two mammalian receptor
subtypes and their ligands have been identified, each of which has an
architecture suggesting they are members of the heptahelical G-protein
coupled receptor superfamily (21-23). One subtype, the GRP receptor,
exhibits selectivity for GRP (21, 22, 24-26), whereas the other, the
NMB receptor, has selectivity for NMB (23, 26, 27). The intracellular
signaling pathways of these two receptors have been characterized, with ligand binding resulting in stimulation of phospholipase C (14, 28-30), protein kinase C activation (14),
[Ca2+]i mobilization (14, 29, 30), and tyrosine
phosphorylation of various intracellular proteins (31-34).
Recently, it has been proposed that an orphan receptor may represent a
third type of mammalian bombesin receptor (35, 36). This 399-amino acid
protein, which was later identified in human tissues (35), was named
bombesin receptor subtype-3 (BRS-3), due to its 51% and 47% amino
acid sequence homology to the GRP receptor and the NMB receptor,
respectively. As opposed to GRP receptors and NMB receptors, which have
widespread distribution in the central nervous system and peripheral
tissues (1, 37, 38), BRS-3 has a pattern of expression limited to
secondary spermatocytes (35), pregnant uterus (36), a few brain regions (36), and human lung (35), breast (39), and epidermal cancer cell lines
(39). In a recent study in which BRS-3-deficient mice were generated by
targeted disruption, the mice were obese, developed hypertension, and
had diabetes, suggesting the BRS-3 receptor is required for regulation
of glucose metabolism, energy balance, and maintenance of blood
pressure (40). To date, the natural ligand for BRS-3 is undiscovered,
and its intracellular signaling mechanisms are largely unknown. This
uncertainty has occurred because no cells have been identified that
natively express sufficient numbers of BRS-3 receptors to allow study
of intracellular coupling, and no high affinity ligands have been
discovered for this receptor. It is known, however, that when the BRS-3
receptor was expressed in Xenopus oocytes (35) or
transfected into BALB 3T3 fibroblasts (41), high concentrations of
various natural (35, 36) or synthetic bombesin analogues (41) could
promote changes in intracellular calcium.
To overcome these problems, we have recently used two different
strategies to create two different cell lines stably expressing this
receptor. These cell lines were used to screen various bombesin peptides for their ability to activate phospholipase C and a synthetic analogue of bombesin,
[D-Phe6,
-Ala11,Phe13,Nle14]Bn-(6-14),
was identified which has high affinity for the BRS-3 receptor (42).
Using an analogue of this peptide that could be radiolabeled,
[D-Tyr6,
-Ala11,Phe13,Nle14]Bn-(6-14),
we demonstrated that both cells lines stably expressing the BRS-3
receptor share the same unique pharmacology for naturally occurring
bombesin peptides and a high affinity for
[D-Phe6,
-Ala11,Phe13,Nle14]Bn-(6-14)
(42).
The purpose of the present study was to examine the intracellular
signaling mechanisms of the BRS-3 receptor using the high affinity
ligand
[D-Phe6,
-Ala11,Phe13,Nle14]Bn-(6-14),
which is an agonist.
 |
MATERIALS AND METHODS |
NCI-H1299 cells were a gift from Herb Oie of the National Cancer
Institute-Navy Medical Oncology Branch, Naval Medical Center, Bethesda,
MD. BALB 3T3 cells were obtained from ATCC, Rockville, MD; Dulbecco's
minimum essential medium (DMEM), RPMI 1640, Dulbecco's phosphate-buffered saline (PBS), fetal bovine serum (FBS), G418 sulfate, 12-O-tetradecanoylphorbol-13-acetate (TPA), and
Tris/HCl were from Life Technologies, Inc.; formic acid, ammonium
formate, disodium tetraborate, EDTA, EGTA, and soybean trypsin
inhibitor were from Sigma; phenylmethylsulfonyl fluoride was from
Fluka, Ronkonkoma, NY; bovine serum albumin (BSA) fraction V was from ICN Biomedicals Inc., Aurora, OH; aprotinin and HEPES were from Boehringer Mannheim; AG 1-X8 resin, thapsigargin, and GF109203X were
from Bio-Rad; monobasic sodium phosphate was from Mallinckrodt; myo-[2-3H]inositol was from NEN Life Science
Products; [2-3H]adenine was from Amersham Pharmacia
Biotech; anti-phosphotyrosine mAb PY20 and anti-FAK mAb were from
Transduction Laboratories, Lexington, KY; horseradish
peroxidase-conjugated secondary antibody was from Pierce; bombesin,
gastrin-releasing peptide, neuromedin B, rhodei-litorin, phyllolitorin,
ranatensin, and litorin were from Bachem, Torrance, CA;
[D-Arg1,D-Trp7,9,Leu11]substance
P and
[D-Pro4,D-Trp7,9,10]substance
P-(4-11) were from Peninsula Laboratories, Belmont, CA;
[Phe13]bombesin, [Ser19]GRP-(18-27) (frog
GRP-10) (43),
[Ser3,Arg10,Phe13]bombesin (44),
D-Nal,Cys,Tyr,D-Trp,Lys,Val,
Cys,Nal-NH2, and [D-Phe6,
-Ala11,Phe13,Nle14]Bn-(6-14)
were gifts from John Taylor of Biomeasure, Inc., Milford, MA. All other
chemicals were reagent grade.
Preparation of Peptides--
The peptides were synthesized by
solid-phase methods as reported previously (45-47). Introduction of
the reduced peptide bond (
) for
[D-Phe6,Leu13,
(CH2NH)-D-Phe14]Bn-(6-14)
and
[(3-Ph-Pr6)-His7,D-Ala11,D-Pro13,
(13-14),Phe14]Bn-(6-14)-NH2
was performed as described previously on 4-methylbenzhdrylamine resin
(Advanced Chem Tech, Louisville, KY) (45, 47).
[D-Phe6]Bn-(6-13) propylamide and
[D-Phe6,Phe13]Bn-(6-13)
propylamide were synthesized in a standard Leu-O-polystyrene resin using tosyl group protection for the imidazole group of His and
cleavage of the desired analogue with 10% propylamine in methanol.
[D-Phe6]Bn-(6-13) methyl ester was also
prepared with Leu-O-polystyrene, and free peptide ester was
removed from the resin by transesterification with 10%
triethylamine/methanol at 40 °C for 48 h. The peptides were
first purified on a Sephadex G-25 column (2.5 × 90 cm) followed by preparative high performance liquid chromatography on a Vydac C18 column (1.5 × 50 cm, 10-15-µm bore size)
(45-47). After rechromatography to achieve
97% purity, the peptides
were characterized by amino acid analysis and matrix-assisted laser
desorption mass spectroscopy (Finnegan, Hemel Hemstead, United
Kingdom).
Preparation and Maintenance of Transfected Cells--
NCI-H1299
cells expressing stably transfected hBRS-3 were obtained using
LipofectAMINE (Life Technologies, Inc.) to introduce human BRS-3
cDNA containing an NH2-terminal flag epitope tag
(5'-GACTACAAGGACGACGATGACAAG-3') subcloned into the expression vectors
pCD2 and pcDNA3 as described previously (42). hBRS-3-transfected
BALB 3T3 fibroblasts were transfected using the calcium phosphate
precipitation method (48) as described previously (42). Both wild-type
BALB 3T3 cells and NCI-H1299 cell lines were grown in DMEM and RPMI
1640 respectively, supplemented with 10% FBS. The transfected cells
were grown in their respective propagation media supplemented with 300 µg/ml G418 sulfate. All cell lines were incubated at 37 °C in a
5% CO2 atmosphere.
Measurement of Inositol Phosphates (IP)--
Cells were
subcultured into 24-well plates (5 × 104 cells/well)
in their respective propagation media. Total [3H]IP was
determined as described previously (30, 49). Briefly, after a 24-h
incubation period at 37 °C, the cells were incubated with 3 µCi/ml
myo-[2-3H]inositol in growth medium
supplemented with 2% FBS for an additional 24 h. Incubation
volumes were 500 µl of assay buffer/well containing 135 mM sodium chloride, 20 mM HEPES (pH 7.4), 2 mM calcium chloride, 1.2 mM magnesium sulfate,
1 mM EGTA, 20 mM lithium chloride, 11.1 mM glucose, and 0.05% BSA (v/v) with or without any of the
peptides studied at 37 °C for 30 min. To determine the time course
of [3H]IP1,
[3H]IP2, and
[3H]IP3 formation, 75-cm2 flasks
of confluent hBRS-3-transfected BALB 3T3 cells labeled with
myo-[2-3H]inositol were scraped, centrifuged
(300 × g, 10 min), resuspended in 10 ml of PBS with 20 mM lithium chloride, and incubated for 10 min (37 °C).
After resuspension in 20 ml of assay buffer, 500-µl aliquots of cell
suspension were added to tubes containing 100 nM
[D-Phe6,
-Ala11,Phe13,Nle14]Bn-(6-14)
and incubated at 37 °C for the indicated times. Experiments were
terminated with 1 ml of ice-cold hydrochloric acid/methanol (0.1%,
v/v). [3H]IP1,
[3H]IP2 and [3H]IP3
were eluted off Dowex AG-1-X8 anion exchange columns as described
previously (50). In the experiments where total [3H]IP
was measured, total [3H]IP was eluted with 2 ml of 1 mM ammonium formate and 100 mM formic acid as
described previously (30, 51). Each of the eluates was collected and
mixed with 10 ml of Hydrofluor scintillation mixture (National
Diagnostics, Atlanta, GA), and the radioactivity was measured in a
scintillation counter.
Intracellular Calcium
([Ca2+]i)--
Cells harvested by scraping
were resuspended in an assay buffer (24.5 mM HEPES (pH
7.4), 98 mM sodium chloride, 6 mM potassium chloride, 2.5 mM monobasic sodium phosphate, 5 mM sodium pyruvate, 5 mM sodium fumarate, 5 mM sodium glutamate, 2 mM glutamine, 11.5 mM glucose, 1.45 mM calcium chloride, 1.15 µM magnesium chloride, 0.01% soybean trypsin inhibitor,
0.2% (v/v) amino acid mixture, and 0.2% BSA) to a concentration of
5 × 106 cells/ml and incubated with 2 µM Fura-2/AM (Molecular Probes, Eugene, OR) for 30 min at
37 °C. After washing two times with assay buffer, 2 ml of cell
suspension were placed in a Delta PTI Scan 1 spectrofluorimeter (Photon
Technology International, South Brunswick, NJ) equipped with a stir bar
and water bath (37 °C). Fluorescence was measured at dual excitation
wavelengths of 340 nm and 380 nm using an emission wavelength of 510 nm. The calcium concentration was calculated using the method of
Grynkiewicz et al. (52).
Immunoprecipitation of Tyrosine-phosphorylated
Proteins--
Quiescent and confluent hBRS-3-transfected BALB 3T3
cells and NCI-H1299 cells grown on 100-mm culture dishes were
preincubated twice with DMEM for 1 h, treated with the indicated
concentrations of
[D-Phe6,
-Ala11,Phe13,Nle14]Bn-(6-14)
or GRP for 10 min at 37 °C, and lysed at 4 °C in 1 ml of lysing
buffer containing 50 mM Tris/HCl (pH 7.5), 150 mM sodium chloride, 1% Triton X-100, 1% deoxycholate,
0.1% (w/v) sodium azide, 1 mM EGTA, 0.4 mM
EDTA, 2.5 µg/ml aprotinin, 2.5 µg/ml leupeptin, 1 mM
phenylmethylsulfonyl fluoride, and 0.2 mM sodium vanadate.
After centrifugation (15,000 × g, 15 min) and adjustment to a protein concentration of 0.5 mg/ml (Bio-Rad protein assay), the supernatants were immunoprecipitated with 4 µg of anti-phosphotyrosine mAb PY20, 4 µg of goat anti-mouse IgG, and 30 µl of protein A-agarose (Upstate Biotechnology, Inc., Lake Placid,
NY) overnight at 4 °C. The immunoprecipitates were washed three
times with PBS and further analyzed by SDS-polyacrylamide gel
electrophoresis and Western blotting.
Western blotting and Measurement of p125FAK Tyrosine
Phosphorylation--
Briefly, p125FAK tyrosine
phosphorylation was determined as described recently (53).
Immunoprecipitates were separated by SDS-polyacrylamide gel
electrophoresis, and the proteins were electrophoretically transferred
to nitrocellulose membranes. After blocking overnight at 4 °C in a
blocking solution (50 mM Tris/HCl (pH 8.0), 2 mM calcium chloride, 80 mM sodium chloride, 5%
non-fat dry milk, 0.05% Tween 20, and 0.02% sodium azide), the
membranes were incubated with anti-p125FAK mAb (1:1000 in
blocking solution) for 2 h at 25 °C. After washing with
blotting solution, the membranes were incubated at 25 °C with
horseradish peroxidase-conjugated secondary antibody (1:10,000 in
blocking solution) for 30 min. Immunoreactive proteins were detected
using the ECL detection system (Pierce). Quantitation of
p125FAK tyrosine phosphorylation was obtained by scanning
densitometry (Molecular Devices, Sunnyvale, CA).
Statistical Analysis--
Data plotting and iterative
curve-fitting was performed with KaleidaGraph graphing software
(Synergy Software, Reading, PA). Analysis of Schild plots (54) and
statistical analysis of the data were performed using Statview version
1.01 (BrainPower, Inc., Calabasas, CA). Student's t test
was used to determine the statistical significance between group
means.
Determination of Changes in Cyclic AMP--
Quiescent and
confluent hBRS-3-transfected BALB 3T3 cells or NCI-H1299 cells grown on
24-well plates were incubated with 500 µl of their respective media
(DMEM or RPMI 1640), which was supplemented with 2% FBS (v/v) and 2 µCi/ml [3H]adenine for 24 h at 37 °C. The
medium was removed, replaced with an equivalent volume of serum-free
medium, and allowed to incubate for 15 min at 37 °C. The medium was
removed and replaced with an equivalent volume of serum-free medium
containing 1% BSA (w/v), 0.5 mM isobutylmethylxanthine,
and the indicated agents at their indicated concentrations. The
reactions were terminated by the addition of 100 µl of stopping
solution (2% SDS (v/v), 5 mM cAMP) followed by 900 µl of
ice-cold Tris (50 mM, pH 7.4). Samples were stored at
20 °C until analyzed.
Determination of the amount of cAMP formation was obtained using a
modified protocol described by Salomon et al. (55). Frozen samples of hBRS-3-transfected BALB 3T3 cells or NCI-H1299 cells were
thawed and added to glass columns containing 1 ml of l:1 (v/v) slurry
of Dowex AG1-X8 anion exchange resin, which had been washed once with 4 ml of 1 N sodium hydroxide, once with 4 ml of 1 N hydrochloric acid, and twice with 10 ml of deionized
water. The columns were washed with 1 ml of deionized water after
addition of sample and stacked over another set of glass columns
containing 1 g of alumina, which had been previously washed with
10 ml of deionized water and 4 ml of 100 mM imidazole (pH
7.2). The samples were eluted with 3 ml onto the alumina columns. As a
final elution step, 4 ml of 0.1 M imidazole was added to
each alumina column. The eluate was collected and mixed with Hydrofluor
scintillation fluid, and the radioactivity was counted.
 |
RESULTS |
We first examined the effect of
[D-Phe6,
-Ala11,Phe13,Nle14]Bn-(6-14),
GRP, and Bn on the release of [3H]inositol phosphates
([3H]IP) in both untransfected and hBRS-3-transfected
cell lines. [D-Phe6,
-Ala11,Phe13,Nle14]Bn-(6-14),
GRP, and Bn elicited a small, but significant elevation of
[3H]IP in untransfected NCI-H1299 cells (Table
I). This result is consistent with
previous studies, which demonstrate that these cells possess a low
density of hGRP and hNMB receptors (35). The hBRS-3-transfected
NCI-H1299 cells exhibited a more robust response to
[D-Phe6,
-Ala11,Phe13,Nle14]Bn-(6-14)
at 10 nM and with each of the three peptides at 1000 nM. The untransfected BALB 3T3 cells, which do not possess
Bn receptors, failed to respond to any of these peptides (Table I). In
the hBRS-3-transfected BALB 3T3 cells, only
[D-Phe6,
-Ala11,Phe13,Nle14]Bn-(6-14)
was capable of eliciting [3H]IP release at both 10 and
1000 nM (Table I).
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Table I
Ability of GRP, NMB, and
[D-Phe6, -Ala11,Phe13,Nle14]Bn-(6-14)
to alter [3H]IP in native and hBRS-3-transfected NCI-H1299
cells and BALB 3T3 cells
Native nontransfected NCI-H1299 cells, BALB 3T3 cells, or
hBRS-3-transfected cell lines were incubated with GRP, Bn, or
[D-Phe6, -Ala11,Phe13,Nle14]Bn-(6-14)
at the above concentrations for 45 min. Results are expressed as the
ratio of total [3H]IP produced in the presence of peptide
(Exp) to that generated in the absence of peptide (Con). Each value
represents the means ± S.E. of at least three experiments
performed in duplicate. The control value in NCI-H1299 nontransfected
and hBRS-3-transfected cells was 1521 ± 237 and 1600 ± 152 dpm, and with 1000 nM
[D-Phe6, -Ala11,Phe13,Nle14]Bn-(6-14)
was 2637 ± 420 and 14,810 ± 2053 dpm, respectively. The
control value in BALB 3T3 nontransfected and hBRS-3-transfected cells
was 2911 ± 227 and 10,473 ± 2700 dpm and with 1000 nM
[D-Phe6, -Ala11,Phe13,Nle14]Bn-(6-14)
was 2919 ± 83 and 34,895 ± 8907 dpm,
respectively.
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The results in Table I indicated that in NCI-H1299 cells, but not in
BALB 3T3 cells, there were low levels of native Bn receptors (GRP
receptor, NMB receptor, or both) that could be stimulated to cause
detectable increases in [3H]IP by various Bn receptor
agonists that could confuse the results. Recent studies (42, 46, 56,
57) show that the potent GRP receptor antagonist
[D-Phe6]Bn-(6-13) methyl ester has low
affinity for the NMB receptor and the hBRS-3 receptor. Therefore, to
assess the extent of GRP receptor interaction with
[D-Phe6,
-Ala11,Phe13,Nle14]Bn-(6-14)
in NCI-H1299 cells, we compared the ability of
[D-Phe6]Bn-(6-13) methyl ester to inhibit a
submaximal dose of
[D-Phe6,
-Ala11,Phe13,Nle14]Bn-(6-14)
in the hBRS-3-transfected cell lines and a submaximal dose of GRP in
the mGRP receptor-transfected BALB 3T3 cells (Fig. 1). In the mGRP receptor transfectants,
[D-Phe6]Bn-(6-13) methyl ester attenuated
[3H]IP formation by GRP in a
concentration-dependent manner. Half-maximal inhibition was
observed at 2.8 ± 1.8 nM, and complete inhibition was
seen at 100 nM (Fig. 1). The [3H]IP release
observed in hBRS-3-transfected NCI-H1299 cells was attenuated 30% by
the antagonist, demonstrating 30% of the maximal stimulation was due
to occupation of GRP receptors. No inhibitory effect was observed in
hBRS-3-transfected BALB 3T3 cells treated with
[D-Phe6]Bn-(6-13) methyl ester (Fig. 1).

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Fig. 1.
Ability of
[D-Phe6]Bn-(6-13) methyl ester to inhibit
[3H]IP formation by
[D-Phe6, -Ala11,Phe13,Nle14]Bn-(6-14)
or GRP in cells transfected with receptors for Bn-like peptides.
BALB 3T3 cells were transfected with the hBRS-3 or the mGRP
receptor, and NCI-H1299 cells were transfected with the hBRS-3
receptor. All cells were treated with
[D-Phe6]Bn-(6-13) methyl ester at the
indicated concentrations for 45 min with either 30 nM
[D-Phe6, -Ala11,Phe13,Nle14]Bn-(6-14)
for the hBRS-3-transfected cells or 30 nM GRP for the mGRP
receptor-transfected cells. Values represent the percent of total
[3H]IP release stimulated by 30 nM
[D-Phe6, -Ala11,Phe13,Nle14]Bn-(6-14)
or GRP alone and are the means ± S.E. from at least three
separate experiments from duplicate determinations. The basal values
were 2632 ± 808, 8544 ± 647, and 465 ± 148 dpm for
the mGRP receptor-transfected BALB 3T3 cells, hBRS-3-transfected BALB
3T3 cells, and hBRS-3-transfected NCI-H1299 cells, respectively.
Stimulated control values were 6261 ± 906, 20,432 ± 3634, and 2900 ± 945 dpm for the mGRP receptor-transfected BALB cells,
hBRS-3-transfected BALB 3T3 cells, and hBRS-3-transfected H1299 cells,
respectively (n = 3).
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Because 100 nM [D-Phe6]Bn-(6-13)
methyl ester caused >95% inhibition at the GRP receptor, and this
concentration has been previously shown not to interact with the BRS-3
receptor (42), we included this concentration in all studies in the
hBRS-3-transfected NCI-H1299 cells to inhibit any stimulation by the
GRP receptor agonists. We next investigated the concentration
dependence of
[D-Phe6,
-Ala11,Phe13,Nle14]Bn-(6-14)
and various Bn-related peptides to elicit [3H]IP
formation in hBRS-3-transfected BALB 3T3 cells and hBRS-3-transfected NCI-H1299 cells (Fig. 2). In
hBRS-3-transfected BALB 3T3 cells and hBRS-3-transfected NCI-H1299
cells,
[D-Phe6,
-Ala11,Phe13,Nle14]Bn-(6-14)
stimulated a concentration-dependent (4- and 9-fold increase, respectively) release of total [3H]IP with an
apparent EC50 of 21 ± 2.1 and 35 ± 5.0 nM, respectively (Fig. 2). In the transfected BALB 3T3
cells, Bn, GRP, and NMB caused no stimulation until concentrations
>100 nM in hBRS-3-transfected BALB 3T3 cells and until
concentrations >10 nM in transfected NCI-H1299 cells (Fig.
2). We examined six other natural Bn-related peptides for agonist
activity, and as shown in Table II, none of these peptides caused a detectable response with concentrations less
than 300 nM. Three synthetic peptides reported to have
agonist properties against BRS-3 (41) were also evaluated. Two of
these, AcNMB-(3-10) and [D-Phe6]Bn-(6-13)
propylamide, stimulated [3H]IP accumulation in a
concentration-dependent manner, causing a detectable effect
at 219 ± 63 and 892 ± 71 nM (Table II) and causing a half-maximal effect at 333 ± 81 nM and
1924 ± 146 nM, respectively (Fig.
3). The efficacy of AcNMB-(3-10),
however, was only 80% of that seen with
[D-Phe6,
-Ala11,Phe13,Nle14]Bn-(6-14)
(Fig. 3). The third synthetic analogue,
[D-Phe6, Phe13]Bn-(6-13)
propylamide, was the least potent, eliciting a detectable increase in
[3H]IP only with concentrations >1000 nM
(Table II, Fig. 3).

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Fig. 2.
Effect of
[D-Phe6, -Ala11,Phe13,Nle14]Bn-(6-14)
and natural Bn-related peptides on [3H]IP formation in
BALB 3T3 cells and NCI-H1299 cells transfected with hBRS-3 receptor.
hBRS-3-transfected BALB 3T3 cells (left) and
hBRS-3-transfected NCI-H1299 cells (right) were incubated
with
[D-Phe6, -Ala11,Phe13,Nle14]Bn-(6-14),
Bn, GRP, or NMB for 45 min at the indicated concentrations. For the
hBRS-3-transfected NCI-H1299 cells, 100 nM
[D-Phe6]Bn-(6-13) methyl ester was included.
Values are expressed as a percent of the total [3H]IP
release stimulated by 1 µM
[D-Phe6, -Ala11,Phe13,Nle14]Bn-(6-14),
and are the means ± S.E. from at least three experiments
performed in duplicate. The basal values for the hBRS-3-transfected
BALB 3T3 cells and hBRS-3-transfected NCI-H1299 cells were 9015 ± 900 and 1530 ± 366 dpm, and the stimulated values for the
hBRS-3-transfected BALB 3T3 cells and hBRS-3-transfected NCI-H1299
cells were 39,750 ± 4213 and 14,525 ± 3729 dpm,
respectively (n = 3).
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Table II
Ability of various synthetic and naturally occurring Bn-related
peptides to stimulate [3H]IP generation in hBRS-3-transfected
BALB 3T3 cells
BALB 3T3 cells transfected with hBRS-3 were incubated with various
concentrations of each peptide (0.1 nM to 10 µM) for 45 min. Results are expressed as the
concentration of peptide capable of stimulating a detectable response
equal to 30% of the maximal response seen with 1000 nM
[D-Phe6, -Ala11,Phe13,Nle14]Bn-(6-14),
and are the means ± S.E. from at least three experiments. The
control and maximal stimulated [3H]IP values were 6104 ± 888 and 21,897 ± 3704 dpm, respectively. Abbreviations:
SAP-Bn, [Ser3,Arg10,Phe13]bombesin (44); Frog
GRP-10, frog gastrin-releasing peptide COOH-terminal decapeptide,
[Ser19]GRP-(18-27) (43); AcNMB-(3-10), acetyl-neuromedin
B-(3-10).
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Fig. 3.
Effect of synthetic Bn analogues on
[3H]IP formation in BALB 3T3 cells transfected with
hBRS-3 receptor. Cells were incubated with
[D-Phe6, -Ala11,Phe13,Nle14]Bn-(6-14),
[D-Phe6]Bn-(6-13) propylamide,
[D-Phe6,Phe13]Bn-(6-13)
propylamide or AcNMB-(3-10) for 45 min at the concentrations
indicated. Values are expressed as a percent of total
[3H]IP release stimulated by 1 µM
[D-Phe6, -Ala11,Phe13,Nle14]Bn-(6-14),
and are the means ± S.E. from at least three experiments
performed in duplicate. The control and 1 µM
[D-Phe6, -Ala11,Phe13,Nle14]Bn-(6-14)
stimulated values were 9303 ± 1059 and 38,015 ± 4167 dpm,
respectively (n = 3).
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We next explored the effects of various GRP receptor antagonists (57)
and NMB receptor antagonists (57, 58) on the ability of
[D-Phe6,
-Ala11,Phe13,Nle14]Bn-(6-14)
to stimulate [3H]IP release in hBRS-3-transfected
NCI-H1299 cells.
[D-Arg11,
-Trp7,9,Leu11]SP,
[D-Pro4,D-Trp7,9,10]SP-(4-11),
[(3-Ph-Pr6)-His7,
-Ala11,D-Pro13,
(13-14),Phe14]Bn-(6-14)-NH2,
and the selective NMB receptor antagonist
D-Nal,Cys,Tyr,D-Trp,Lys, Val,Cys,Nal-NH2 (58) were capable of attenuating
[D-Phe6,
-Ala11,Phe13,Nle14]Bn-(6-14)
by
50%, with
D-Nal,Cys,Tyr,D-Trp,Lys,Val,Cys,Nal-NH2 being the most potent (Fig. 4). The
inhibition caused by the substance P analogues and
D-Nal,Cys,Tyr,D-Trp,Lys,Val,Cys,Nal-NH2
was examined in detail and found to be
concentration-dependent (Fig. 4). Of these three,
D-Nal,Cys,Tyr,D-Trp,Lys,Val,Cys,Nal-NH2
exhibited the highest potency with half-maximal inhibition at 1.1 ± 0.3 µM. In the BALB 3T3 cells,
D-Nal,Cys,Tyr,D-Trp,Lys,Val,Cys,Nal-NH2 caused a rightward parallel shift of the
[D-Phe6,
-Ala11,Phe13,Nle14]Bn-(6-14)
dose-response curve for stimulation of [3H]IP (data not
shown). Plotting these data in the form of Schild (54) gave an equation
with a slope of 0.94, which was not significantly different from unity.
Calculation of the affinity of
D-Nal,Cys,Tyr,D-Trp,Lys,Val,Cys,Nal-NH2 for the hBRS-3 receptor from these data gave an affinity of 504 ± 146 nM.

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Fig. 4.
Ability of various Bn receptor antagonists to
inhibit [3H]IP formation by
[D-Phe6, -Ala11,Phe13,Nle14]Bn-(6-14)
in hBRS-3-transfected NCI-H1299 cells. H1299 cells transfected
with hBRS-3 receptor were treated with 100 nM
[D-Phe6, -Ala11,Phe13,Nle14]Bn-(6-14),
100 nM [D-Phe6]Bn-(6-13) methyl
ester, and
[D-Arg1,D-Trp7,9,Leu11]SP,
[D-Pro4,D-Trp7,9,10]SP-(4-11),
or D-Nal,
Cys,Tyr,D-Trp,Lys,Val,Cys,Nal-NH2 at the
concentrations indicated for 45 min. Values represent the percent of
total [3H]IP release stimulated by 100 nM
[D-Phe6, -Ala11,Phe13,Nle14]Bn-(6-14)
(basal dpm = 1331 ± 175, stimulated dpm = 12,411 ± 2096), and are the means ± S.E. from at least three
experiments performed in duplicate.
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To determine the ability of the hBRS-3 receptor to increase different
isomers of inositol phosphates, we examined the time course of
[3H]IP1, [3H]IP2,
and [3H]IP3 formation in the
hBRS-3-transfected BALB 3T3 cells stimulated with 100 nM
[D-Phe6,
-Ala11,Phe13,Nle14]Bn-(6-14)
(Fig. 5).
[D-Phe6,
-Ala11,Phe13,Nle14]Bn-(6-14)
stimulated a time-dependent increase in
[3H]IP1, [3H]IP2,
and [3H]IP3. The maximal increase of
[3H]IP3 was most rapidly reached.
[D-Phe6,
-Ala11,Phe13,Nle14]Bn-(6-14)
caused a maximal increase in [3H]IP3 (15 s),
followed by [3H]IP2 (2 min) and
[3H]IP1 at 30 min. The magnitude of
stimulation of [3H]IP3 and
[3H]IP2 both decreased with time, whereas
[3H]IP1 stimulation continued to increase
until 60 min (Fig. 5).

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Fig. 5.
Effect of
[D-Phe6, -Ala11,Phe13,Nle14]Bn-(6-14)
on the time course of [3H]IP1
(top), [3H]IP2
(middle), and [3H]IP3
(bottom) accumulation in hBRS-3-transfected BALB 3T3
cells. The transfected cells were treated with 100 nM
[D-Phe6, -Ala11,Phe13,Nle14]Bn-(6-14)
for the times indicated, and the isomers were separated by column
chromatography as described under "Materials and Methods." The
values represent the mean dpm ± S.E. (dpm in the presence of
100 nM
[D-Phe6, -Ala11,Phe13,Nle14]Bn-(6-14)
minus the unstimulated dpm obtained from at least three experiments
performed in triplicate. The maximal counts for
[3H]IP1 (30 min),
[3H]IP2 (2 min), and
[3H]IP3 (15 s) were 42,509 ± 5225, 2632 ± 456, and 1552 ± 118 dpm, respectively, while the
corresponding unstimulated controls were 19,358 ± 2602, 872 ± 233, and 884 ± 31 dpm, respectively (n = 4).
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To examine the ability of hBRS-3 receptor activation to cause changes
in cytosolic calcium ([Ca2+]i), we examined the
effect of
[D-Phe6,
-Ala11,Phe13,Nle14]Bn-(6-14)
on Fura-2/AM-loaded hBRS-3-transfected NCI-H1299 cells and BALB 3T3
cells (Fig. 6).
[D-Phe6,
-Ala11,Phe13,Nle14]Bn-(6-14)
stimulated a 1.8- and 3-fold increase in [Ca2+]i
in hBRS-3-transfected BALB 3T3 cells and hBRS-3-transfected NCI-H1299
cells, and a 2-fold increase in [Ca2+]i in the
wild-type, untransfected, NCI-H1299 cells (Fig. 6). The kinetics of
[Ca2+]i release were similar in both transfected
cell lines, reaching maximal levels in 10 s (Fig. 6). The GRP
receptor antagonist, [D-Phe6]Bn-(6-13)
methyl ester, completely inhibited the ability of
[D-Phe6,
-Ala11,Phe13,Nle14]Bn-(6-14)
to elicit changes in [Ca2+]i in the untransfected
NCI-H1299 cells (Fig. 6, left panel) and attenuated the
response in the transfected NCI-H1299 cells by 23% (Fig. 6,
middle panel). The antagonist had no effect on the ability
of
[D-Phe6,
-Ala11,Phe13,Nle14]Bn-(6-14)
to stimulate [Ca2+]i in transfected BALB 3T3
cells (Fig. 6, right panel). To determine the contribution
of extracellular calcium to the [Ca2+]i
transient, cells were stimulated with
[D-Phe6,
-Ala11,Phe13,Nle14]Bn-(6-14)
in the presence of the Ca2+-chelating agent EGTA. In the
absence of extracellular Ca2+, the magnitude of the
response was reduced only by 15% and the return to basal levels was
more rapid than that seen in Ca2+-containing buffer (Fig.
7).

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Fig. 6.
Effect of
[D-Phe6, -Ala11,Phe13,Nle14]Bn-(6-14)
on [Ca2+]i in untransfected H1299 cells or
hBRS-3-transfected NCI-H1299 and BALB 3T3 cells. Untransfected
H1299 cells (left), hBRS-3-transfected BALB 3T3 cells
(center), and hBRS-3-transfected NCI-H1299 cells
(right) were loaded with Fura-2/AM and treated with 100 nM
[D-Phe6, -Ala11,Phe13,Nle14]Bn-(6-14)
in the presence ( ) or absence ( ) of 3 µM
[D-Phe6]Bn-(6-13) methyl ester. The figure
depicts the results from a typical experiment performed at least three
times.
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Fig. 7.
The effect of extracellular
Ca2+ on
[D-Phe6, -Ala11,Phe13,Nle14]Bn-(6-14)-induced
Ca2+ release in hBRS-transfected BALB 3T3 cells.
hBRS-3-transfected BALB 3T3 cells were loaded with Fura-2/AM and
assayed under conditions outlined under "Materials and Methods."
The cells were stimulated with 100 nM
[D-Phe6, -Ala11,Phe13,Nle14]Bn-(6-14)
in the presence or absence of 1.45 mM EGTA. The tracing is
typical of an experiment performed three times.
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Recent studies have demonstrated that a number of neuropeptides,
without intrinsic tyrosine kinase activity, can stimulate tyrosine
phosphorylation of a number of proteins including the cytosolic focal
adhesion kinase (p125FAK) (32, 33, 59-63). Therefore, we
studied the effect of
[D-Phe6,
-Ala11,Phe13,Nle14]Bn-(6-14)
on tyrosine phosphorylation of p125FAK. In both the
hBRS-3-transfected BALB 3T3 cells (Fig.
8) and the hBRS-3-transfected NCI-H1299
cells (Fig. 9), 100 nM
[D-Phe6,
-Ala11,Phe13,Nle14]Bn-(6-14)
stimulated a 3- and 3.5-fold increase in tyrosine phosphorylation of
p125FAK, respectively. In hBRS-3-transfected BALB 3T3
cells, this increase was unaffected by the GRP receptor antagonist
[D-Phe6]Bn-(6-13) methyl ester (Fig. 8).
Furthermore, in these cells, GRP failed to stimulate
p125FAK phosphorylation (Fig. 8). In the hBRS-3-transfected
NCI-H1299 cells, both 100 nM GRP and 100 nM
[D-Phe6,
-Ala11,Phe13,Nle14]Bn-(6-14)
stimulated p125FAK phosphorylation to the same extent (Fig.
9). In the hBRS-3-transfected NCI-H1299 cells,
[D-Phe6]Bn-(6-13) methyl ester completely
abolished the GRP-mediated effect, but failed to inhibit the
phosphorylation seen with
[D-Phe6,
-Ala11,Phe13,Nle14]Bn-(6-14)
(Fig. 9). The phosphorylation induced by
[D-Phe6,
-Ala11,Phe13,Nle14]Bn-(6-14)
was concentration-dependent in both hBRS-3-transfected NCI-H1299 cells and hBRS-3-transfected BALB cells (Fig.
10). A maximal 4-fold stimulation in
both transfected cell lines was obtained with 100 nM
[D-Phe6,
-Ala11,Phe13,Nle14]Bn-(6-14),
and the EC50 values for hBRS-3-transfected BALB 3T3 and
NCI-H1299 cells were 0.7 ± 0.2 nM and 0.2 ± 0.1 nM, respectively.

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Fig. 8.
Effect of
[D-Phe6, -Ala11,Phe13,Nle14]Bn-(6-14),
GRP, and the GRP receptor antagonist
[D-Phe6]Bn-(6-13) methyl ester on tyrosine
phosphorylation of p125FAK in hBRS-3-transfected BALB 3T3
cells. hBRS-3-transfected BALB 3T3 cells were incubated with 100 nM GRP or
[D-Phe6, -Ala11,Phe13,Nle14]Bn-(6-14)
in the presence or absence of 300 nM
[D-Phe6]Bn-(6-13) methyl ester. The
upper panel shows a typical Western blot for
p125FAK in cells treated with (+) or without ( ) the
various peptides. The lower panel represents the mean
p125FAK tyrosine phosphorylation expressed as the amount
phosphorylated in the various treatment groups compared with the
control untreated level. Data are means ± S.E. from at least
three experiments.
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Fig. 9.
Effect of
[D-Phe6, -Ala11,Phe13,Nle14]Bn-(6-14),
GRP, and the GRP receptor antagonist
[D-Phe6]Bn-(6-13) methyl ester on tyrosine
phosphorylation of p125FAK in hBRS-3-transfected NCI-H1299
cells. hBRS-3-transfected NCI-H1299 cells were incubated with 100 nM GRP or
[D-Phe6, -Ala11,Phe13,Nle14]Bn-(6-14)
in the presence or absence of 300 nM
[D-Phe6]Bn-(6-13) methyl ester. The
upper panel shows a typical Western blot for
p125FAK in cells treated with (+) or without ( ) the
various peptides. The lower panel represents the mean
p125FAK tyrosine phosphorylation expressed as the amount of
phosphorylated p125FAK in the various treatment groups
compared with the untreated control. Data are means ± S.E. from at least three experiments. NS, not
significant.
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Fig. 10.
Dose-response effect of
[D-Phe6, -Ala11,Phe13,Nle14]Bn-(6-14)
on stimulation of tyrosine phosphorylation of p125FAK in
cells transfected with the hBRS-3 receptor. hBRS-3-transfected
NCI-H1299 (top) or BALB 3T3 cells (bottom) were
treated with
[D-Phe6, -Ala11,Phe13,Nle14]Bn-(6-14)
at the concentrations indicated. For the NCI-H1299 cells, 300 nM [D-Phe6]Bn-(6-13) methyl
ester was included. Values represent the means ± S.E. from at
least three experiments. In the top panels above each graph
is a Western blot from a typical experiment performed at least three
times. Data are expressed as a percentage of the maximal increase in
tyrosine phosphorylation caused by 100 nM
[D-Phe6, -Ala11,Phe13,Nle14]Bn-(6-14).
The maximal value represented a 4.0 ± 1.8-fold increase in the
hBRS-3-transfected NCI-H1299 cells and a 4.1 ± 1.5-fold increase
in the hBRS-3-transfected BALB 3T3 cells.
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To explore the relationship between the ability of hBRS-3 receptor
activation to stimulate phospholipase C activity resulting in protein
kinase C (PKC) activation and changes in [Ca2+]i
and its ability to stimulate changes in p125FAK
phosphorylation, we examined the effect of the selective PKC inhibitor
GF109203X (64) and the Ca2+/ATPase inhibitor thapsigargin
(65) on tyrosine phosphorylation of p125FAK stimulated by
[D-Phe6,
-Ala11,Phe13,Nle14]Bn-(6-14)
(Fig. 11). In hBRS-3-transfected BALB
3T3 cells, both [D-Phe6,
-Ala11,Phe13,Nle14]Bn-(6-14)
(100 nM) and TPA (100 nM) stimulated a 3.5-fold
increase in tyrosine phosphorylation of p125FAK (Fig. 11).
GF109203X (5 µM) abolished the effect of TPA but did not
diminish the p125FAK phosphorylation seen with
[D-Phe6,
-Ala11,Phe13,Nle14]Bn-(6-14).
Thapsigargin (100 nM), when added 30 min prior to assay,
completely inhibited the ability of
[D-Phe6,
-Ala11,Phe13,Nle14]Bn-(6-14)
to stimulate [Ca2+]i (Fig. 11, inset),
but failed to inhibit
[D-Phe6,
-Ala11,Phe13,Nle14]Bn-(6-14)-induced
p125FAK phosphorylation (Fig. 11). Furthermore, the
combination of GF109203X and thapsigargin had had no effect on
phosphorylation alone and had no significant effect on tyrosine
phosphorylation of p125FAK induced by hBRS-3 receptor
activation (Fig. 11).

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Fig. 11.
Effect of thapsigargin alone or
with GF109203X on
[D-Phe6, -Ala11,Phe13,Nle14]Bn-(6-14)
stimulation of tyrosine phosphorylation of
p125FAK. hBRS-3-transfected BALB 3T3 cells were
pretreated for 1 h at 37 °C in the presence or absence of 5 µM GF109203X (GFX). During the final 30 min
of pretreatment, 0.1 µM thapsigargin (TG) was
added to the samples indicated. Control cells received an equivalent
volume of Me2SO. Cells were incubated for another 10 min
with no additions (control) or with 0.1 µM
[D-Phe6, -Ala11,Phe13,Nle14]Bn-(6-14)
or 0.1 µM TPA. p125FAK phosphorylation was
determined as described under "Materials and Methods." The
top panel shows a single experiment representative of two
others. The lower panel is the quantitation of
p125FAK tyrosine phosphorylation determined by scanning
densitometry. The values are mean ± S.E. from three experiments and
are expressed as the ratio of total p125FAK tyrosine
phosphorylation in the presence of
[D-Phe6, -Ala11,Phe13,Nle14]Bn-(6-14)
(Exp) to that in the absence of
[D-Phe6, -Ala11,Phe13,Nle14]Bn-(6-14)
(Con). Inset, effect of thapsigargin on
[D-Phe6, -Ala11,Phe13,Nle14]Bn-(6-14)-induced
changes in [Ca2+]i in hBRS-3-transfected BALB
3T3 cells. Fura-2-loaded cells were incubated in the absence or
presence of 0.1 µM thapsigargin for 30 min, and 0.1 µM
[D-Phe6, -Ala11,Phe13,Nle14]Bn-(6-14)
was added. [Ca2+]i was continuously
measured in a spectrofluorimeter as described under "Materials and
Methods." This result is representative of three others.
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Since it had been shown previously (66, 67) that activation of the
hBRS-3-related receptor, the GRP receptor in Swiss 3T3 fibroblasts,
could stimulate increases in cyclic AMP, the ability of
[D-Phe6,
-Ala11,Phe13,Nle14]Bn-(6-14)
and various agonists known to activate adenylate cyclase via receptor
activation were studied (Table III). In
the hBRS-3-transfected NCI-H1299 cells, forskolin, a direct activator
of adenylate cyclase, PACAP-27, and PACAP-38, stimulated a significant
release of cAMP, whereas
[D-Phe6,
-Ala11,Phe13,Nle14]Bn-(6-14),
vasopressin, and epinephrine had no stimulatory effect. We also
examined the ability of
[D-Phe6,
-Ala11,Phe13,Nle14]Bn-(6-14)
and various agonists known to attenuate stimulated adenylate cyclase
activity via receptor activation by determining its ability to inhibit
forskolin-induced cAMP formation in hBRS-3-transfected BALB 3T3 cells
(Table IV). Neither
[D-Phe6,
-Ala11,Phe13,Nle14]Bn-(6-14),
bombesin, dopamine, nor neuropeptide Y at concentrations of 3 µM were able to inhibit the elevation of cAMP seen with
forskolin, although 3 µM serotonin caused a significant
decrease in cAMP (Table IV).
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Table III
Comparison of the ability of
[D-Phe6, -Ala11,Phe13,Nle14]Bn-(6-14)
and other agents to elevate cyclic AMP levels in hBRS-3-transfected
NCI-H1299 cells
hBRS-3-transfected NCI-H1299 cells were incubated with each of the
indicated agents at the above concentrations for 30 min. Results are
expressed as the ratio of total [3H]cAMP in the presence of
agonists (Exp) to that in the absence of agonists (Con). Each value
represents the means ± S.E. from at least three experiments
performed in duplicate. The control values were 500 ± 78 cpm.
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Table IV
Comparison of the ability of
[D-Phe6, -Ala11,Phe13,Nle14]Bn-(6-14)
and other agents to inhibit elevated cyclic AMP levels in
hBRS-3-transfected NCI-H1299 cells
hBRS-3-transfected BALB 3T3 cells were incubated with each of the
indicated agents at the above concentrations for 30 min in the presence
of 25 µM forskolin. Results are expressed as the ratio of
total [3H]cAMP in the presence of agonists (Exp) to that in
the absence of agonists and forskolin (Con). Each value represents the
means ± S.E. from at least three experiments performed in
duplicate. The basal value was 162 ± 18 cpm, and the maximally
stimulated value (forskolin alone) was 631 ± 61 cpm.
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DISCUSSION |
Although the BRS-3 receptor was identified several years ago in
human (35) and guinea pig (36), a lack of cell lines expressing physiologically relevant levels of this protein, as well as a lack of a
ligand, have impeded studies of the biological role of BRS-3 receptors.
To gain further insight into the pharmacology of BRS-3 receptors, we
created two cell lines whose signaling sequelae and receptor coupling
might resemble those seen in cells expressing native, functional BRS-3
receptors. We chose BALB 3T3 fibroblasts as one candidate, because
these cells are devoid of receptors for bombesin-related peptides (24,
49). Furthermore, both GRP receptors and NMB receptors have been
successfully transfected into this cell line, and exhibit intracellular
signaling and a pharmacology similar to cells containing native mGRP,
hGRP, hNMB, and rNMB receptors (24, 30, 49). However, it is unknown whether hBRS-3-transfected, murine BALB 3T3 cells have a signaling repertoire identical to human cells natively expressing BRS-3 receptors. Therefore, we included the human non-small cell lung cancer
cell line NCI-H1299 in our study, which natively expresses low levels
of hBRS-3 receptor (68, 69), assuming that the necessary elements for
hBRS-3 receptor coupling and intracellular signaling were likely
extant.
Our results show that, in hBRS-3-transfected BALB 3T3 cells, the new
ligand
[D-Phe6,
-Ala11,Phe13,Nle14]Bn-(6-14)
interacts and behaves as an agonist at hBRS-3 receptors. This
conclusion is reached because untransfected BALB 3T3 cells did not
respond to
[D-Phe6,
-Ala11,Phe13,Nle14]Bn-(6-14)
in either the [3H]IP or [Ca2+]i
assay; however,
[D-Phe6,
-Ala11,Phe13,Nle14]Bn-(6-14)
stimulated increases in both [3H]IP release and
[Ca2+]i in hBRS-3-transfected BALB 3T3 cells, and
NCI-H1299 cells under assay conditions, where GRP receptor activity was inhibited.
A number of results demonstrate that hBRS-3 receptors are coupled to
phospholipase C activation, which in turn causes mobilization of
intracellular calcium. First, we found that
[D-Phe6,
-Ala11,Phe13,Nle14]Bn-(6-14)
elicited total [3H]IP release in both hBRS-3-transfected
cell lines under conditions in which no stimulation was seen in
untransfected cells. Second, this agonist ligand stimulated release of
[3H]IP1, [3H]IP2
and [3H]IP3 isomers in hBRS-3-transfected
BALB 3T3 cells, consistent with an increase in phospholipase C activity
and subsequent metabolism of inositol phosphates. Third,
[D-Phe6,
-Ala11,Phe13,Nle14]Bn-(6-14)
stimulated a rapid rise of cytosolic calcium in both hBRS-3
transfectants under conditions where no change was seen in
untransfected cells. Fourth, hBRS-3 receptor-activated increases in
cytosolic calcium were only minimally inhibited by removal of
extracellular calcium, demonstrating release was primarily from an
IP3-sensitive intracellular calcium pool. The ability of
BRS-3 activation to alter cytosolic calcium is consistent with previous
findings with the BRS-3 receptor (35, 41). When the hBRS-3 receptor was
expressed in Xenopus oocytes by injecting hBRS-3 sense
mRNA (35), the hBRS-3 receptor coupled to calcium-activated chloride channels, because high concentrations of GRP or NMB (10 µM) caused changes in chloride current. In contrast,
uninjected oocytes, or those injected with hBRS-3 receptor antisense
mRNA, showed no changes with these peptides. Furthermore, in an
earlier study, when hBRS-3 was transfected into BALB 3T3 cells (41), some GRP- and NMB-related peptides caused changes in cytosolic calcium
at high concentrations.
Our studies demonstrate that like a number of other neuropeptide
receptors (32, 33, 60-63), the hBRS-3 receptor can also stimulate
tyrosine phosphorylation of the cytosolic focal adhesion kinase
(p125FAK), which in a number of cells, has been shown to be
required for the promotion of focal adhesions which are important in
cell growth and motility (70, 71). In the presence of
[D-Phe6,
-Ala11,Phe13,Nle14]Bn-(6-14),
tyrosine phosphorylation of p125FAK was stimulated in a
concentration-dependent fashion in hBRS-3-transfected BALB
3T3 cells.
[D-Phe6,
-Ala11,Phe13,Nle14]Bn-(6-14)
also caused a concentration-dependent increase in
p125FAK tyrosine phosphorylation under conditions in which
the activation of GRP receptors was completely inhibited, in
hBRS-3-transfected NCI-H1299 cells. This is an important control
because recent studies demonstrate that agonist activation of the GRP
receptor as well as the NMB receptor cause rapid phosphorylation of
both p125FAK and paxillin (34, 59, 60). Because the studies
were performed in these cells under experimental conditions in which
[D-Phe6,
-Ala11,Phe13,Nle14]Bn-(6-14)
gave no stimulation of cellular function (changes in Ca2+,
[3H]IP accumulation) in untransfected NCI-H1299 cells, it
is unlikely that activation of either of the other native bombesin
receptors was causing the increase in p125FAK
phosphorylation seen with the novel ligand in hBRS-3-transfected NCI-H1299 cells.
Recent studies show that for some G protein-coupled receptors such as
those for angiotensin II (72, 73), cholecystokinin (74), epinephrine
(75), bradykinin (33), and endothelin-1 (73, 76), but not for others
such as those for GRP (71) or NMB (31), depletion of Ca2+
or inhibition of PKC activity resulted in a reduction of
p125FAK tyrosine phosphorylation in the presence of
agonist. These results demonstrate that, with some G protein-coupled
receptors, but not others, activation of the phospholipase C cascade is
required for stimulation of tyrosine phosphorylation of
p125FAK. Furthermore, p125FAK has a PKC
phosphorylation sequence (77), and activation of PKC by phorbol ester
or diacylglycerol has been shown to stimulate p125FAK
tyrosine phosphorylation (71). A number of our findings support the
conclusion that, with hBRS-3 receptor activation, the stimulation of
tyrosine phosphorylation of p125FAK is independent of PKC
activation and changes in cytosolic calcium. First, the PKC inhibitor
GF109203X had no inhibitory effect on [D-Phe6,
-Ala11,Phe13,Nle14]Bn-(6-14)-stimulated
p125FAK tyrosine phosphorylation under conditions where it
completely inhibited TPA-stimulated increases. Second, complete
inhibition of the ability of hBRS-3 activation to increase
[Ca2+]i by depletion of intracellular calcium by
thapsigargin failed to attenuate hBRS-3 receptor-mediated
p125FAK tyrosine phosphorylation.
A number of studies have shown that the combination of calcium
mobilization and PKC activation can have a synergistic effect on
cellular responses such as amylase release from pancreatic acini (78),
protein phosphorylation (79), or pepsinogen release from chief cells
(80). Our results suggest that this synergistic effect is not important
in mediating stimulation of p125FAK tyrosine
phosphorylation upon hBRS-3 receptor activation because the combination
of GF109203X, at a concentration that blocked TPA-induced
p125FAK tyrosine phosphorylation and thapsigargin at a
concentration that inhibited
[D-Phe6,
-Ala11,Phe13,Nle14]Bn-(6-14)-induced
increases in cytosolic calcium, had no effect on hBRS-3 receptor
activation of p125FAK tyrosine phosphorylation. Therefore,
stimulation of tyrosine phosphorylation of p125FAK by
hBRS-3 receptor activation is not altered by simultaneous activation of
both limbs of the phospholipase C cascade, which is similar to that
described for NMB receptors (53), but not for other receptors such as
for thrombin in platelets (75) or CCKA receptors in
pancreatic acini (74).
A number of G protein receptors are coupled to both phospholipase C
(81-84) and adenylate cyclase, either through Gs or
Gi. Elevation of cAMP has been associated with activation
of the hBRS-3-related receptor, the GRP receptor in Swiss 3T3 cells
(66, 67), but not with activation of NMB receptors (29, 30). In the
present study, activation of hBRS-3 receptors in hBRS-3-transfected
H1299 cells did not result in stimulation of increases in cAMP. Because PACAP-27 and PACAP-38 were capable of stimulating an increase in cAMP,
it is unlikely that the NCI-H1299 cells possessed inadequate Gs. Therefore, the failure of hBRS-3 receptors to mediate
adenylate cyclase activity in these cells cannot be explained on the
basis of insufficient Gs availability. In addition,
forskolin stimulated a significant cAMP response in the
hBRS-3-transfected NCI-H1299 cell line, demonstrating that adenylate
cyclase could be directly activated. The possibility that hBRS-3
receptor activation could inhibit cAMP by coupling to Gi is
also unlikely because
[D-Phe6,
-Ala11,Phe13,Nle14]Bn-(6-14)
was incapable of inhibiting adenylate cyclase activation by forskolin
in the hBRS-3-transfected BALB 3T3 cells. These latter results support
the conclusion the hBRS-3, in contrast to the closely related GRP
receptor, does not stimulate changes in cyclic AMP as a transduction
cascade.
Based on its 51% amino acid identity to the GRP receptor and 47%
amino acid identity with the NMB receptor (35), it was proposed that
BRS-3 might be related to these receptors. The ability of
bombesin-related peptides to interact with the hBRS-3 receptor has both
similarities and differences to what is seen with GRP and NMB
receptors. First, it is similar to the GRP and NMB receptors in that it
is coupled to phospholipase C activation. In all species examined,
including human, rat, and mouse tissues, both the GRP and NMB receptors
are coupled to phospholipase C (9, 14, 24, 28-30, 67). Second, it
resembles both GRP and NMB receptors in its ability to stimulate
tyrosine phosphorylation of such proteins as p125FAK.
Third, it demonstrates similar stoichiometric intracellular coupling of
these two intracellular pathways to what is seen with the GRP and NMB
receptors. With each of these receptors, the dose-response curve for
the ability of agonists to stimulate inositol phosphate formation was
to the right of its ability to cause tyrosine phosphorylation of
p125FAK (59, 71). However, the hBRS-3 receptor and the GRP
and NMB receptors differ in the stoichiometric relationships of these two dose-responses to each other and to receptor occupation (59, 71,
85). With each of these three receptors, phospholipase C activation is
closely coupled to receptor occupation, whereas submaximal receptor
occupation gives maximal p125FAK tyrosine phosphorylation
(9, 53, 71, 86). These receptor subtypes differ in that the receptor
spareness for tyrosine phosphorylation is greater for the hBRS-3
receptor than the GRP or NMB receptor.
The pharmacology of the hBRS-3 receptor, both in terms of its affinity
for agonists and many receptor antagonists, has a number of differences
from both the GRP receptor and the NMB receptor, as well as the
recently described bombesin receptor subtype 4 (BB4) (43).
It differs in that none of the nine naturally occurring peptides that
are members of the bombesin family had a potency >500 nM
for activating phospholipase C through the hBRS-3 receptor. In
contrast, at least four of these peptides interact in the nanomolar range to activate phospholipase C through the GRP receptor (Bn, GRP,
[Phe13]Bn, and litorin) (9, 25), two peptides with the
NMB receptor (NMB, litorin) (9, 25, 29), and three peptides have
nanomolar binding affinities for the BB4 receptor (Bn,
[Phe13]Bn, GRP) (43). This suggests either its natural
ligand is not a bombesin-like peptide, or it has a completely novel
sequence differing significantly from the existing naturally occurring bombesin-related peptides. The pharmacology of various synthetic analogues of bombesin and neuromedin B were also found to differ between the hBRS-3 receptor and the known bombesin receptor subtypes. In a previous study (42) of 27 synthetic analogues of Bn, GRP or NMB,
which had high affinity for GRP, NMB, or BB4 receptors, only
[D-Phe6,
-Ala11,Phe13,Nle14]Bn-(6-14)
had a high affinity at the hBRS-3 receptor. The present study
demonstrates this peptide functions as a high affinity agonist at this
receptor activating phospholipase C, inducing changes in cytosolic
calcium concentrations and stimulating p125FAK tyrosine
phosphorylation. Recently, Wu et al. (41) reported three
synthetic peptides, acetyl-NMB-(3-10),
[D-Phe6,Phe13]Bn-(6-13)
propylamide, and [D-Phe6]Bn-(6-13)
propylamide, were all agonists stimulating
[Ca2+]i mobilization in hBRS-3-transfected BALB
3T3 cells and that
[D-Phe6,Phe13]Bn-(6-13)
propylamide was particularly potent. Our results have both similarities
and differences from this study (41). Our results were similar in that
we found each of these three peptides had agonist activity at hBRS-3
receptors. Our results were also similar in the potency of
AcNMB-(3-10) for stimulating changes in [Ca2+]i
in their study (i.e. 219 nM) and for stimulating
[3H]IP in our study (i.e. 333 nM).
However, our results differed significantly in the potencies of
[D-Phe6]Bn-(6-13) propylamide and
[D-Phe6,Phe13]Bn-(6-13)
propylamide, which we found were much lower than they reported
(20-2000 fold), and both peptides were less efficacious than reported
previously (41). The reasons for the discrepancies between the two
studies are not clear. In the previous study (41), because of the
potency of NMB analogues, it was proposed that the hBRS-3 receptor had
a binding site more like the NMB receptor than the GRP receptor. The
finding of a submicromolar agonist potency of AcNMB-(3-10) for hBRS-3,
as well as our discovery that D-Nal,Cys,Tyr,D-Trp,Lys,Val,Cys,Nal-NH2,
a selective NMB receptor antagonist, also inhibited agonist activity at
the hBRS-3 receptor, might be considered to be supportive of this
proposal. However, NMB had a low affinity for hBRS-3 in our study, and
none of the other naturally occurring peptides with a Phe13
substitution, such as [Phe13]Bn, litorin, phyllolitorin,
and rhodei-litorin, had high affinity for hBRS-3. This does not exclude
the possibility that a Phe13 plays a role in selectivity
for BRS-3, since
[D-Phe6,
-Ala11,Phe13,Nle14]Bn-(6-14)
and all of the Phe13-containing peptides, except
[Ser3,Arg10,Phe13]bombesin, were
slightly more potent than the Leu13-containing peptides GRP
and Bn.
Six different classes of GRP or NMB receptor antagonists have
been described (46, 57, 87, 88). None of these functioned as a potent
receptor antagonist of the hBRS-3 receptor. However, members of three
classes of low affinity antagonists for bombesin receptors, including
the GRP and NMB receptor antagonists,
[D-Pro4,D-Trp7,9,10]SP-(4-11)
and
[D-Arg1,D-Trp7,9,Leu11]SP
(56, 89-91), and a selective NMB receptor antagonist, the somatostatin
octapeptide analogue
D-Nal,Cys,Tyr,D-Trp,Lys,Val,Cys,Nal-NH2 (58), did function as antagonists of the hBRS-3 receptor. The latter
somatostatin analogue was the most potent with an affinity of 504 nM, and pharmacological analysis demonstrated it functions as a competitive hBRS-3 receptor antagonist. In a previous study (58),
this analogue was reported to be selective for the NMB receptor and did
not interact with the GRP receptor. However, our data demonstrate this
compound also functions as a hBRS-3 receptor antagonist with slightly
lower affinity than seen with NMB receptors (i.e. 216 nM) (58). Additional structure-function studies will be
needed to determine if somatostatin analogues can be identified which
are more selective for hBRS-3 receptors than NMB receptors.
Nevertheless, the availability of these three classes of low affinity
antagonists will be useful either as starting points to develop more
potent antagonists, or in studies of hBRS-3 receptor cell biology aimed
at determining its role in various physiological or pharmacological
processes.
In conclusion, using the novel ligand
[D-Phe6,
-Ala11,Phe13,Nle14]Bn-(6-14),
we demonstrated that hBRS-3 activation stimulates phospholipase C
activity and tyrosine kinase activity in a similar manner in two cell
lines stably transfected with the hBRS-3 receptor. In contrast to all
naturally occurring bombesin- or NMB-related peptides or synthetic
analogues tested, only this novel ligand functioned as a high affinity
agonist. These results demonstrate that the pharmacology of hBRS-3 is
different from any other Bn receptors, and the native ligand likely is
not related to any of the naturally occurring Bn peptides currently
described. None of the high affinity, selective antagonists for the
other Bn receptors interact with the hBRS-3 receptors, although three
classes of low affinity antagonists did function as hBRS-3 receptor
antagonists. The availability of these hBRS-3-transfected cells, the
discovery of the potent agonist
[D-Phe6,
-Ala11,Phe13,Nle14]Bn-(6-14),
and three classes of low affinity Bn receptor antagonists that also
function as hBRS-3 receptor antagonists should prove valuable in the
search for the natural ligand for hBRS-3 and in further studies
investigating its importance in physiological and pathological
processes.