Proc. Natl. Acad. Sci. USAVol. 92, pp. 6205-6209, June 1995Cell Biology

Cloning of a receptor for amphibian [Phe13Jbombesin distinctfrom the receptor for gastrin-releasing peptide: Identificationof a fourth bombesin receptor subtype (BB4)

(Bombina orientalis/frog)

SRINIVASA R. NAGALLA*t, BRENDA J. BARRY*, KIM C. CRESWICK*, PETER EDENt, JOHN T. TAYLOR4,AND ELIOT R. SPINDEL**Division of Neuroscience, Oregon Regional Primate Research Center, Beaverton, OR 97006; and *Biomeasure Incorporated, Milford, MA 01757

Communicated by Charles H. Sawyer, University of California School of Medicine, Los Angeles, CA, March 27, 1995 (received for reviewJanuary 17, 1995)

ABSTRACT Bombesin is a tetradecapeptide originally iso-lated from frog skin and demonstrated to have a wide range ofactions in mammals. Based on structural homology and similarbiological activities, gastrin-releasing peptide (GRP) has beenconsidered the mammalian equivalent of bombesin. We previ-ously reported that frogs have both GRP and bombesin, whichtherefore are distinct peptides. We now report the cloning of abombesin receptor subtype (BB4) that has higher affinity forbombesin than GRP. PCR was used to amplify cDNAs related tothe known bombesin receptors from frog brain. Sequence anal-ysis of the amplified cDNAs revealed 3 classes of receptorsubtypes. Based on amino acid homology, two classes were clearlythe amphibian homologs of the GRP and neuromedin B recep-tors. The third class was unusual and a full-length clone wasisolated from a Bombina oriknkis brain cDNA library. Expres-sion of the receptor in Xenopus oocytes demonstrated that thereceptor responded to picomolar concentrations of [Phe13] -bombesin, the form of bombesin most prevalent in frog brain.The relative rank potency of bombesin-like peptides for thisreceptor was [Phe13]bombesin > [Leu13bombesin > GRP >neuromedin B. In contrast, the rank potency for the GRPreceptor is GRP > [Leu'3]bombesin > [Phe13]bombesin >neuromedin B. Transient expression in CHOP cells gave a K; for[Phe13]bombesin of 0.2 nM versus a K; of 2.1 nM for GRP.Distribution analysis showed that this receptor was expressedonly in brain, consistent with the distribution of [Phel3]-bombesin. Thus, based on distribution and affinity, this bomb-esin receptor is the receptor for [Phe&3]bombesin. Phylogeneticanalysis suggests that this receptor separated prior to separationoftheGRP and neuromedin B receptors; thus, BB4 receptors andtheir cognate ligands may also exist in mammals.

Bombesin, a tetradecapeptide, was isolated from the skin ofthe frog Bombina bombina by Anastasi et al. (1) in 1971.Amphibian bombesin was found to have multiple effects inmammals (2, 3) and bombesin-like immunoreactivity wasobserved in mammalian brain, gastrointestinal (GI) tract, andlung (4-6). In 1979, using gastrin release as a bioassayMcDonald and co-workers (7) isolated from porcine stomacha 27-amino acid peptide homologous to the C terminus ofbombesin and named it gastrin-releasing peptide (GRP). GRPis widely distributed in mammals; it acts as a neurotransmitterin brain, a paracrine hormone in GI tract, and a growth factorin developing lung (8-10). Because GRP reproduces all ofbombesin's biologic effects, GRP was initially considered to bemammalian bombesin (see Fig. 1). In 1992, Nagalla et al. (11)showed that GRP and bombesin are clearly distinct peptides,in that frogs have both GRP and bombesin, and phylogenetic

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

analysis suggests that the peptides separated prior to thevertebrate radiation. This suggests that GRP and bombesin willhave different receptors and that mammals, like frogs, mayhave bombesin-related peptides (BRPs) distinct from GRP.Neuromedin B (NMB) is a second mammalian bombesin-likepeptide. NMB was originally isolated from porcine spinal cord(12) and like GRP is widely distributed in brain and GI tract.Making matters more complex, bombesin does not exist in

just a single form. In all frogs studied to date, there aremultiple forms of bombesin (13, 14). In Bombina orientalis,[Leul3]bombesin (the prototypic bombesin) is found in skin,GI tract, and oocytes; [Phe'3]bombesin predominates in brainand [Ser3,Arg9,Phel3]bombesin (SAP bombesin) is present inlung and GI tract (Fig. 1) (14). Similarly in Xenopus laevis andPhyllomedusa sauvagei there are both Phe and Leu forms ofbombesin (13, 14, 16) with distinct distributions. These datasuggest that the different forms of bombesin will have differentphysiologic roles and, most likely, distinct receptors. We havedesignated these multiple forms of bombesin as BRPs.Three mammalian bombesin receptor subtypes have been

characterized to date. The GRP-preferring subtype was clonedby Spindel et al. (17) and by Battey et al. (18); the NMBreceptor was cloned by Wada et al. (19); and a third subtype,designated BRS-3, was cloned by Fathi et al. (20) and byGorbulev et al. (21). All the bombesin receptors characterizedto date are guanine nucleotide binding protein (G protein)coupled, have seven hydrophobic domains, and activate phos-pholipase C to increase inositol 1,4,5-trisphosphate, diacyl-glycerol, and intracellular calcium. A second nomenclaturesystem for the bombesin receptors is also in use. In this system,the NMB receptor is known as the BB1 receptor, the GRPreceptor is the BB2 receptor, and the BRS-3 receptor is theBB3 receptor (22). Because of the high homology betweenbombesin-like peptides, there is potential for cross-talk be-tween ligands and receptors. To begin to study the role ofbombesin distinct from GRP, we undertook to clone thereceptors for the BRPs.§

MATERIALS AND METHODSAnimals and Reagents. Frogs (B. orientalis) were obtained

from California Zoological Supply (Santa Ana), and albino Xlaevis were from Xenopus 1 (Ann Arbor, MI). Recombinantaequorin was generously provided by 0. Shimomura (23). Bomb-esin, GRP, and NMB peptides were obtained from Peninsula

Abbreviations: GI, gastrointestinal; GRP, gastrin-releasing peptide;NMB, neuromedin B; BRP, bombesin-related peptide; SAP bombesin,[Ser3,Arg9,Phe13]bombesin; RT, reverse transcription; cRNA, com-plementary RNA; G protein, guanine nucleotide binding protein.tTo whom reprint requests should be addressed.§The sequences reported in this paper have been deposited in theGenBank data base [accession nos. L39358 (frog BB4 receptor),L39359 (frog GRP receptor), and L39360 (frog NMB receptor)].

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1 14Leu13 bombesin pGlu-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met.NH2Phe13 bombesin pGlu-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His P Met.NH2SAP-bombesin pGlu-GlnE Leu-Gly-Asn-Gln-Trp A Val-Gly-His Phe-Met.NH2Frog GRP-10 GlyiSer His Trp-Ala-Val-Gly-His-Leu-Met.NH2Human GRP-10 Gly-Asn His Trp-Ala-Val-Gly-His-Leu-Met.NH2

FIG. 1. Structures of the three distinct forms of bombesin in B. orientalis (14), GRP-10, and human GRP-10. GRP-10 is the C-terminaldecapeptide of GRP, contains full biological activity, and is present in all species examined to date (11, 15). pGlu, pyroglutamate; NH2, C-terminalamide. Residues differing from [Leul3]bombesin are boxed.

Laboratories; [Phe13]bombesin and SAP bombesin were gener-ously provided by Biomeasure. CHOP cells [Chinese hamsterovary fibroblasts modified to express papovavirus large T antigen(24)] were a gift of James W. Dennis (Samuel Lunenfeld Re-search Institute, Toronto) and were grown in Dulbecco's modi-fied Eagle's medium supplemented with 10% fetal bovine serum.

Reverse Transcription (RT) PCR. Total RNAwas prepared asdescribed (13). 5 ,ug of total RNAwas reverse transcribed with 25pmol of oligo(dT)18, 200 units of Moloney mouse leukemia virusreverse transcriptase (BRL), and Sx buffer (250 mM Tris HCl,pH 8.3/375 mM KCI/25 mM MgCl2/50 mM dithiothreitol/2.5mM dNTPs in 20 ,ul total vol at 37°C). The entire 20-,ul reactionmixture was then used in a 100-pl PCRwith 100 pmol of 5' primerTCIGTKGGKGTKTCIGTSTT (I, inosine; K, G or T; S, G or C)and 100 pmol of 3' primer GCRAAIGGRTTIACRCAR-GARTT. PCR was performed at 92°C for 1 min, 55°C for 2 min,and 72°C for 3 min for 35 cycles, using 2.5 units of Taq polymerase(Promega) as described (13). Fifteen microliters of the reactionmixture was separated on a 1% agarose gel, blotted, and hybrid-ized to an internal primer (TGYTGGYTXCCXAAYCA). Fordistribution analysis, RT-PCR was performed similarly with the5' and 3' primers described above, but PCR amplifications wereelectrophoresed in triplicate and probed with primers specific foreach receptor as follows: BB4, TGCTGAAGAGCACACT; frogGRP, AGTGGAGGGAAACGTG; frog NMB, TGGAGAG-TACAGCGAG. Hybridization was in 6x SSC/5x Denhardt'ssolution/0.1% SDS. Hybridization and washing were performed

Frog BB4Human GRPHuman NMBHuman BRS-3

Frog BB4Human GRPHuman NMBHuman BRS-3

Frog BB4Human GRPHuman NMBHuman BRS-3

Frog BB4Human GRPHuman NMBHuman BRS-3

Frog BB4Human GRPHuman NMSHuman BRS-3

Frog BB4Human GRPHuman NMBHuman BRS-3

Frog BB4Human GRPHuman NSHHuman BRS-3

at 50°C for the BB4 primer, at 52°C for the GRP receptor primer,and at 55°C for the NMB receptor primer.cDNA Library Screening and RNA Blot Analysis. The B.

orientalis brain cDNA library was prepared in A ZAPII (Strata-gene) from 50 pooled brain samples as described (13), and 5X 105 plaque-forming units were screened with a complemen-tary RNA (cRNA) probe generated from the partial BB4cDNA isolated by RT-PCR. Hybridizing clones were plaquepurified and excised into plasmids, and two full-length cloneswere sequenced on both strands by the double-strandeddideoxynucleotide termination method (13). For RNA blotanalysis, 3 ,ug of poly(A)+ RNA was prepared from the tissuesshown, resolved on a 1.5% formaldehyde agarose gel, blotted,and probed with the BB4 cRNA probe as described (13).Xenopus Oocyte Expression. Functional expression of the

frog BB4 receptor in oocytes was performed as described (25).In brief,X laevis oocytes were coinjected with 5 ng of in vitrotranscribed receptor RNA and the calcium photoprotein ae-quorin and then incubated for 18 hr in nutritive medium (17).Single oocytes were placed in a luminometer (Berthold LB253)and challenged with various ligands at 10-6-10-10 M. Ligand-induced increases in intracellular calcium cause a proportionalincrease in light emission by aequorin, which is recorded by theluminometer.

Receptor Binding Assays. For receptor expression, the full-length BB4 receptor was subcloned into pcDNA1-neo (Invitro-gen). Expression constructs for the human GRP and NMBreceptors (also in a modified pcDNA1-neo) were generously

MALNDCFLLNLEVDHFMHCNISSHSADL PVNDDWSHP ------------ GILYVIPAVYGM SKS--LSNL SVT ------- TGANES SVPEGWERDFLPASDGT TTETELVIR IPSLYLMAQRQ ---- PHSPNQTLISITNDTESSS SVVSNDNTN-KGWSGDNS PGIEALCAIYITYA

~~~~~~~~II

VIISGILNTILIK]V[FFKIKSM~QTVPNTIFITSLAiFG DLLLLLrc PD SR IDWVIILIGL I NI TILIIKIIFICTVKSIMIRN VPNILFIISSLALIGDLLLLI krC|A|PVDA|SR|Y|LADR|W|LLIIITVIGLLL GNIIMILIVIKIIFIITNSAIMIRS VNPNIIFFIISNILAIAIGDLLLLL k CIVIPVDAlSRIYIFFDEIWIM IVIISVGIL NA ILIKVFFKTKSMQT V PNIFITSF LAFGDLLLL CVPVDATH LAEGL

III IVGRA|GCKIISF IQLTSVGVSVFTLTV STDRYRAIKLQLTSD VLKTCGKA|VCVWIIGRIGCKIL IFF II QLTSVGVSVFTLTA SADRYKH LMKIICILKAIAFIIWIIIIIGIKV IGCKLIPVIQLTSVGVSVFTLTA SADRGYRALLRTCVKA|MGIIWIVVVSIGRIGCKIV5LVSSFIR RLTSVGVSVFTLTI SAIYSKAJVKKVKPJLERQ_PSNAJI LKTCVIKAIGCVWJIV5J

MLL(APEAV SWDLYEFGSSEKNTT EA APYP|VSEKILQETHLICFLVFYIVPLSIISAM L L IAII IP E AIV S ID L H P F H E E S T NQ T I S A P Y PH S N E L H P K I H S M A S F L VIF Y V IIP LIS I I SIVVLL|A|VIPEA|V S|EVARI-SSLD|N|SS|FTATA I P|Y PIQTDELHPK IH S|VL IFLVIYFLIIPLA|IISIM IFAJLIPEAIIF SINVY TF RD PN KNJMT WES JT SIY PIV SK KLLQEIIHSILL CIFL VIF Y IIPLSIIISIV

YYFL[IKT YKST FiMiAEEHTHAR KIESRKRVAKTVLVLVALFAVCWLPNHIMLLRIYYIYFIIAIKN I|QSA ALPVEGNHVK KQII|E|S|RKR|L|A|KT|VLV|F|V|GL FIAF|CWILPNH|VI|Y|L|Y|R IIYY|YHIIAIKT IK SAH|N|L|P|GIE|YNE|H|TK KQ|M|E|T|RKR|L|A|KI|VLV|F|V|GC|F|I FICWIFIPNHIIL|Y|MIYIRISIIYY.SLL5A~RT ~YKSTLNIPTEEQSHJARK E R RA T L LALA C LN LY Y

VIIFTYHSAVNS FA F[ ]LSATIFAWVE[ ALR SCVNP FALYWLSR[FRQHH KK ]VYCCKTEPLHPYHYSE-V DT MSILIHIFVTSIC AIRILLIAFTGTSCVNPFALYILILSIKESF|RKR ,NTQLLCCOPGK QIIRFNYNE-IDP ~LGHMIVTLVARVLSFG TSCVNPFALYILILSESFIRRHlFISQLCCGRXSYQERFTSQTYVDP JAM HFIFTIFS RVLAFS SCVNPFALYWLSKSFQKH KA IQJLFCJCKAERPEP

T K S D P Q - - - - - - - - - - - - - - - - - - - Q Y H N W N Y R - C E - R Q H P D V MS H S - - T GR ST T C M T S L K ST N P - - - S VAT F S - L IN GN IC HER Y V LGTSYLLSSSAV RMTSLKSNAK --- NMVTNSVLLNGHSMKQEMAMPVADTSLTTLAVMGTVPGTGSIQMSEIS VTSFTGCSVKQAEDRF

54485155

114108111115

174168171175

234228230235

294288290295

354347349355

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FIG. 2. Comparison of deduced amino acid sequences of the frog BB4 receptor, human GRP, NMB, and BRS-3 receptors. Seven predictedtransmembrane domains are overlined. 0, Consensus sites for N-linked glycosylation; *, consensus sites for protein kinase C phosphorylation.

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Table 1. Amino acid identity (%) of bombesin receptor subtypes

Frog Human Human HumanBB4 GRP NMB BRS-3

Frog GRP 59 83 55 58Frog NMB 48 60 80 51Frog BB4 - 61 56 70P. sauvagei 86 61 53 68R. pipiens 88 58 52 70

Amino acid alignment as calculated by DNASTAR AALIGN. HumanGRP (27), NMB (28), and BRS-3 (20) receptors have been reported;P. sauvagei and R. pipiens are BB4-related receptor fragments asdescribed in the text.

provided by J. Battey. Plasmids were transiently transfected intoCHOP cells by using calcium phosphate (24). Cells were har-vested at 48 hr and resuspended in the binding buffer (120 mMNaCl/7.2 mM KCI/6.0 mM pyruvate/7.1 mM fumarate/14.0mM glucose/0.01% soybean trypsin inhibitor/25 mM Hepes/0.5mM CaCl2/1.2 mM MgCl2/2.1 mM KH2PO4/0.03% L-glutamine/0.2% bovine serum albumin/0.1% bacitracin) at aconcentration of 1 X 106 cells per ml. Cells (500 ,ul) wereincubated at 22°C with 50 pM [125I-Tyr4]bombesin (2000 Ci/mmol; 1 Ci = 37 GBq; New England Nuclear) and variousconcentrations of unlabeled peptides. Nonsaturable binding wasthe amount of radioactivity associated with the cells in thepresence of 1 ,uM unlabeled peptide and was <10% in all theexperiments. All values are reported as saturable binding (totalbinding minus nonsaturable) and are means ± SEM of threeexperiments performed in triplicate. Analysis of the data wasperformed with the LIGAND program and by the method ofChengand Prusoff (26).

RESULTSCloning of Frog BB4 Receptor. Alignment of the known

mammalian bombesin receptors showed greatest homology intransmembrane domains III and VII. Therefore, degenerateprimers corresponding to Ser-Val-Gly-Val-Ser-Val-Phe con-served in transmembrane domain III and Asn-Ser-Cys-Val-Asn-Pro-Phe conserved in transmembrane domain VII weresynthesized as described. RNA from B. orientalis skin, stom-ach, and brain was reverse transcribed and amplified with theseprimers. The PCR products were electrophoresed and blotted,and the 600-bp hybridizing product was subcloned. Sequenceanalysis of multiple cDNAs revealed three major classes ofreceptor fragments. Amino acid alignment with the threemammalian receptors shows that two of the receptor frag-ments were highly homologous (>80%) to mammalian GRPand NMB receptors and likely represented fragments of thefrog GRP and NMB receptors. The third class had lowerhomology (<70%) and appeared to encode a receptor subtypewe designated as the bombesin BB4 receptor.The partial cDNA was used to screen a B. orientalis brain

cDNA library and 12 positive clones were isolated. The full-lengthcDNA encodes a protein of377 amino acids with a predicted massof 42.3 kDa. Hydropathy analysis predicts seven putative trans-membrane domains consistent with a G-protein-coupled recep-tor (Fig. 2). There are four potential N-linked glycosylation sites:two in the extracellular N terminus, one in the extracellular loopbetween the fourth and fifth transmembrane domains, and one in

the extracellular loop between the sixth and seventh transmem-brane domains. There are two potential protein kinase C phos-phorylation sites: one (Ser264) is located in the third cytoplasmicloop (Fig. 2) similar to the other members in this family and asecond site (Ser335) is located in the C-terminal tail.Comparison of the frog BB4 receptor with the mammalian

receptors showed 56%, 61%, and 70% amino acid identity tothe human GRP, NMB, and BRS-3 receptors, respectively(Table 1). By contrast, the fragments of the putative frog GRPand NMB receptors were 80% and 83% homologous to thehuman GRP and NMB receptors, respectively. Partial cDNAsisolated from the brain tissue of two other species of frogs(Rana pipiens and P. sauvagei) revealed receptor fragmentshighly homologous to the B. orientalis BB4 receptor (Table 1).Phylogenetic analysis (Fig. 3) of all the bombesin receptorsubtypes isolated to date showed four branches: 1, the GRPfamily; 2, the NMB family; 3, the BRP family; and 4, the BRS-3family. The phylogenetic tree of the receptors is in goodagreement with the predicted tree derived from the prohor-mones of bombesin-like peptides (13).

Distribution Analysis. The distribution of BB4 mRNA wasdetermined by high-stringency Northern blot analysis (Fig. 4A).The apparent size of the BB4 mRNA was 2.3 kb and it wasdetected only in the brain. To map expression with more sensi-tivity and for comparison to frog GRP receptor and NMBreceptor expression, RT-PCR was used. RT-PCR, like Northernblot analysis, showed BB4 expression only in brain (Fig. 4B). Incontrast, GRP receptor mRNA was detected at relatively highlevels in stomach and at lower levels in brain, kidney, and lung.Frog NMB receptor mRNA expression was higher in brain andlower in kidney (72-hr exposure showed low levels of expressionin stomach; data not shown). Expression in brain was furtheranalyzed in dissected regions (Fig. 4C). BB4 expression wasrestricted primarily to cortex and forebrain and was at low levelsin the midbrain. By contrast, frog GRP receptor and NMBreceptor were expressed in all brain regions examined.

Functional Expression of the BB4 Receptor. Pharmacologyof the frog BB4 receptor was determined by expression inXenopus oocytes and CHOP cells. Oocytes injected with BB4receptor transcripts showed strong responses to nanomolarconcentrations of bombesin-like peptides. Responses to 1 nM[Phe13]bombesin, [Leu13]bombesin, GRP, andNMB are shownin Fig. SA. Agonist potency was [Phe13]bombesin >[Leu13]bombesin > GRP > NMB. Oocytes injected with frogBB4 or human GRP receptor transcripts showed log-linearresponses to [Phe13]bombesin (Fig. 5B). However, the BB4receptor responded to 100 pM [Phe13]bombesin, whereas thehuman GRP receptor did not. One hundred nanomolar[D-Phe6]bombesin-(6-13) propylamide, a specific GRP recep-tor antagonist (30), only partially blocked the response of theBB4 receptor to 3 nM [Phe13]bombesin but completely blockedthe response of the GRP receptor (Fig. SB). Antagonist aloneat 100 nM showed weak agonist activity on both receptors(data not shown). Because oocyte expression does not give aquantitative measure of binding affinity, Ki values were deter-mined by binding assays on CHOP cells transiently transfectedwith the BB4, GRP, and NMB receptors using [125I-Tyr4]-bombesin as a tracer. Consistent with the oocyte data, affin-ities for the frog BB4 receptor were [Phe13]bombesin >[Leu13]bombesin > GRP > NMB (Fig. SC). The apparent

Rat ORP

Human GRP

B.o. GRPRat NMB

Human NMB

B.o NMB

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FIG. 3. Phylogenetic tree de-rived from multiple comparisonsof the known bombesin receptorsubtypes by the method of Hein(29), as implemented by DNASTARin MEGALIGN. Sequences are thesame as analyzed in Table 1 (P.sauvagei and R. pipiens se-quences; S.R.N., unpublisheddata). B.o., B. orientalis.0

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A

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FIG. 4. (A) Northern blot analysis of BB4 mRNA expression in B. orientalis. Three micrograms of poly(A)+ RNA from the tissues shown wasresolved on a 1.5% formaldehyde agarose gel, processed, and probed with the BB4 cRNA probe. Blot was exposed for 72 hr at -80°C. (B) RT-PCRanalysis of frog BB4, GRP, and NMB receptor subtypes in B. orientalis. A pair of degenerate primers that hybridizes to all three forms was usedto amplify 5 ,ug of total RNA as described. Aliquots (15 ,lI) of the PCR amplification were resolved on 1% agarose gels in triplicate and probedwith internal gene-specific primers. Blots were exposed for 18 hr at -80°C. (C) Expression of frog (f) BB4, GRP, and NMB receptor RNAs inB. orientalis nervous system. RT-PCR analysis was performed as described above.

dissociation constants (K1) as shown in Table 2 were[Phel3]bombesin = 0.2 nM, [Leul3]bombesin = 0.9 nM, GRP= 2.0 nM. By contrast, the human GRP receptor had a Ki of1.8 nM for GRP and a Kd of 3.6 nM for [Phel3]bombesin. Thehuman NMB receptor showed highest affinity to NMB (1.2nM) followed by [Phet3]bombesin (1.8 nM). [D-Phe6]Bombesin-(6-13) propylamide bound with equal affinity to both the BB4and GRP receptors (Ki = 2 nM).

DISCUSSIONThe presence of both bombesin and GRP in frog brainsuggested that distinct receptors for those peptides wouldalso be present in frog brain. In this study, we have charac-terized a bombesin receptor subtype present in B. orientalisthat has high affinity for one form of bombesin ([Phe13]-bombesin) and shown that this receptor is distinct from thefrog GRP receptor.

Sequence analysis of partial cDNAs cloned from the skin,stomach, and brain of Bombina revealed three major receptor

A

C)

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subtypes (Table 1). High amino acid identity suggested thattwo of the frog receptor subtypes were the amphibian ho-mologs of the GRP and NMB receptors. The frog GRPreceptor fragment had 83% identity with the human GRPreceptor; the frog NMB receptor fragment had 80% homologywith the human NMB receptor. By contrast, the third class ofreceptor fragments showed lower homology and appeared torepresent a bombesin receptor subtype that we designatedBB4. A full-length BB4 receptor cDNA was isolated from frogbrain and showed overall amino acid identity with the threehuman bombesin receptors as follows: 56% to NMB, 61% toGRP, and 70% to BRS-3. Homology was strongest in trans-membrane domains II, III, and VII (60-89%) and mostdivergent in the N terminus. Similar to other receptors in thefamily a conserved protein kinase C phosphorylation site islocated in the third cytoplasmic loop (28, 32) and a secondpotential site is in the C-terminal cytoplasmic tail. Proteinkinase C phosphorylation sites in the C terminus of the GRPreceptor have been shown to be important in receptor inter-

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FIG. 5. (A) Luminometric assay of functional expression of the frog BB4 receptor in Xenopus oocytes. Oocytes were coinjected with 5 ng offrog BB4 receptor RNA transcribed from a cDNA encoding the receptor and the calcium photoprotein aequorin (17). After 24 hr, the eggs werechallenged with 1 nM ligand and the light output was measured over 120 sec in a luminometer. (B) Response of frog BB4 (hatched box) and humanGRP (solid box) receptors expressed in oocytes to [PheO3]bombesin. Results are expressed as peak light output over 120 sec. For antagonistexperiments, eggs were exposed to 100 nM [D-Phe6]bombesin-(6-13) propylamide for 20 sec followed by injection of 3 nM [Phel3]bombesin. Resultsrepresent means + SEM of three separate experiments. (C) Competitive inhibition of [125I-Tyr4]bombesin binding to frog BB4 receptors transientlyexpressed in CHOP cells. Whole cells expressing BB4 receptor were incubated with 50 pM [1251-Tyr4]bombesin and the indicated concentrationsof [Phel3]bombesin (m), [Leul3]bombesin (0), GRP (-), and NMB (O). Results are expressed as percentage maximal specific binding in the absenceof competitor (means ± SEM of three experiments performed in triplicate).

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Table 2. Binding affinities (Ki) of bombesin-like peptides to bombesin receptor subtypes[Leu13]- [Phel3]- SAP Human

Receptor Bombesin Bombesin bombesin GRP NMB AntagonistFrog BB4 0.9 ± 0.2 0.2 ± 0.05 100 ± 30 2.1 ± 0.4 30 ± 6 2.0 ± 0.2Human GRP 2.9 ± 0.5 3.6 ± 0.3 300 ± 50 1.8 ± 0.4 220 ± 25 2.2 ± 0.6Human NMB 14 ± 0.5 1.8 ± 0.3 300 ± 50 15 ± 0.4 1.2 ± 0.2 ND

Ki values were determined from the data shown in Fig. SC by the method of Cheng and Prusoff (31) and the LIGAND program.Values are means ± SEM of three experiments. ND, not determined.

nalization (33). This suggests a conservation of receptorregulatory mechanisms.

Phylogenetic analysis of the known bombesin receptor sub-types shows four branches: GRP receptor, NMB receptor,BRS-3, and BB4 (Fig. 3). The BRS-3 branch shows greatestevolutionary distance and suggests that BRS-3 ligands may bequite distinct from the known bombesin-like peptides. TheBB4 branch appears to have evolved prior to the GRP receptorand NMB receptor branches. This suggests that mammals willlikely have BB4 receptors and as yet undiscovered bombesin-like peptides that are the cognate ligands.

Expression in Xenopus oocytes demonstrates that the BB4cDNA encodes a functional receptor that responds to bomb-esin-like peptides (Fig. 5). The rank potency of agonists is[Phe13]bombesin > [Leu13]bombesin > GRP > NMB. This isin contrast to the GRP receptor, which shows greatest affinityfor GRP. Binding studies show that the BB4 receptor binds[Phe13]bombesin with a Ki of 0.2 nM. By contrast, the humanGRP receptor binds [Phe13]bombesin with a Ki of 3.6 nM, andthe human NMB receptor binds [Phe'3]bombesin with anaffinity of 1.8 nM. The BB4 receptor is also clearly differentfrom the BRS-3 receptor since that receptor shows no responseto [Phe13]bombesin (or to any other known bombesin-likepeptides at nanomolar doses). Thus, the BB4 receptor ispharmacologically distinct from the GRP, NMB, and BRS-3receptors. The high affinity of [Phe13]bombesin for the BB4receptor suggests that it is the endogenous ligand. This isfurther supported by the distribution of BB4 and [Phe13]_bombesin expression. RNA blot analysis and RT-PCR showBB4 expression only in brain (Fig. 3). Similarly, [Phe13]-bombesin is found only in brain (14). Thus, on the basis ofligand affinity and distribution [Phe13]bombesin is the likelyligand for this bombesin receptor subtype.The BB4 and GRP receptors have similar affinities of -2.0

nM for the GRP antagonist [D-Phe6]bombesin-(6-13) propyl-amide. However, the affinity of the BB4 receptor for its likelyendogenous ligand, [Phe13]bombesin, is 0.2 nM. This makesthe antagonist relatively less effective in blocking the responseof the BB4 receptor to [Phe13]bombesin. This is consistent withthe data shown in Fig. SB in which the antagonist completelyblocks the response of the GRP receptor to [Phe13]bombesinbut only partially blocks the response of the BB4 receptor. Theantagonist is also a weak partial agonist for the BB4 receptor,consistent with its known weak partial agonist for the GRPreceptor (17).

Given that [Leu13]bombesin and SAP bombesin are ex-pressed in lung and GI tract, but the BB4 receptor is not, it islikely that receptors for those peptides distinct from the BB4receptor exist. Given the high homology between [Leu13]-bombesin, [Phe13]bombesin, and SAP bombesin, the receptorsfor these peptides are likely to be highly homologous to theBB4 receptor. The possibility of a high affinity, mammalian[Leu13]bombesin receptor is further supported by the findingsof Vigna et al. (34), who observed that bombesin binds to antralG cells with a Ki of 0.89 nM.

In summary, we have identified a bombesin receptor subtype(BB4) with high affinity for the BRPs distinct from GRP. Theendogenous ligand for this receptor appears to be [Phe13]-bombesin. This further supports the hypothesis that GRP andbombesin are distinct peptides with specific receptors and distinct

physiologic roles. We predict that mammals, like frogs, will havea BRP distinct from GRP and a cognate BB4 receptor.

We wish to thank Drs. Robert T. Jensen, Samuel Mantey, andRichard V. Benya for advice on binding assays; Dr. James F. Battey forproviding human GRP and NMB receptor expression constructs; 0.Shimomura, Y. Kishi, and S. Inouye for provision of recombinantaequorin; and Kalama Taylor for assistance with cell culture. This workwas supported by National Institutes of Health Grants CA39237,CA53584, and RR00163; by the cell culture and molecular biologycores of National Institute of Child Health and Human DevelopmentPopulation Research Center Grant P30-HD18185; and by grants fromthe Council on Tobacco Research and Biomeasure Inc.

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