Ability of Various Bombesin Receptor Agonists and Antagonists to Alter Intracellular Signaling of the Human Orphan Receptor BRS-3*

Richard R. RyanDagger , H. Christian WeberDagger , Wei HouDagger , Eduardo Sainz§, Samuel A. ManteyDagger , James F. Battey§, David H. Coy, and Robert T. JensenDagger parallel

From the Dagger  Digestive Diseases Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, § NIDCD, National Institutes of Health, Rockville, Maryland 20892, and  Peptide Research Laboratories, Tulane University Medical Center, New Orleans, Louisiana 70112

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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,beta -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,beta -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,beta -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
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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,beta -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,beta -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,beta -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,beta -Ala11,Phe13,Nle14]Bn-(6-14), which is an agonist.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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,beta -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 (psi ) for [D-Phe6,Leu13,psi (CH2NH)-D-Phe14]Bn-(6-14) and [(3-Ph-Pr6)-His7,D-Ala11,D-Pro13,psi (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,beta -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,beta -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
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

We first examined the effect of [D-Phe6,beta -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,beta -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,beta -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,beta -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,beta -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,beta -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,beta -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,beta -Ala11,Phe13,Nle14]Bn-(6-14) was 2919 ± 83 and 34,895 ± 8907 dpm, respectively.

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,beta -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,beta -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,beta -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,beta -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,beta -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).

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,beta -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,beta -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,beta -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,beta -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,beta -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,beta -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,beta -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,beta -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,beta -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,beta -Ala11,Phe13,Nle14]Bn-(6-14) stimulated values were 9303 ± 1059 and 38,015 ± 4167 dpm, respectively (n = 3).

We next explored the effects of various GRP receptor antagonists (57) and NMB receptor antagonists (57, 58) on the ability of [D-Phe6,beta -Ala11,Phe13,Nle14]Bn-(6-14) to stimulate [3H]IP release in hBRS-3-transfected NCI-H1299 cells. [D-Arg11,beta -Trp7,9,Leu11]SP, [D-Pro4,D-Trp7,9,10]SP-(4-11), [(3-Ph-Pr6)-His7,beta -Ala11,D-Pro13,psi (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,beta -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,beta -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,beta -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,beta -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,beta -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.

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,beta -Ala11,Phe13,Nle14]Bn-(6-14) (Fig. 5). [D-Phe6,beta -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,beta -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,beta -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,beta -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 Delta dpm ± S.E. (dpm in the presence of 100 nM [D-Phe6,beta -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).

To examine the ability of hBRS-3 receptor activation to cause changes in cytosolic calcium ([Ca2+]i), we examined the effect of [D-Phe6,beta -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,beta -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,beta -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,beta -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,beta -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,beta -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,beta -Ala11,Phe13,Nle14]Bn-(6-14) in the presence (bullet ) or absence (open circle ) 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,beta -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,beta -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.

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,beta -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,beta -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,beta -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,beta -Ala11,Phe13,Nle14]Bn-(6-14) (Fig. 9). The phosphorylation induced by [D-Phe6,beta -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,beta -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,beta -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,beta -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,beta -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,beta -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,beta -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,beta -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,beta -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.

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,beta -Ala11,Phe13,Nle14]Bn-(6-14) (Fig. 11). In hBRS-3-transfected BALB 3T3 cells, both [D-Phe6,beta -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,beta -Ala11,Phe13,Nle14]Bn-(6-14). Thapsigargin (100 nM), when added 30 min prior to assay, completely inhibited the ability of [D-Phe6,beta -Ala11,Phe13,Nle14]Bn-(6-14) to stimulate [Ca2+]i (Fig. 11, inset), but failed to inhibit [D-Phe6,beta -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,beta -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,beta -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,beta -Ala11,Phe13,Nle14]Bn-(6-14) (Exp) to that in the absence of [D-Phe6,beta -Ala11,Phe13,Nle14]Bn-(6-14) (Con). Inset, effect of thapsigargin on [D-Phe6,beta -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,beta -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.

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,beta -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,beta -Ala11,Phe13,Nle14]Bn-(6-14), vasopressin, and epinephrine had no stimulatory effect. We also examined the ability of [D-Phe6,beta -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,beta -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,beta -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,beta -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.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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,beta -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,beta -Ala11,Phe13,Nle14]Bn-(6-14) in either the [3H]IP or [Ca2+]i assay; however, [D-Phe6,beta -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,beta -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,beta -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,beta -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,beta -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,beta -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,beta -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,beta -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,beta -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,beta -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,beta -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,beta -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,beta -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.

    FOOTNOTES

* 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.

parallel To whom correspondence and reprint requests should be addressed: NIH/NIDDK/DDB, Bldg. 10, Rm. 9C-103, 10 Center Dr., MSC 1804, Bethesda, MD 20892-1804. Tel.: 301-496-4201; Fax: 301-402-0600.

1 The abbreviations used are: Bn, bombesin; GRP, gastrin-releasing peptide; NMB, neuromedin B; PKC, protein kinase C; SP, substance P; DMEM, Dulbecco's modified Eagle's medium; IP, inositol phosphate; BB4, bombesin receptor subtype 4; PBS, phosphate-buffered saline; FBS, fetal bovine serum; mAb, monoclonal antibody; TPA, 12-O-tetradecanoylphorbol-13-acetate; BSA, bovine serum albumin.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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