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

(Received for publication, July 22, 1997, and in revised form, January 19, 1998)

Richard R. Ryan‡, H. Christian Weber‡, Wei Hou‡, Eduardo Sainz§, Samuel A. Mantey‡,James F. Battey§, David H. Coy¶, and Robert T. Jensen‡i

From the ‡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

Bombesin (Bn) receptor subtype 3 (BRS-3) is an or-phan receptor that is a predicted member of the hepta-helical G-protein receptor family and so named becauseit shares a 50% amino acid homology with receptors forthe mammalian bombesin-like peptides neuromedin B(NMB) and gastrin-releasing peptide. In a recent tar-geted disruption study, in which BRS-3-deficient micewere generated, the mice developed obesity, diabetes,and hypertension. To date, BRS-3’s natural ligand re-mains unknown, its pharmacology unclear, and cellularbasis of action undetermined. Furthermore, there arefew tissues or cell lines found that express sufficientlevels of BRS-3 protein for study. To define the intracel-lular signaling properties of BRS-3, we examined theability of [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14), a newlydiscovered peptide with high affinity for BRS-3, andvarious Bn receptor agonists and antagonists to altercellular function in hBRS-3-transfected BALB 3T3 cellsand hBRS-3-transfected NCI-H1299 non-small cell lungcancer cells, which natively express very low levels ofhBRS-3. This ligand stimulated a 4–9-fold increase in[3H]inositol phosphate formation in both cell lines un-der conditions where it caused no stimulation in un-transfected cells and also stimulated an increase in[3H]IP1, [3H]IP2, and 3H]IP3. The elevation of [3H]IP wasconcentration-dependent, with an EC50 of 20–35 nM inboth cell lines. [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14)stimulated a 2–3-fold increase in [Ca21]i, a 3-fold in-crease in tyrosine phosphorylation of p125FAK with anEC50 of 0.2–0.7 nM, but failed to either stimulate in-creases in cyclic AMP or inhibit forskolin-stimulatedincreases. None of nine naturally occurring Bn peptidesor three synthetic Bn analogues reported to activatehBRS-3 did so with high affinity. No high affinity Bnreceptor antagonists had high affinity for the hBRS-3receptor, although two low affinity antagonists for gas-trin-releasing peptide and NMB receptors, [D-Arg1,D-Trp7,9,Leu11]substance P and [D-Pro4,D-Trp7,9,10]substanceP-(4–11), inhibited hBRS-3 receptor activation. The NMBreceptor-specific antagonist D-Nal,Cys,Tyr,D-Trp,Lys,Val,Cys,Nal-NH2 inhibited hBRS-3 receptor activation in acompetitive fashion (Ki 5 0.5 mM). Stimulation of p125FAK

tyrosine phosphorylation by hBRS-3 activation was notinhibited by the protein kinase C inhibitor, GF109203X, orthapsigargin, alone or in combination. These results showthat hBRS-3 receptor activation increases phospholipase

C activity, which causes generation of inositol phosphatesand changes in [Ca21]i and is also coupled to tyrosinekinase activation, but is not coupled to adenylate cyclaseactivation or inhibition. hBRS-3 receptor activation re-sults in tyrosine phosphorylation of p125FAK, and it is notdependent on activation of either limb of the phospho-lipase C cascade. Although the natural ligand is not aknown bombesin-related peptide, the availability of[D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14), which functions asa high affinity agonist in conjunction with hBRS-3-trans-fected cell lines and the recognition of three classes ofreceptor antagonists including one with affinity of 0.5 mM,should provide important tools to assist in the identifica-tion of its natural ligand, the development of more potentselective receptor antagonists and agonists, and furtherexploration of the signaling properties of the hBRS-3receptor.

The mammalian bombesin (Bn)1-like peptides gastrin-re-leasing peptide (GRP) and neuromedin B (NMB) contribute todiverse 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 ofpancreatic secretion (6), stimulation of gastrointestinal hor-mone release (7–9), and macrophage activation (10). Thesepeptides also have important developmental effects (11, 12)and potent growth effects (13–15), causing proliferation of nor-mal cells (13, 14, 16, 17) and various tumor cell lines (15, 16,18–20). To date, two mammalian receptor subtypes and theirligands have been identified, each of which has an architecturesuggesting they are members of the heptahelical G-proteincoupled receptor superfamily (21–23). One subtype, the GRPreceptor, exhibits selectivity for GRP (21, 22, 24–26), whereasthe other, the NMB receptor, has selectivity for NMB (23, 26,27). The intracellular signaling pathways of these two recep-tors have been characterized, with ligand binding resulting instimulation of phospholipase C (14, 28–30), protein kinase Cactivation (14), [Ca21]i mobilization (14, 29, 30), and tyrosinephosphorylation of various intracellular proteins (31–34).

Recently, it has been proposed that an orphan receptor mayrepresent a third type of mammalian bombesin receptor (35,36). This 399-amino acid protein, which was later identified inhuman tissues (35), was named bombesin receptor subtype-3(BRS-3), due to its 51% and 47% amino acid sequence homology

* The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked“advertisement” in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.

i 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-releasingpeptide; 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-tetra-decanoylphorbol-13-acetate; BSA, bovine serum albumin.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 273, No. 22, Issue of May 29, pp. 13613–13624, 1998Printed in U.S.A.

This paper is available on line at http://www.jbc.org 13613

by guest on April 9, 2019

http://ww

w.jbc.org/

Dow

nloaded from

to the GRP receptor and the NMB receptor, respectively. Asopposed to GRP receptors and NMB receptors, which havewidespread distribution in the central nervous system andperipheral tissues (1, 37, 38), BRS-3 has a pattern of expressionlimited to secondary spermatocytes (35), pregnant uterus (36),a few brain regions (36), and human lung (35), breast (39), andepidermal cancer cell lines (39). In a recent study in whichBRS-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 ofglucose metabolism, energy balance, and maintenance of bloodpressure (40). To date, the natural ligand for BRS-3 is undis-covered, and its intracellular signaling mechanisms are largelyunknown. This uncertainty has occurred because no cells havebeen identified that natively express sufficient numbers ofBRS-3 receptors to allow study of intracellular coupling, and nohigh affinity ligands have been discovered for this receptor. Itis known, however, that when the BRS-3 receptor was ex-pressed in Xenopus oocytes (35) or transfected into BALB 3T3fibroblasts (41), high concentrations of various natural (35, 36)or synthetic bombesin analogues (41) could promote changes inintracellular calcium.

To overcome these problems, we have recently used twodifferent strategies to create two different cell lines stablyexpressing this receptor. These cell lines were used to screenvarious bombesin peptides for their ability to activate phospho-lipase C and a synthetic analogue of bombesin, [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14), was identified which has high affinityfor the BRS-3 receptor (42). Using an analogue of this peptide thatcould be radiolabeled, [D-Tyr6,b-Ala11,Phe13,Nle14]Bn-(6–14), wedemonstrated that both cells lines stably expressing the BRS-3receptor share the same unique pharmacology for naturally occur-ring bombesin peptides and a high affinity for [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14) (42).

The purpose of the present study was to examine the intra-cellular signaling mechanisms of the BRS-3 receptor using thehigh affinity ligand [D-Phe6,b-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 CancerInstitute-Navy Medical Oncology Branch, Naval Medical Center, Be-thesda, MD. BALB 3T3 cells were obtained from ATCC, Rockville, MD;Dulbecco’s minimum essential medium (DMEM), RPMI 1640, Dulbec-co’s phosphate-buffered saline (PBS), fetal bovine serum (FBS), G418sulfate, 12-O-tetradecanoylphorbol-13-acetate (TPA), and Tris/HClwere from Life Technologies, Inc.; formic acid, ammonium formate,disodium tetraborate, EDTA, EGTA, and soybean trypsin inhibitorwere from Sigma; phenylmethylsulfonyl fluoride was from Fluka,Ronkonkoma, NY; bovine serum albumin (BSA) fraction V was fromICN Biomedicals Inc., Aurora, OH; aprotinin and HEPES were fromBoehringer Mannheim; AG 1-X8 resin, thapsigargin, and GF109203Xwere from Bio-Rad; monobasic sodium phosphate was from Mallinck-rodt; myo-[2-3H]inositol was from NEN Life Science Products;[2-3H]adenine was from Amersham Pharmacia Biotech; anti-phospho-tyrosine mAb PY20 and anti-FAK mAb were from Transduction Labo-ratories, Lexington, KY; horseradish peroxidase-conjugated secondaryantibody was from Pierce; bombesin, gastrin-releasing peptide, neuro-medin B, rhodei-litorin, phyllolitorin, ranatensin, and litorin were fromBachem, Torrance, CA; [D-Arg1,D-Trp7,9,Leu11]substance P and[D-Pro4,D-Trp7,9,10]substance P-(4–11) were from Peninsula Laborato-ries, 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,b-Ala11,Phe13,Nle14]Bn-(6–14) were giftsfrom John Taylor of Biomeasure, Inc., Milford, MA. All other chemicalswere reagent grade.

Preparation of Peptides—The peptides were synthesized by solid-phase methods as reported previously (45–47). Introduction of thereduced peptide bond (c) for [D-Phe6,Leu13,c(CH2NH)-D-Phe14]Bn-(6–14) and [(3-Ph-Pr6)-His7,D-Ala11,D-Pro13,c(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 synthe-sized in a standard Leu-O-polystyrene resin using tosyl group protec-tion for the imidazole group of His and cleavage of the desired analoguewith 10% propylamine in methanol. [D-Phe6]Bn-(6–13) methyl esterwas also prepared with Leu-O-polystyrene, and free peptide ester wasremoved from the resin by transesterification with 10% triethylamine/methanol at 40 °C for 48 h. The peptides were first purified on aSephadex G-25 column (2.5 3 90 cm) followed by preparative highperformance liquid chromatography on a Vydac C18 column (1.5 3 50cm, 10–15-mm bore size) (45–47). After rechromatography to achieve$97% purity, the peptides were characterized by amino acid analysisand matrix-assisted laser desorption mass spectroscopy (Finnegan,Hemel Hemstead, United Kingdom).

Preparation and Maintenance of Transfected Cells—NCI-H1299 cellsexpressing stably transfected hBRS-3 were obtained using Lipo-fectAMINE (Life Technologies, Inc.) to introduce human BRS-3 cDNAcontaining an NH2-terminal flag epitope tag (59-GACTACAAGGAC-GACGATGACAAG-39) subcloned into the expression vectors pCD2 andpcDNA3 as described previously (42). hBRS-3-transfected BALB 3T3fibroblasts were transfected using the calcium phosphate precipitationmethod (48) as described previously (42). Both wild-type BALB 3T3cells and NCI-H1299 cell lines were grown in DMEM and RPMI 1640respectively, supplemented with 10% FBS. The transfected cells weregrown in their respective propagation media supplemented with 300mg/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 3 104 cells/well) in their respective propagationmedia. Total [3H]IP was determined as described previously (30, 49).Briefly, after a 24-h incubation period at 37 °C, the cells were incubatedwith 3 mCi/ml myo-[2-3H]inositol in growth medium supplemented with2% FBS for an additional 24 h. Incubation volumes were 500 ml of assaybuffer/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 withoutany of the peptides studied at 37 °C for 30 min. To determine the timecourse of [3H]IP1, [3H]IP2, and [3H]IP3 formation, 75-cm2 flasks ofconfluent hBRS-3-transfected BALB 3T3 cells labeled with myo-[2-3H]inositol were scraped, centrifuged (300 3 g, 10 min), resuspended in10 ml of PBS with 20 mM lithium chloride, and incubated for 10 min(37 °C). After resuspension in 20 ml of assay buffer, 500-ml aliquots ofcell suspension were added to tubes containing 100 nM [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14) and incubated at 37 °C for the indicatedtimes. Experiments were terminated with 1 ml of ice-cold hydrochloricacid/methanol (0.1%, v/v). [3H]IP1, [3H]IP2 and [3H]IP3 were eluted offDowex AG-1-X8 anion exchange columns as described previously (50).In the experiments where total [3H]IP was measured, total [3H]IP waseluted with 2 ml of 1 mM ammonium formate and 100 mM formic acid asdescribed previously (30, 51). Each of the eluates was collected andmixed with 10 ml of Hydrofluor scintillation mixture (National Diag-nostics, Atlanta, GA), and the radioactivity was measured in a scintil-lation counter.

Intracellular Calcium ([Ca21]i)—Cells harvested by scraping wereresuspended in an assay buffer (24.5 mM HEPES (pH 7.4), 98 mM

sodium chloride, 6 mM potassium chloride, 2.5 mM monobasic sodiumphosphate, 5 mM sodium pyruvate, 5 mM sodium fumarate, 5 mM

sodium glutamate, 2 mM glutamine, 11.5 mM glucose, 1.45 mM calciumchloride, 1.15 mM magnesium chloride, 0.01% soybean trypsin inhibitor,0.2% (v/v) amino acid mixture, and 0.2% BSA) to a concentration of 5 3106 cells/ml and incubated with 2 mM Fura-2/AM (Molecular Probes,Eugene, OR) for 30 min at 37 °C. After washing two times with assaybuffer, 2 ml of cell suspension were placed in a Delta PTI Scan 1spectrofluorimeter (Photon Technology International, South Brun-swick, NJ) equipped with a stir bar and water bath (37 °C). Fluores-cence was measured at dual excitation wavelengths of 340 nm and 380nm using an emission wavelength of 510 nm. The calcium concentrationwas calculated using the method of Grynkiewicz et al. (52).

Immunoprecipitation of Tyrosine-phosphorylated Proteins—Quies-cent and confluent hBRS-3-transfected BALB 3T3 cells and NCI-H1299cells grown on 100-mm culture dishes were preincubated twice withDMEM for 1 h, treated with the indicated concentrations of [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14) or GRP for 10 min at 37 °C, and lysed at4 °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) sodiumazide, 1 mM EGTA, 0.4 mM EDTA, 2.5 mg/ml aprotinin, 2.5 mg/mlleupeptin, 1 mM phenylmethylsulfonyl fluoride, and 0.2 mM sodiumvanadate. After centrifugation (15,000 3 g, 15 min) and adjustment to

Cell Signaling by the BRS-3 Receptor13614

by guest on April 9, 2019

http://ww

w.jbc.org/

Dow

nloaded from

a protein concentration of 0.5 mg/ml (Bio-Rad protein assay), the su-pernatants were immunoprecipitated with 4 mg of anti-phosphotyrosinemAb PY20, 4 mg of goat anti-mouse IgG, and 30 ml of protein A-agarose(Upstate Biotechnology, Inc., Lake Placid, NY) overnight at 4 °C. Theimmunoprecipitates were washed three times with PBS and furtheranalyzed by SDS-polyacrylamide gel electrophoresis and Westernblotting.

Western blotting and Measurement of p125FAK Tyrosine Phosphoryl-ation—Briefly, p125FAK tyrosine phosphorylation was determined asdescribed recently (53). Immunoprecipitates were separated by SDS-polyacrylamide gel electrophoresis, and the proteins were electro-phoretically transferred to nitrocellulose membranes. After blockingovernight 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 incubatedwith anti-p125FAK mAb (1:1000 in blocking solution) for 2 h at 25 °C.After washing with blotting solution, the membranes were incubated at25 °C with horseradish peroxidase-conjugated secondary antibody (1:10,000 in blocking solution) for 30 min. Immunoreactive proteins weredetected using the ECL detection system (Pierce). Quantitation ofp125FAK tyrosine phosphorylation was obtained by scanning densitom-etry (Molecular Devices, Sunnyvale, CA).

Statistical Analysis—Data plotting and iterative curve-fitting wasperformed with KaleidaGraph graphing software (Synergy Software,Reading, PA). Analysis of Schild plots (54) and statistical analysis of thedata were performed using Statview version 1.01 (BrainPower, Inc.,Calabasas, CA). Student’s t test was used to determine the statisticalsignificance between group means.

Determination of Changes in Cyclic AMP—Quiescent and confluenthBRS-3-transfected BALB 3T3 cells or NCI-H1299 cells grown on 24-well plates were incubated with 500 ml of their respective media(DMEM or RPMI 1640), which was supplemented with 2% FBS (v/v)and 2 mCi/ml [3H]adenine for 24 h at 37 °C. The medium was removed,replaced with an equivalent volume of serum-free medium, and allowedto incubate for 15 min at 37 °C. The medium was removed and replacedwith an equivalent volume of serum-free medium containing 1% BSA(w/v), 0.5 mM isobutylmethylxanthine, and the indicated agents at theirindicated concentrations. The reactions were terminated by the addi-tion of 100 ml of stopping solution (2% SDS (v/v), 5 mM cAMP) followedby 900 ml of ice-cold Tris (50 mM, pH 7.4). Samples were stored at220 °C until analyzed.

Determination of the amount of cAMP formation was obtained usinga modified protocol described by Salomon et al. (55). Frozen samples ofhBRS-3-transfected BALB 3T3 cells or NCI-H1299 cells were thawedand added to glass columns containing 1 ml of l:1 (v/v) slurry of DowexAG1-X8 anion exchange resin, which had been washed once with 4 mlof 1 N sodium hydroxide, once with 4 ml of 1 N hydrochloric acid, andtwice with 10 ml of deionized water. The columns were washed with 1ml of deionized water after addition of sample and stacked over anotherset of glass columns containing 1 g of alumina, which had been previ-ously 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 toeach alumina column. The eluate was collected and mixed with Hy-drofluor scintillation fluid, and the radioactivity was counted.

RESULTS

We first examined the effect of [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14), GRP, and Bn on the release of [3H]i-nositol phosphates ([3H]IP) in both untransfected and hBRS-3-transfected cell lines. [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14),GRP, and Bn elicited a small, but significant elevation of [3H]IPin untransfected NCI-H1299 cells (Table I). This result is con-sistent with previous studies, which demonstrate that thesecells possess a low density of hGRP and hNMB receptors (35).The hBRS-3-transfected NCI-H1299 cells exhibited a more ro-bust response to [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14) at 10nM and with each of the three peptides at 1000 nM. The un-transfected BALB 3T3 cells, which do not possess Bn receptors,failed to respond to any of these peptides (Table I). In thehBRS-3-transfected BALB 3T3 cells, only [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14) was capable of eliciting [3H]IP re-lease at both 10 and 1000 nM (Table I).

The results in Table I indicated that in NCI-H1299 cells, butnot in BALB 3T3 cells, there were low levels of native Bnreceptors (GRP receptor, NMB receptor, or both) that could bestimulated to cause detectable increases in [3H]IP by variousBn receptor agonists that could confuse the results. Recentstudies (42, 46, 56, 57) show that the potent GRP receptorantagonist [D-Phe6]Bn-(6–13) methyl ester has low affinity forthe NMB receptor and the hBRS-3 receptor. Therefore, to as-sess the extent of GRP receptor interaction with [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14) in NCI-H1299 cells, we comparedthe ability of [D-Phe6]Bn-(6–13) methyl ester to inhibit a sub-maximal dose of [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14) in thehBRS-3-transfected cell lines and a submaximal dose of GRP inthe mGRP receptor-transfected BALB 3T3 cells (Fig. 1). In themGRP receptor transfectants, [D-Phe6]Bn-(6–13) methyl esterattenuated [3H]IP formation by GRP in a concentration-de-pendent manner. Half-maximal inhibition was observed at2.8 6 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, demon-strating 30% of the maximal stimulation was due to occupationof 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).

TABLE IAbility of GRP, NMB, and [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14) to alter [3H]IP in native and hBRS-3-transfected NCI-H1299 cells and

BALB 3T3 cellsNative nontransfected NCI-H1299 cells, BALB 3T3 cells, or hBRS-3-transfected cell lines were incubated with GRP, Bn, or [D-Phe6,b-

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 ofpeptide (Exp) to that generated in the absence of peptide (Con). Each value represents the means 6 S.E. of at least three experiments performedin duplicate. The control value in NCI-H1299 nontransfected and hBRS-3-transfected cells was 1521 6 237 and 1600 6 152 dpm, and with 1000nM [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14) was 2637 6 420 and 14,810 6 2053 dpm, respectively. The control value in BALB 3T3 nontransfectedand hBRS-3-transfected cells was 2911 6 227 and 10,473 6 2700 dpm and with 1000 nM [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14) was 2919 6 83 and34,895 6 8907 dpm, respectively.

Cell line

Total [3H]IP (Exp/Con)

GRP Bn [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14)

10 nM 1000 nM 10 nM 1000 nM 10 nM 1000 nM

I. NCI-H1299 cellsNon-transfected 1.1 6 0.2 1.5 6 0a,c 1.2 6 0.1 1.4 6 0.1b 1.5 6 0.1a 1.7 6 0.1a

hBRS-3-transfected 1.2 6 0.1 2.3 6 0.3b,c 1.7 6 0.3a 2.8 6 0.2a,c 3.6 6 0.9a 9.2 6 0.5a,d

II. BALB 3T3 cellsNon-transfected 0.9 6 0.1 0.9 6 0.1 0.9 6 0.1 1.0 6 0.1 1.0 6 0.1 1.0 6 0.1hBRS-3-transfected 0.9 6 0 1.1 6 0 0.9 6 0 1.0 6 0 1.9 6 0.2a 3.3 6 0a,d

a Significantly greater (p , 0.01) than untreated cells.b Significantly greater (p , 0.05) than untreated cells.c Significantly greater (p , 0.05) than 10 nM same peptide.d Significantly greater (p , 0.01) than 10 nM same peptide.

Cell Signaling by the BRS-3 Receptor 13615

by guest on April 9, 2019

http://ww

w.jbc.org/

Dow

nloaded from

Because 100 nM [D-Phe6]Bn-(6–13) methyl ester caused.95% inhibition at the GRP receptor, and this concentrationhas been previously shown not to interact with the BRS-3receptor (42), we included this concentration in all studies inthe hBRS-3-transfected NCI-H1299 cells to inhibit any stimu-lation by the GRP receptor agonists. We next investigated theconcentration dependence of [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14) and various Bn-related peptides to elicit [3H]IP forma-tion in hBRS-3-transfected BALB 3T3 cells and hBRS-3-trans-fected NCI-H1299 cells (Fig. 2). In hBRS-3-transfected BALB3T3 cells and hBRS-3-transfected NCI-H1299 cells, [D-Phe6,b-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 6 2.1 and 35 6 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 untilconcentrations .10 nM in transfected NCI-H1299 cells (Fig. 2).We examined six other natural Bn-related peptides for agonistactivity, and as shown in Table II, none of these peptidescaused a detectable response with concentrations less than 300nM. Three synthetic peptides reported to have agonist proper-ties against BRS-3 (41) were also evaluated. Two of these,AcNMB-(3–10) and [D-Phe6]Bn-(6–13) propylamide, stimu-lated [3H]IP accumulation in a concentration-dependent man-ner, causing a detectable effect at 219 6 63 and 892 6 71 nM

(Table II) and causing a half-maximal effect at 333 6 81 nM and1924 6 146 nM, respectively (Fig. 3). The efficacy of AcNMB-(3–10), however, was only 80% of that seen with [D-Phe6,b-

Ala11,Phe13,Nle14]Bn-(6–14) (Fig. 3). The third synthetic ana-logue, [D-Phe6, Phe13]Bn-(6–13) propylamide, was the leastpotent, eliciting a detectable increase in [3H]IP only with con-centrations .1000 nM (Table II, Fig. 3).

We next explored the effects of various GRP receptor antag-onists (57) and NMB receptor antagonists (57, 58) on the abilityof [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14) to stimulate [3H]IPrelease in hBRS-3-transfected NCI-H1299 cells. [D-Arg11,b-Trp7,9,Leu11]SP, [D-Pro4,D-Trp7,9,10]SP-(4–11), [(3-Ph-Pr6)-His7,b-Ala11,D-Pro13,c(13–14),Phe14]Bn-(6–14)-NH2, and theselective NMB receptor antagonist D-Nal,Cys,Tyr,D-Trp,Lys,Val,Cys,Nal-NH2 (58) were capable of attenuating [D-Phe6,b-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). Theinhibition caused by the substance P analogues and D-Nal,Cy-s,Tyr,D-Trp,Lys,Val,Cys,Nal-NH2 was examined in detail andfound to be concentration-dependent (Fig. 4). Of these three,D-Nal,Cys,Tyr,D-Trp,Lys,Val,Cys,Nal-NH2 exhibited the high-est potency with half-maximal inhibition at 1.1 6 0.3 mM. In theBALB 3T3 cells, D-Nal,Cys,Tyr,D-Trp,Lys,Val,Cys,Nal-NH2

caused a rightward parallel shift of the [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14) dose-response curve for stimula-tion of [3H]IP (data not shown). Plotting these data in the formof Schild (54) gave an equation with a slope of 0.94, which wasnot significantly different from unity. Calculation of the affin-ity of D-Nal,Cys,Tyr,D-Trp,Lys,Val,Cys,Nal-NH2 for thehBRS-3 receptor from these data gave an affinity of 504 6 146nM.

To determine the ability of the hBRS-3 receptor to increasedifferent isomers of inositol phosphates, we examined the timecourse of [3H]IP1, [3H]IP2, and [3H]IP3 formation in the hBRS-3-transfected BALB 3T3 cells stimulated with 100 nM

[D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14) (Fig. 5). [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14) stimulated a time-dependent in-crease in [3H]IP1, [3H]IP2, and [3H]IP3. The maximal increaseof [3H]IP3 was most rapidly reached. [D-Phe6,b-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 30min. The magnitude of stimulation of [3H]IP3 and [3H]IP2 bothdecreased with time, whereas [3H]IP1 stimulation continued toincrease until 60 min (Fig. 5).

To examine the ability of hBRS-3 receptor activation to causechanges in cytosolic calcium ([Ca21]i), we examined the effect of[D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14) on Fura-2/AM-loadedhBRS-3-transfected NCI-H1299 cells and BALB 3T3 cells (Fig.6). [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14) stimulated a 1.8-and 3-fold increase in [Ca21]i in hBRS-3-transfected BALB 3T3cells and hBRS-3-transfected NCI-H1299 cells, and a 2-foldincrease in [Ca21]i in the wild-type, untransfected, NCI-H1299cells (Fig. 6). The kinetics of [Ca21]i release were similar inboth transfected cell lines, reaching maximal levels in 10 s (Fig.6). The GRP receptor antagonist, [D-Phe6]Bn-(6–13) methylester, completely inhibited the ability of [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14) to elicit changes in [Ca21]i in theuntransfected NCI-H1299 cells (Fig. 6, left panel) and attenu-ated the response in the transfected NCI-H1299 cells by 23%(Fig. 6, middle panel). The antagonist had no effect on theability of [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14) to stimulate[Ca21]i in transfected BALB 3T3 cells (Fig. 6, right panel). Todetermine the contribution of extracellular calcium to the[Ca21]i transient, cells were stimulated with [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14) in the presence of the Ca21-che-lating agent EGTA. In the absence of extracellular Ca21, themagnitude of the response was reduced only by 15% and thereturn to basal levels was more rapid than that seen in Ca21-containing buffer (Fig. 7).

FIG. 1. Ability of [D-Phe6]Bn-(6–13) methyl ester to inhibit[3H]IP formation by [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14) orGRP in cells transfected with receptors for Bn-like peptides.BALB 3T3 cells were transfected with the hBRS-3 or the mGRP recep-tor, 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 indi-cated concentrations for 45 min with either 30 nM [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14) for the hBRS-3-transfected cells or 30 nM

GRP for the mGRP receptor-transfected cells. Values represent thepercent of total [3H]IP release stimulated by 30 nM [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14) or GRP alone and are the means 6 S.E.from at least three separate experiments from duplicate determina-tions. The basal values were 2632 6 808, 8544 6 647, and 465 6 148dpm for the mGRP receptor-transfected BALB 3T3 cells, hBRS-3-trans-fected BALB 3T3 cells, and hBRS-3-transfected NCI-H1299 cells, re-spectively. Stimulated control values were 6261 6 906, 20,432 6 3634,and 2900 6 945 dpm for the mGRP receptor-transfected BALB cells,hBRS-3-transfected BALB 3T3 cells, and hBRS-3-transfected H1299cells, respectively (n 5 3).

Cell Signaling by the BRS-3 Receptor13616

by guest on April 9, 2019

http://ww

w.jbc.org/

Dow

nloaded from

Recent studies have demonstrated that a number of neu-ropeptides, without intrinsic tyrosine kinase activity, can stim-ulate tyrosine phosphorylation of a number of proteins includ-ing the cytosolic focal adhesion kinase (p125FAK) (32, 33, 59–63). Therefore, we studied the effect of [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14) on tyrosine phosphorylation ofp125FAK. 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,b-Ala11,Phe13,Nle14]Bn-(6–14) stimulated a 3- and 3.5-fold increase in tyrosine phosphorylation of p125FAK, respec-tively. In hBRS-3-transfected BALB 3T3 cells, this increasewas unaffected by the GRP receptor antagonist [D-Phe6]Bn-(6–13) methyl ester (Fig. 8). Furthermore, in these cells, GRPfailed to stimulate p125FAK phosphorylation (Fig. 8). In thehBRS-3-transfected NCI-H1299 cells, both 100 nM GRP and100 nM [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14) stimulatedp125FAK 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,b-Ala11,Phe13,Nle14]Bn-(6–14) (Fig. 9). The phosphorylation in-duced by [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14) was concen-tration-dependent in both hBRS-3-transfected NCI-H1299 cellsand hBRS-3-transfected BALB cells (Fig. 10). A maximal 4-foldstimulation in both transfected cell lines was obtained with 100nM [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14), and the EC50 values

FIG. 2. Effect of [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14) and naturalBn-related peptides on [3H]IP forma-tion in BALB 3T3 cells and NCI-H1299cells 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,b-Ala11,Phe13,Nle14]Bn-(6–14), Bn, GRP, orNMB for 45 min at the indicated concentra-tions. For the hBRS-3-transfected NCI-H1299 cells, 100 nM [D-Phe6]Bn-(6–13)methyl ester was included. Values are ex-pressed as a percent of the total [3H]IP re-lease stimulated by 1 mM [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14), and are themeans 6 S.E. from at least three experi-ments performed in duplicate. The basal val-ues for the hBRS-3-transfected BALB 3T3cells and hBRS-3-transfected NCI-H1299cells were 9015 6 900 and 1530 6 366 dpm,and the stimulated values for the hBRS-3-transfected BALB 3T3 cells and hBRS-3-transfected NCI-H1299 cells were 39,750 64213 and 14,525 6 3729 dpm, respectively(n 5 3).

TABLE IIAbility of various synthetic and naturally occurring Bn-related

peptides to stimulate [3H]IP generation in hBRS-3-transfected BALB3T3 cells

BALB 3T3 cells transfected with hBRS-3 were incubated with variousconcentrations of each peptide (0.1 nM to 10 mM) for 45 min. Results areexpressed as the concentration of peptide capable of stimulating adetectable response equal to 30% of the maximal response seen with1000 nM [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14), and are the means 6S.E. from at least three experiments. The control and maximal stimu-lated [3H]IP values were 6104 6 888 and 21,897 6 3704 dpm, respec-tively. Abbreviations: SAP-Bn, [Ser3,Arg10,Phe13]bombesin (44); FrogGRP-10, frog gastrin-releasing peptide COOH-terminal decapeptide,[Ser19]GRP-(18–27) (43); AcNMB-(3–10), acetyl-neuromedin B-(3–10).

Agonist Total [3H]IP potency

nM

[D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14) 8.6 6 1.2Neuromedin B 4848 6 572Bombesin 5955 6 146Gastrin-releasing peptide 5045 6 467Phyllolitorin 1787 6 367Litorin 475 6 88Rhodei-litorin 604 6 19[Phe13]Bn 3386 6 19SAP-Bn .10,000Frog GRP-10 .10,000[D-Phe6]Bn-(6–13)propylamide 892 6 71[D-Phe6,Phe13]Bn-(6–13)propylamide .10,000AcNMB-(3–10) 219 6 63

FIG. 3. Effect of synthetic Bn analogues on [3H]IP formation inBALB 3T3 cells transfected with hBRS-3 receptor. Cells wereincubated with [D-Phe6,b-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 asa percent of total [3H]IP release stimulated by 1 mM [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14), and are the means 6 S.E. from at leastthree experiments performed in duplicate. The control and 1 mM

[D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14) stimulated values were 9303 61059 and 38,015 6 4167 dpm, respectively (n 5 3).

Cell Signaling by the BRS-3 Receptor 13617

by guest on April 9, 2019

http://ww

w.jbc.org/

Dow

nloaded from

for hBRS-3-transfected BALB 3T3 and NCI-H1299 cells were0.7 6 0.2 nM and 0.2 6 0.1 nM, respectively.

To explore the relationship between the ability of hBRS-3receptor activation to stimulate phospholipase C activity re-sulting in protein kinase C (PKC) activation and changes in[Ca21]i and its ability to stimulate changes in p125FAK phos-phorylation, we examined the effect of the selective PKC inhib-itor GF109203X (64) and the Ca21/ATPase inhibitor thapsigar-gin (65) on tyrosine phosphorylation of p125FAK stimulated by[D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14) (Fig. 11). In hBRS-3-transfected BALB 3T3 cells, both [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14) (100 nM) and TPA (100 nM) stim-ulated a 3.5-fold increase in tyrosine phosphorylation ofp125FAK (Fig. 11). GF109203X (5 mM) abolished the effect ofTPA but did not diminish the p125FAK phosphorylation seenwith [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14). Thapsigargin(100 nM), when added 30 min prior to assay, completely inhib-ited the ability of [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14) tostimulate [Ca21]i (Fig. 11, inset), but failed to inhibit [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14)-induced p125FAK phosphorylation(Fig. 11). Furthermore, the combination of GF109203X andthapsigargin had had no effect on phosphorylation alone andhad no significant effect on tyrosine phosphorylation ofp125FAK induced by hBRS-3 receptor activation (Fig. 11).

Since it had been shown previously (66, 67) that activation ofthe hBRS-3-related receptor, the GRP receptor in Swiss 3T3fibroblasts, could stimulate increases in cyclic AMP, the abilityof [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14) and various agonistsknown to activate adenylate cyclase via receptor activation

were studied (Table III). In the hBRS-3-transfected NCI-H1299cells, forskolin, a direct activator of adenylate cyclase, PACAP-27, and PACAP-38, stimulated a significant release of cAMP,whereas [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14), vasopressin,and epinephrine had no stimulatory effect. We also examinedthe ability of [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14) and vari-ous agonists known to attenuate stimulated adenylate cyclaseactivity via receptor activation by determining its ability toinhibit forskolin-induced cAMP formation in hBRS-3-trans-fected BALB 3T3 cells (Table IV). Neither [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14), bombesin, dopamine, nor neu-ropeptide Y at concentrations of 3 mM were able to inhibit theelevation of cAMP seen with forskolin, although 3 mM serotonincaused a significant decrease in cAMP (Table IV).

DISCUSSION

Although the BRS-3 receptor was identified several yearsago in human (35) and guinea pig (36), a lack of cell linesexpressing physiologically relevant levels of this protein, aswell as a lack of a ligand, have impeded studies of the biologicalrole of BRS-3 receptors. To gain further insight into the phar-macology of BRS-3 receptors, we created two cell lines whosesignaling sequelae and receptor coupling might resemble thoseseen in cells expressing native, functional BRS-3 receptors. Wechose BALB 3T3 fibroblasts as one candidate, because these

FIG. 4. Ability of various Bn receptor antagonists to inhibit[3H]IP formation by [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14) inhBRS-3-transfected NCI-H1299 cells. H1299 cells transfectedwith hBRS-3 receptor were treated with 100 nM [D-Phe6,b-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 for45 min. Values represent the percent of total [3H]IP release stimulatedby 100 nM [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14) (basal dpm 5 1331 6175, stimulated dpm 5 12,411 6 2096), and are the means 6 S.E. fromat least three experiments performed in duplicate.

FIG. 5. Effect of [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14) on thetime course of [3H]IP1 (top), [3H]IP2 (middle), and [3H]IP3 (bot-tom) accumulation in hBRS-3-transfected BALB 3T3 cells. Thetransfected cells were treated with 100 nM [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14) for the times indicated, and the isomerswere separated by column chromatography as described under “Mate-rials and Methods.” The values represent the mean Ddpm 6 S.E. (dpmin the presence of 100 nM [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14) minusthe unstimulated dpm obtained from at least three experiments per-formed in triplicate. The maximal counts for [3H]IP1 (30 min), [3H]IP2 (2min), and [3H]IP3 (15 s) were 42,509 6 5225, 2632 6 456, and 1552 6118 dpm, respectively, while the corresponding unstimulated controlswere 19,358 6 2602, 872 6 233, and 884 6 31 dpm, respectively (n 5 4).

Cell Signaling by the BRS-3 Receptor13618

by guest on April 9, 2019

http://ww

w.jbc.org/

Dow

nloaded from

cells are devoid of receptors for bombesin-related peptides (24,49). Furthermore, both GRP receptors and NMB receptors havebeen successfully transfected into this cell line, and exhibitintracellular signaling and a pharmacology similar to cellscontaining native mGRP, hGRP, hNMB, and rNMB receptors(24, 30, 49). However, it is unknown whether hBRS-3-trans-fected, murine BALB 3T3 cells have a signaling repertoireidentical to human cells natively expressing BRS-3 receptors.Therefore, we included the human non-small cell lung cancercell line NCI-H1299 in our study, which natively expresses lowlevels of hBRS-3 receptor (68, 69), assuming that the necessaryelements for hBRS-3 receptor coupling and intracellular sig-naling were likely extant.

Our results show that, in hBRS-3-transfected BALB 3T3cells, the new ligand [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14) in-teracts and behaves as an agonist at hBRS-3 receptors. Thisconclusion is reached because untransfected BALB 3T3 cellsdid not respond to [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14) ineither the [3H]IP or [Ca21]i assay; however, [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14) stimulated increases in both[3H]IP release and [Ca21]i in hBRS-3-transfected BALB 3T3cells, and NCI-H1299 cells under assay conditions, where GRPreceptor activity was inhibited.

A number of results demonstrate that hBRS-3 receptors arecoupled to phospholipase C activation, which in turn causesmobilization of intracellular calcium. First, we found that[D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14) elicited total [3H]IP re-lease in both hBRS-3-transfected cell lines under conditions inwhich 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, con-sistent with an increase in phospholipase C activity and sub-sequent metabolism of inositol phosphates. Third, [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14) stimulated a rapid rise of cytosoliccalcium in both hBRS-3 transfectants under conditions whereno change was seen in untransfected cells. Fourth, hBRS-3receptor-activated increases in cytosolic calcium were onlyminimally inhibited by removal of extracellular calcium, dem-onstrating release was primarily from an IP3-sensitive intra-

cellular calcium pool. The ability of BRS-3 activation to altercytosolic calcium is consistent with previous findings with theBRS-3 receptor (35, 41). When the hBRS-3 receptor was ex-pressed in Xenopus oocytes by injecting hBRS-3 sense mRNA(35), the hBRS-3 receptor coupled to calcium-activated chloridechannels, because high concentrations of GRP or NMB (10 mM)caused changes in chloride current. In contrast, uninjectedoocytes, or those injected with hBRS-3 receptor antisensemRNA, showed no changes with these peptides. Furthermore,in an earlier study, when hBRS-3 was transfected into BALB3T3 cells (41), some GRP- and NMB-related peptides causedchanges in cytosolic calcium at high concentrations.

Our studies demonstrate that like a number of other neu-ropeptide receptors (32, 33, 60–63), the hBRS-3 receptor canalso stimulate tyrosine phosphorylation of the cytosolic focaladhesion kinase (p125FAK), which in a number of cells, hasbeen shown to be required for the promotion of focal adhesionswhich are important in cell growth and motility (70, 71). In thepresence of [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14), tyrosinephosphorylation of p125FAK was stimulated in a concentration-dependent fashion in hBRS-3-transfected BALB 3T3 cells.[D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14) also caused a concen-tration-dependent increase in p125FAK tyrosine phosphoryla-tion under conditions in which the activation of GRP receptorswas completely inhibited, in hBRS-3-transfected NCI-H1299cells. This is an important control because recent studies dem-onstrate that agonist activation of the GRP receptor as well asthe NMB receptor cause rapid phosphorylation of both p125FAK

and paxillin (34, 59, 60). Because the studies were performed inthese cells under experimental conditions in which [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14) gave no stimulation of cellularfunction (changes in Ca21, [3H]IP accumulation) in untrans-fected NCI-H1299 cells, it is unlikely that activation of either ofthe other native bombesin receptors was causing the increasein p125FAK phosphorylation seen with the novel ligand inhBRS-3-transfected NCI-H1299 cells.

Recent studies show that for some G protein-coupled recep-tors 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 Ca21 or inhibition of PKC activity resulted in areduction of p125FAK tyrosine phosphorylation in the presenceof agonist. These results demonstrate that, with some G pro-tein-coupled receptors, but not others, activation of the phos-pholipase C cascade is required for stimulation of tyrosinephosphorylation of p125FAK. Furthermore, p125FAK has a PKCphosphorylation sequence (77), and activation of PKC by phor-bol ester or diacylglycerol has been shown to stimulate p125FAK

tyrosine phosphorylation (71). A number of our findings sup-port the conclusion that, with hBRS-3 receptor activation, thestimulation of tyrosine phosphorylation of p125FAK is inde-pendent of PKC activation and changes in cytosolic calcium.First, the PKC inhibitor GF109203X had no inhibitory effect on[D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14)-stimulated p125FAK ty-rosine phosphorylation under conditions where it completelyinhibited TPA-stimulated increases. Second, complete inhibi-tion of the ability of hBRS-3 activation to increase [Ca21]i bydepletion of intracellular calcium by thapsigargin failed to at-tenuate hBRS-3 receptor-mediated p125FAK tyrosinephosphorylation.

A number of studies have shown that the combination ofcalcium mobilization and PKC activation can have a synergis-tic effect on cellular responses such as amylase release frompancreatic acini (78), protein phosphorylation (79), or pepsino-gen release from chief cells (80). Our results suggest that thissynergistic effect is not important in mediating stimulation of

FIG. 6. Effect of [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14) on[Ca21]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 100nM [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14) in the presence (●) or ab-sence (E) of 3 mM [D-Phe6]Bn-(6–13) methyl ester. The figure depicts theresults from a typical experiment performed at least three times.

Cell Signaling by the BRS-3 Receptor 13619

by guest on April 9, 2019

http://ww

w.jbc.org/

Dow

nloaded from

p125FAK tyrosine phosphorylation upon hBRS-3 receptor acti-vation because the combination of GF109203X, at a concentra-tion that blocked TPA-induced p125FAK tyrosine phosphoryla-tion and thapsigargin at a concentration that inhibited[D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14)-induced increases in cy-tosolic calcium, had no effect on hBRS-3 receptor activation ofp125FAK tyrosine phosphorylation. Therefore, stimulation oftyrosine phosphorylation of p125FAK by hBRS-3 receptor acti-vation is not altered by simultaneous activation of both limbs ofthe phospholipase C cascade, which is similar to that describedfor NMB receptors (53), but not for other receptors such as forthrombin in platelets (75) or CCKA receptors in pancreaticacini (74).

A number of G protein receptors are coupled to both phos-pholipase C (81–84) and adenylate cyclase, either through Gs

or Gi. Elevation of cAMP has been associated with activation ofthe hBRS-3-related receptor, the GRP receptor in Swiss 3T3cells (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 in-

creases in cAMP. Because PACAP-27 and PACAP-38 werecapable of stimulating an increase in cAMP, it is unlikely thatthe NCI-H1299 cells possessed inadequate Gs. Therefore, thefailure of hBRS-3 receptors to mediate adenylate cyclase activ-ity in these cells cannot be explained on the basis of insufficientGs availability. In addition, forskolin stimulated a significantcAMP response in the hBRS-3-transfected NCI-H1299 cell line,demonstrating that adenylate cyclase could be directly acti-vated. The possibility that hBRS-3 receptor activation couldinhibit cAMP by coupling to Gi is also unlikely because[D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14) was incapable of inhib-iting adenylate cyclase activation by forskolin in the hBRS-3-transfected BALB 3T3 cells. These latter results support theconclusion the hBRS-3, in contrast to the closely related GRPreceptor, does not stimulate changes in cyclic AMP as a trans-duction cascade.

Based on its 51% amino acid identity to the GRP receptorand 47% amino acid identity with the NMB receptor (35), it wasproposed that BRS-3 might be related to these receptors. Theability of bombesin-related peptides to interact with the

FIG. 7. The effect of extracellularCa21 on [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14)-induced Ca21 release in hBRS-transfected BALB 3T3 cells. hBRS-3-transfected BALB 3T3 cells were loadedwith Fura-2/AM and assayed under condi-tions outlined under “Materials and Meth-ods.” The cells were stimulated with 100 nM

[D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14) inthe presence or absence of 1.45 mM EGTA.The tracing is typical of an experiment per-formed three times.

FIG. 8. Effect of [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14), GRP,and the GRP receptor antagonist[D-Phe6]Bn-(6–13) methyl ester on ty-rosine phosphorylation of p125FAK inhBRS-3-transfected BALB 3T3 cells.hBRS-3-transfected BALB 3T3 cells wereincubated with 100 nM GRP or [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14) in the pres-ence or absence of 300 nM [D-Phe6]Bn-(6–13) methyl ester. The upper panel shows atypical Western blot for p125FAK in cellstreated with (1) or without (2) the vari-ous peptides. The lower panel representsthe mean p125FAK tyrosine phosphoryla-tion expressed as the amount phosphoryl-ated in the various treatment groups com-pared with the control untreated level.Data are means 6 S.E. from at least threeexperiments.

Cell Signaling by the BRS-3 Receptor13620

by guest on April 9, 2019

http://ww

w.jbc.org/

Dow

nloaded from

hBRS-3 receptor has both similarities and differences to whatis seen with GRP and NMB receptors. First, it is similar to theGRP and NMB receptors in that it is coupled to phospholipaseC activation. In all species examined, including human, rat,and mouse tissues, both the GRP and NMB receptors arecoupled to phospholipase C (9, 14, 24, 28–30, 67). Second, itresembles both GRP and NMB receptors in its ability to stim-ulate tyrosine phosphorylation of such proteins as p125FAK.Third, it demonstrates similar stoichiometric intracellular cou-pling of these two intracellular pathways to what is seen withthe GRP and NMB receptors. With each of these receptors, thedose-response curve for the ability of agonists to stimulateinositol phosphate formation was to the right of its ability tocause tyrosine phosphorylation of p125FAK (59, 71). However,the hBRS-3 receptor and the GRP and NMB receptors differ inthe stoichiometric relationships of these two dose-responses toeach other and to receptor occupation (59, 71, 85). With each ofthese three receptors, phospholipase C activation is closelycoupled to receptor occupation, whereas submaximal receptoroccupation gives maximal p125FAK tyrosine phosphorylation(9, 53, 71, 86). These receptor subtypes differ in that the recep-tor spareness for tyrosine phosphorylation is greater for thehBRS-3 receptor than the GRP or NMB receptor.

The pharmacology of the hBRS-3 receptor, both in terms ofits affinity for agonists and many receptor antagonists, has anumber of differences from both the GRP receptor and theNMB receptor, as well as the recently described bombesinreceptor subtype 4 (BB4) (43). It differs in that none of the ninenaturally occurring peptides that are members of the bombesinfamily had a potency .500 nM for activating phospholipase Cthrough the hBRS-3 receptor. In contrast, at least four of thesepeptides interact in the nanomolar range to activate phospho-lipase C through the GRP receptor (Bn, GRP, [Phe13]Bn, andlitorin) (9, 25), two peptides with the NMB receptor (NMB,litorin) (9, 25, 29), and three peptides have nanomolar bindingaffinities for the BB4 receptor (Bn, [Phe13]Bn, GRP) (43). Thissuggests either its natural ligand is not a bombesin-like pep-tide, or it has a completely novel sequence differing signifi-cantly from the existing naturally occurring bombesin-relatedpeptides. The pharmacology of various synthetic analogues ofbombesin and neuromedin B were also found to differ betweenthe hBRS-3 receptor and the known bombesin receptor sub-types. In a previous study (42) of 27 synthetic analogues of Bn,

FIG. 10. Dose-response effect of [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14) on stimulation of tyrosine phosphorylation of p125FAK incells transfected with the hBRS-3 receptor. hBRS-3-transfectedNCI-H1299 (top) or BALB 3T3 cells (bottom) were treated with[D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14) at the concentrations indicated.For the NCI-H1299 cells, 300 nM [D-Phe6]Bn-(6–13) methyl ester wasincluded. Values represent the means 6 S.E. from at least three exper-iments. In the top panels above each graph is a Western blot from atypical experiment performed at least three times. Data are expressedas a percentage of the maximal increase in tyrosine phosphorylationcaused by 100 nM [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14). The maximalvalue represented a 4.0 6 1.8-fold increase in the hBRS-3-transfectedNCI-H1299 cells and a 4.1 6 1.5-fold increase in the hBRS-3-trans-fected BALB 3T3 cells.

FIG. 9. Effect of [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14), GRP, and theGRP receptor antagonist [D-Phe6]Bn-(6 –13) methyl ester on tyrosinephosphorylation of p125FAK in hBRS-3-transfected NCI-H1299 cells. hBRS-3-transfected NCI-H1299 cells were incu-bated with 100 nM GRP or [D-Phe6,b-Ala-11,Phe13,Nle14]Bn-(6–14) in the presenceor absence of 300 nM [D-Phe6]Bn-(6–13)methyl ester. The upper panel shows atypical Western blot for p125FAK in cellstreated with (1) or without (2) thevarious peptides. The lower panelrepresents the mean p125FAK tyrosinephosphorylation expressed as the amountof phosphorylated p125FAK in the varioustreatment groups compared with the unt-reated control. Data are means 6 S.E.from at least three experiments. NS, notsignificant.

Cell Signaling by the BRS-3 Receptor 13621

by guest on April 9, 2019

http://ww

w.jbc.org/

Dow

nloaded from

GRP or NMB, which had high affinity for GRP, NMB, or BB4

receptors, only [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14) had ahigh affinity at the hBRS-3 receptor. The present study dem-onstrates this peptide functions as a high affinity agonist atthis receptor activating phospholipase C, inducing changes incytosolic calcium concentrations and stimulating p125FAK ty-rosine phosphorylation. Recently, Wu et al. (41) reported threesynthetic peptides, acetyl-NMB-(3–10), [D-Phe6,Phe13]Bn-(6–13) propylamide, and [D-Phe6]Bn-(6–13) propylamide, were allagonists stimulating [Ca21]i mobilization in hBRS-3-trans-fected BALB 3T3 cells and that [D-Phe6,Phe13]Bn-(6–13) pro-pylamide was particularly potent. Our results have both simi-larities and differences from this study (41). Our results weresimilar in that we found each of these three peptides hadagonist activity at hBRS-3 receptors. Our results were alsosimilar in the potency of AcNMB-(3–10) for stimulating

changes in [Ca21]i in their study (i.e. 219 nM) and for stimulat-ing [3H]IP in our study (i.e. 333 nM). However, our resultsdiffered significantly in the potencies of [D-Phe6]Bn-(6–13) pro-pylamide and [D-Phe6,Phe13]Bn-(6–13) propylamide, which wefound were much lower than they reported (20–2000 fold), andboth peptides were less efficacious than reported previously(41). The reasons for the discrepancies between the two studiesare not clear. In the previous study (41), because of the potencyof NMB analogues, it was proposed that the hBRS-3 receptorhad a binding site more like the NMB receptor than the GRPreceptor. The finding of a submicromolar agonist potency ofAcNMB-(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 re-ceptor antagonist, also inhibited agonist activity at the hBRS-3receptor, might be considered to be supportive of this proposal.However, NMB had a low affinity for hBRS-3 in our study, and

FIG. 11. Effect of thapsigargin aloneor with GF109203X on [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14) stimula-tion of tyrosine phosphorylation ofp125FAK. hBRS-3-transfected BALB3T3 cells were pretreated for 1 h at 37 °Cin the presence or absence of 5 mM

GF109203X (GFX). During the final 30min of pretreatment, 0.1 mM thapsigargin(TG) was added to the samples indicated.Control cells received an equivalent vol-ume of Me2SO. Cells were incubated foranother 10 min with no additions (con-trol) or with 0.1 mM [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14) or 0.1 mM TPA. p125FAK

phosphorylation was determined as de-scribed under “Materials and Methods.”The top panel shows a single experimentrepresentative of two others. The lowerpanel is the quantitation of p125FAK tyro-sine phosphorylation determined by scan-ning densitometry. The values are mean6 S.E. from three experiments and areexpressed as the ratio of total p125FAK

tyrosine phosphorylation in the presenceof [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14)(Exp) to that in the absence of [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14) (Con). Inset,effect of thapsigargin on [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14)-induced changesin [Ca21]i in hBRS-3-transfected BALB3T3 cells. Fura-2-loaded cells were incu-bated in the absence or presence of 0.1 mM

thapsigargin for 30 min, and 0.1 mM

[D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14) wasadded. [Ca21]i was continuously meas-ured in a spectrofluorimeter as describedunder “Materials and Methods.” This re-sult is representative of three others.

Cell Signaling by the BRS-3 Receptor13622

by guest on April 9, 2019

http://ww

w.jbc.org/

Dow

nloaded from

none of the other naturally occurring peptides with a Phe13

substitution, such as [Phe13]Bn, litorin, phyllolitorin, and rho-dei-litorin, had high affinity for hBRS-3. This does not excludethe possibility that a Phe13 plays a role in selectivity for BRS-3,since [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14) and all of thePhe13-containing peptides, except [Ser3,Arg10,Phe13]bombesin,were slightly more potent than the Leu13-containing peptidesGRP and Bn.

Six different classes of GRP or NMB receptor antagonists havebeen described (46, 57, 87, 88). None of these functioned as a potentreceptor antagonist of the hBRS-3 receptor. However, members ofthree 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), anda selective NMB receptor antagonist, the somatostatin octapeptideanalogue D-Nal,Cys,Tyr,D-Trp,Lys,Val,Cys,Nal-NH2 (58), did func-tion as antagonists of the hBRS-3 receptor. The latter somatostatinanalogue was the most potent with an affinity of 504 nM, andpharmacological analysis demonstrated it functions as a competi-tive hBRS-3 receptor antagonist. In a previous study (58), thisanalogue was reported to be selective for the NMB receptor and didnot interact with the GRP receptor. However, our data demon-strate this compound also functions as a hBRS-3 receptor antago-nist with slightly lower affinity than seen with NMB receptors (i.e.216 nM) (58). Additional structure-function studies will be needed todetermine if somatostatin analogues can be identified which aremore selective for hBRS-3 receptors than NMB receptors. Never-theless, the availability of these three classes of low affinity antag-

onists will be useful either as starting points to develop more potentantagonists, or in studies of hBRS-3 receptor cell biology aimed atdetermining its role in various physiological or pharmacologicalprocesses.

In conclusion, using the novel ligand [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14), we demonstrated that hBRS-3activation stimulates phospholipase C activity and tyrosinekinase activity in a similar manner in two cell lines stablytransfected with the hBRS-3 receptor. In contrast to all natu-rally occurring bombesin- or NMB-related peptides or syntheticanalogues tested, only this novel ligand functioned as a highaffinity agonist. These results demonstrate that the pharma-cology of hBRS-3 is different from any other Bn receptors, andthe native ligand likely is not related to any of the naturallyoccurring Bn peptides currently described. None of the highaffinity, selective antagonists for the other Bn receptors inter-act with the hBRS-3 receptors, although three classes of lowaffinity antagonists did function as hBRS-3 receptor antago-nists. The availability of these hBRS-3-transfected cells, thediscovery of the potent agonist [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14), and three classes of low affinity Bn receptor antago-nists that also function as hBRS-3 receptor antagonists shouldprove valuable in the search for the natural ligand for hBRS-3and in further studies investigating its importance in physio-logical and pathological processes.

REFERENCES

1. Tache, Y., Melchiorri, P., and Negri, L. (eds) (1988) Ann. N. Y. Acad. Sci. 547,1–541

2. Bunnett, N. (1994) in Gut Peptides (Walsh, J. H., and Dockray, G. J., eds) pp.423–445, Raven Press, Ltd., New York

3. Brown, M. R., Carver, K., and Fisher, L. A. (1988) Ann. N. Y. Acad. Sci. 547,174–182

4. McCoy, J. G., and Avery, D. D. (1990) Peptides 11, 595–6075. Albers, H. E., Liou, S. Y., Stopa, E. G., and Zoeller, R. T. (1991) J. Neurosci. 11,

846–8516. Jensen, R. T., Coy, D. H., Saeed, Z. A., Heinz-Erian, P., Mantey, S., and

Gardner, J. D. (1988) Ann. N. Y. Acad. Sci. 547, 138–1497. Ghatei, M. A., Jung, R. T., Stevenson, J. C., Hillyard, C. J., Adrian, T. E., Lee,

Y. C., Christofides, N. D., Sarson, D. L., Mashiter, K., MacIntyre, I., andBloom, S. R. (1982) J. Clin. Endocrinol. Metab. 54, 980–985

8. Severi, C., Jensen, R. T., Erspamer, V., D’Arpino, L., Coy, D. H., Torsoli, A.,and Delle Fave, G. (1991) Am. J. Physiol. 260, G683–G690

9. Jensen, R. T. (1994) in Physiology of the Gastrointestinal Tract (Johnson, L. R.,Jacobsen, E. D., Christensen, J., Alpers, D. H., and Walsh, J. H. eds) 3rdEd., pp. 1377–1446, Raven Press, New York

10. DelaFuente, M., DelRio, M., and Hernanz, A. (1993) J. Neuroimmunol. 48,143–150

11. Hill, D. J., and McDonald, T. J. (1992) Endocrinology 130, 2811–281912. Sunday, M. E., Hua, J., Reyes, B., Masui, H., and Torday, J. S. (1993) Anat.

Rec. 236, 25–3213. Willey, J. C., Lechner, J. F., and Harris, C. C. (1984) Exp. Cell Res. 153,

245–24814. Rozengurt, E. (1988) Ann. N. Y. Acad. Sci. 547, 277–29215. Moody, T. W., and Jensen, R. T. (1996) in Growth Factors and Cytokines in

Health and Disease (LeRoith, D., and Bondy, C. eds) pp. 491–535, JAI Press,Inc., Greenwich, CT

16. Seglen, P. O., Skomedal, H., Saeter, G., Schwartze, P. E., and Nesland, J. M.(1989) Carcinogenesis 10, 21–29

17. Endo, T., Fukue, H., Kanaya, M., Mizunuma, M., Fujii, M., Yamamoto, H.,Tanaka, S., and Hashimoto, M. (1991) J. Endocrinol. 131, 313–318

18. Cuttitta, F., Carney, D. N., Mulshine, J., Moody, T. W., Fedorko, J., Fischler,A., and Minna, J. D. (1985) Nature 316, 823–826

19. Bologna, M., Festuccia, C., Muzi, P., Biordi, L., and Ciomei, M. (1989) Cancer63, 1714–1720

20. Schrey, M. P., Patel, K. V., and Tezapsidis, N. (1992) Cancer Res. 52,1786–1790

21. Battey, J. F., Way, J. M., Corjay, M. H., Shapira, H., Kusano, K., Harkins, R.,Wu, J. M., Slattery, T., Mann, E., and Feldman, R. I. (1991) Proc. Natl.Acad. Sci. U. S. A. 88, 395–399

22. Spindel, E. R., Giladi, E., Brehm, P., Goodman, R. H., and Segerson, T. P.(1990) Mol. Endocrinol. 4, 1956–1963

23. Wada, E., Way, J., Shapira, H., Kusano, K., Lebacq-Verheyden, A. M., Coy, D.,Jensen, R., and Battey, J. (1991) Neuron 6, 421–430

24. Benya, R. V., Kusui, T., Pradhan, T. K., Battey, J. F., and Jensen, R. T. (1995)Mol. Pharmacol. 47, 10–20

25. Lin, J. T., Coy, D. H., Mantey, S. A., and Jensen, R. T. (1996) Eur. J.Pharmacol. 294, 55–69

26. Wang, L. H., Mantey, S. A., Lin, J. T., Frucht, H., and Jensen, R. T. (1993)Biochim. Biophys. Acta 1175, 232–242

27. von Schrenck, T., Heinz-Erian, P., Moran, T., Mantey, S. A., Gardner, J. D.,and Jensen, R. T. (1989) Am. J. Physiol. 256, G747–G758

28. Kroog, G. S., Jensen, R. T., and Battey, J. F. (1995) Med. Res. Rev. 15, 389–417

TABLE IIIComparison of the ability of [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14)

and other agents to elevate cyclic AMP levels in hBRS-3-transfectedNCI-H1299 cells

hBRS-3-transfected NCI-H1299 cells were incubated with each of theindicated agents at the above concentrations for 30 min. Results areexpressed as the ratio of total [3H]cAMP in the presence of agonists(Exp) to that in the absence of agonists (Con). Each value represents themeans 6 S.E. from at least three experiments performed in duplicate.The control values were 500 6 78 cpm.

Agent added (100 nM)Total [3H]cAMP (Exp/Con) in

hBRS-3-transfectedNCI-H1299 cells

[D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14) 1.1 6 0.1PACAP-27 16 6 4a

PACAP-38 16 6 2a

Vasopressin 1.3 6 0.2Epinephrine 2.7 6 0.2Forskolin (25 mM) 25 6 5a

a Significantly greater (p , 0.01) than untreated cells.

TABLE IVComparison of the ability of [D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14)and other agents to inhibit elevated cyclic AMP levels in hBRS-3-

transfected NCI-H1299 cellshBRS-3-transfected BALB 3T3 cells were incubated with each of the

indicated agents at the above concentrations for 30 min in the presenceof 25 mM forskolin. Results are expressed as the ratio of total [3H]cAMPin the presence of agonists (Exp) to that in the absence of agonists andforskolin (Con). Each value represents the means 6 S.E. from at leastthree experiments performed in duplicate. The basal value was 162 618 cpm, and the maximally stimulated value (forskolin alone) was631 6 61 cpm.

Agent added (3 mM)Total [3H]cAMP (Exp/Con) in

hBRS-3-transfectedBALB 3T3 cells

Forskolin alone 2.6 6 0.3[D-Phe6,b-Ala11,Phe13,Nle14]Bn-(6–14) 2.5 6 0.6Bombesin 3.9 6 0.6Dopamine 2.1 6 0.6Serotonin 1.7 6 0.3a

Neuropeptide Y 2.2 6 0.4a Significantly less (p , 0.02) than cells treated with forskolin alone.

Cell Signaling by the BRS-3 Receptor 13623

by guest on April 9, 2019

http://ww

w.jbc.org/

Dow

nloaded from

29. Wang, L. H., Battey, J. F., Wada, E., Lin, J. T., Mantey, S., Coy, D. H., andJensen, R. T. (1992) Biochem. J. 286, 641–648

30. Benya, R. V., Wada, E., Battey, J. F., Fahti, Z., Wang, L. H., Mantey, S. A., Coy,D. H., and Jensen, R. T. (1992) Mol. Pharmacol. 42, 1058–1068

31. Tsuda, T., Kusui, T., Hou, W., Benya, R. V., Akeson, M. A., Kroog, G. S.,Battey, J. F., and Jensen, R. T. (1997) Mol. Pharmacol. 51, 721–732

32. Zachary, I., Sinnett-Smith, J., and Rozengurt, E. (1992) J. Biol. Chem. 267,19031–19034

33. Leeb-Lundberg, L. M. F., and Song, X. H. (1991) J. Biol. Chem. 266,7746–7749

34. Lach, E. B., Broad, S., and Rozengurt, E. (1995) Cell Growth Differ. 6,1427–1435

35. Fathi, Z., Corjay, M. H., Shapira, H., Wada, E., Benya, R., Jensen, R., Viallet,J., Sausville, E. A., and Battey, J. F. (1993) J. Biol. Chem. 268, 5979–5984

36. Gorbulev, V., Akhundova, A., Buchner, H., and Fahrenholz, F. (1992) Eur.J. Biochem. 208, 405–410

37. Battey, J., and Wada, E. (1991) Trends Neurosci. 14, 524–52738. Vigna, S. R., Mantyh, C. R., Giraud, A. S., Soll, A. H., Walsh, J. H., and

Mantyh, P. W. (1987) Gastroenterology 93, 1287–129539. Gorbulev, V., Akhundova, A., Grzeschik, K. H., and Fahrenholz, F. (1994)

FEBS Lett. 340, 260–26440. Ohki-Hamazaki, H., Watase, K., Yamamoto, K., Ogura, H., Yamano, M.,

Yamada, K., Maeno, H., Imaki, J., Kikuyama, S., Wada, E., and Wada, K.(1997) Nature 390, 165–169

41. Wu, J. M., Nitecki, D. E., Biancalana, S., and Feldman, R. I. (1996) Mol.Pharmacol. 50, 1355–1363

42. Mantey, S. A., Weber, H. C., Sainz, E., Akeson, M., Ryan, R. R., Pradhan, T. K.,Searles, R. P., Spindel, E. R., Battey, J. F., Coy, D. H., and Jensen, R. T.(1997) J. Biol. Chem. 272, 26062–26071

43. Nagalla, S. R., Barry, B. J., Creswick, K. C., Eden, P., Taylor, J. T., andSpindel, E. R. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 6205–6209

44. Nagalla, S. R., Barry, B. J., Falick, A. M., Gibson, B. W., Taylor, J. E., Dong,J. Z., and Spindel, E. R. (1996) J. Biol. Chem. 271, 7731–7737

45. Sasaki, Y., and Coy, D. H. (1987) Peptides 8, 119–12146. Wang, L. H., Coy, D. H., Taylor, J. E., Jiang, N. Y., Moreau, J. P., Huang, S. C.,

Frucht, H., Haffar, B. M., and Jensen, R. T. (1990) J. Biol. Chem. 265,15695–15703

47. Coy, D. H., Heinz-Erian, P., Jiang, N. Y., Sasaki, Y., Taylor, J., Moreau, J. P.,Wolfrey, W. T., Gardner, J. D., and Jensen, R. T. (1988) J. Biol. Chem. 263,5056–5060

48. Davis, L. G., Kuehl, W. M., and Battey, J. F. (1994) Basic Methods in MolecularBiology, Appleton & Lange, Norwalk, CT

49. Benya, R. V., Fathi, Z., Pradhan, T., Battey, J. F., Kusui, T., and Jensen, R. T.(1994) Mol. Pharmacol. 46, 235–245

50. Rowley, W. H., Sato, S., Huang, S. C., Collado-Escobar, D. M., Beaven, M. A.,Wang, L. H., Martinez, J., Gardner, J. D., and Jensen, R. T. (1990) Am. J.Physiol. 259, G655–G665

51. Benya, R. V., Kusui, T., Battey, J. F., and Jensen, R. T. (1994) J. Biol. Chem.269, 11721–11728

52. Grynkiewicz, G., Poenie, M., and Tsien, R. Y. (1985) J. Biol. Chem. 260,3440–3450

53. Tsuda, T., Kusui, T., and Jensen, R. T. (1997) Biochemistry 36, 16328–1633754. Schild, H. O. (1949) Br. J. Pharmacol. 4, 277–28055. Salomon, Y., Londos, C., and Rodbell, M. (1974) Anal. Biochem. 58, 541–54856. von Schrenck, T., Wang, L. H., Coy, D. H., Villanueva, M. L., Mantey, S., and

Jensen, R. T. (1990) Am. J. Physiol. 259, G468–G47357. Jensen, R. T., and Coy, D. H. (1991) Trends Pharmacol. Sci. 12, 13–1958. Orbuch, M., Taylor, J. E., Coy, D. H., Mrozinski, J. E., Jr., Mantey, S. A.,

Battey, J. F., Moreau, J., and Jensen, R. T. (1993) Mol. Pharmacol. 44,841–850

59. Kusui, T., Tsuda, T., Benya, R. V., and Jensen, R. T. (1997) Gastroenterology108, A984

60. Zachary, I., Gil, J., Lenhmann, W., Sinnett-Smith, J., and Rozengurt, E. (1991)Proc. Natl. Acad. Sci. U. S. A. 88, 4577–4581

61. Zachary, I., Sinnett-Smith, J., and Rozengurt, E. (1991) J. Biol. Chem. 266,24126–24133

62. Schieffer, B., Bernstein, K. E., and Marrero, M. B. (1996) J. Mol. Med. 74,85–91

63. Turner, C. E., Pietras, K. M., Taylor, D. S., and Molloy, C. J. (1995) J. Cell. Sci.108, 333–342

64. Toullec, D., Pianetti, P., Coste, H., Bellevergue, P., Grand-Perret, T., Ajakane,M., Baudet, V., Boissin, P., Boursier, E., Loriolle, F., Duhamel, L., Charon,D., and Kirilovsky, J. (1991) J. Biol. Chem. 266, 15771–15781

65. Thastrup, O. (1990) Agents Actions 29, 8–1566. Millar, J. B. A., and Rozengurt, E. (1988) J. Cell. Physiol. 137, 214–22267. Garcia, L. J., Pradhan, T. K., Weber, H. C., Moody, T. W., and Jensen, R. T.

(1997) Biochim. Biophys. Acta 1356, 343–35468. Corjay, M. H., Dobrzanski, D. J., Way, J. M., Viallet, J., Shapira, H., Worland,

P., Sausville, E. A., and Battey, J. F. (1991) J. Biol. Chem. 266,18771–18779

69. Fathi, Z., Way, J. W., Corjay, M. H., Viallet, J., Sausville, E. A., and Battey,J. F. (1996) J. Cell. Biochem. Suppl. 24, 237–246

70. Seckl, M., and Rozengurt, E. (1993) J. Biol. Chem. 268, 9548–955471. Sinnett-Smith, J., Zachary, I., Valverde, A. M., and Rozengurt, E. (1993)

J. Biol. Chem. 268, 14261–1426872. Huckle, W. R., Dy, R. C., and Earp, H. S. (1992) Proc. Natl. Acad. Sci. U. S. A.

89, 8837–884173. Force, T., Kyriakis, J. M., Avruch, J., and Bonventre, J. V. (1991) J. Biol.

Chem. 266, 6650–665674. Garcia, L. J., Rosado, J. A., Gonzalez, A., and Jensen, R. T. (1997) Biochem. J.

327, 461–47275. Shattil, S. J., Haimovich, B., Cunningham, M., Lipfert, L., Parsons, J. T.,

Ginsberg, M. H., and Brugge, J. S. (1994) J. Biol. Chem. 269, 14738–1474576. Saville, M. K., Graham, A., Malarkey, K., Paterson, A., Gould, G. W., and

Plevin, R. (1994) Biochem. J. 301, 407–41477. Schaller, M. D., Borgman, C. A., Cobb, B. S., Vines, R. R., Reynolds, A. B., and

Parsons, J. T. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 5192–519678. Williams, J. A., and Yule, D. I. (1993) in Pancreas: Biology, Pathobiology, and

Disease (Go, V. L. W., DiMagno, E. P., Gardner, J. D., Lebenthal, E., Reber,H. A., and Scheele, G. A., eds) pp. 167–189, Raven Press, New York

79. Itoh, H., Shimomura, A., Okubo, S., Ichikawa, K., Ito, M., Konishi, T., andNakano, T. (1993) Am. J. Physiol. 265, C1319–C1324

80. Raufman, J. P., Kasbekar, D. K., Jensen, R. T., and Gardner, J. D. (1983)Am. J. Physiol. 245, G525–G530

81. Eguchi, S., Hirata, Y., Imai, T., and Marumo, F. (1993) Endocrinology 132,524–529

82. Jelinek, L. J., Lok, S., Rosenberg, G. B., Smith, R. A., Grant, F. J., Biggs, S.,Bensch, P. A., Kuijper, J. L., Sheppard, P. O., and Sprecher, C. A. (1993)Science 259, 1614–1616

83. DelValle, J., Wang, L., Gantz, I., and Yamada, T. (1992) Am. J. Physiol. 263,G967–G972

84. Deutsch, P. J., and Sun, Y. (1992) J. Biol. Chem. 267, 5108–511385. Mantey, S. A., Weber, H. C., Sainz, E., Ryan, R. R., Pradhan, T. K., Spindel,

E. R., Battey, J. F., Coy, D. C., and Jensen, R. T. (1997) Gastroenterology112, A1168

86. Rozengurt, E., and Sinnett-Smith, J. (1983) Proc. Natl. Acad. Sci. U. S. A. 80,2936–2940

87. Wang, L. H., Coy, D. H., Taylor, J. E., Jiang, N. Y., Kim, S. H., Moreau, J. P.,Huang, S. C., Mantey, S. A., Frucht, H., and Jensen, R. T. (1990)Biochemistry 29, 616–622

88. Leban, J. J., Kull, F. C., Jr., Landavazo, A., Stockstill, B., and McDermed, J. D.(1993) Proc. Natl. Acad. Sci. U. S. A. 90, 1922–1926

89. Lin, J. T., Coy, D. H., Mantey, S. A., and Jensen, R. T. (1995) J. Pharmacol.Exp. Ther. 275, 285–295

90. Jensen, R. T., Jones, S. W., Folkers, K., and Gardner, J. D. (1984) Nature 309,61–63

91. Jensen, R. T., Heinz-Erian, P., Mantey, S., Jones, S. W., and Gardner, J. D.(1988) Am. J. Physiol. 254, G883–G890

Cell Signaling by the BRS-3 Receptor13624

by guest on April 9, 2019

http://ww

w.jbc.org/

Dow

nloaded from

James F. Battey, David H. Coy and Robert T. JensenRichard R. Ryan, H. Christian Weber, Wei Hou, Eduardo Sainz, Samuel A. Mantey,

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

doi: 10.1074/jbc.273.22.136131998, 273:13613-13624.J. Biol. Chem. 

  http://www.jbc.org/content/273/22/13613Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/273/22/13613.full.html#ref-list-1

This article cites 86 references, 40 of which can be accessed free at

by guest on April 9, 2019

http://ww

w.jbc.org/

Dow

nloaded from