Tyrosine 220 in the 5th Transmembrane Domain of the NeuromedinB Receptor Is Critical for the High Selectivity of the PeptoidAntagonist PD168368*

Received for publication, July 10, 2000, and in September 22, 2000Published, JBC Papers in Press, September 29, 2000, DOI 10.1074/jbc.M006059200

Kenji Tokita‡, Simon J. Hocart§, Tatsuro Katsuno‡, Samuel A. Mantey‡, David H. Coy§, andRobert T. Jensen‡¶

From the ‡Digestive Diseases Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892-1804 and the§Department of Medicine, Peptide Research Laboratories, Tulane University Health Sciences Center,New Orleans, Louisiana 70112

Peptoid antagonists are increasingly being describedfor G protein-coupled receptors; however, little isknown about the molecular basis of their binding. Re-cently, the peptoid PD168368 was found to be a potentselective neuromedin B receptor (NMBR) antagonist. Toinvestigate the molecular basis for its selectivity for theNMBR over the closely related receptor for gastrin-re-leasing peptide (GRPR), we used a chimeric receptorapproach and a site-directed mutagenesis approach.Mutated receptors were transiently expressed in Balb3T3. The extracellular domains of the NMBR were notimportant for the selectivity of PD168368. However, sub-stitution of the 5th upper transmembrane domain(uTM5) of the NMBR by the comparable GRPR domainsdecreased the affinity 16-fold. When the reverse studywas performed by substituting the uTM5 of NMBR intothe GRPR, a 9-fold increase in affinity occurred. Each ofthe 4 amino acids that differed between NMBR andGRPR in the uTM5 region were exchanged, but only thesubstitution of Phe220 for Tyr in the NMBR caused adecrease in affinity. When the reverse study was per-formed to attempt to demonstrate a gain of affinity inthe GRPR, the substitution of Tyr219 for Phe caused anincrease in affinity. These results suggest that the hy-droxyl group of Tyr220 in uTM5 of NMBR plays a criticalrole for high selectivity of PD168368 for NMBR overGRPR. Receptor and ligand modeling suggests that thehydroxyl of the Tyr220 interacts with nitrophenyl groupof PD168368 likely primarily by hydrogen bonding. Thisresult shows the selectivity of the peptoid PD168368,similar to that reported for numerous non-peptide ana-logues with other G protein-coupled receptors, is pri-marily dependent on interaction with transmembraneamino acids.

Recently the “peptoid” approach was described for the designof low molecular weight, nonpeptide ligands (peptoid), usingthe chemical structure of mammalian neuropeptides as a start-ing point (1, 2). These peptoids may act as either agonists orantagonists at neuropeptide receptors. To date, several classes

of peptoid antagonists for receptors of gastrointestinal (GI)1

hormones/neurotransmitters have been described including forcholecystokinin (3–5), somatostatin (6), tachykinins (7–9), orbombesin (10) receptors. They have been proven useful in help-ing to examine the role of these receptors in mediating variousphysiological and pathophysiological processes and they maybe useful as therapeutic agents in such conditions as panicattacks (1, 2, 5). Whereas there have been a number of studiesof the molecular basis of action of nonpeptide antagonists forvarious GI hormone/neurotransmitter receptors (11–15) withinthe heptahelical G protein-coupled receptors (GPCRs), almostnothing is known about the molecular basis of action for pep-toid antagonists. For most small molecule ligands for GPCRssuch as nonpeptide antagonists, transmembrane regions playan important role in determining the high affinity binding (16,17). However, the essential receptor domains for the high se-lectivity of peptoid binding are still unclear.

Neuromedin B (NMB) and gastrin-releasing peptide (GRP),mammalian homologues of the amphibian tetradecapeptidebombesin, are small amidated peptides with structurally re-lated carboxyl termini (18). These peptides mediate a spectrumof biological activities by binding to two structurally and phar-macologically distinct receptors, the NMB receptor (NMBR)and GRP receptor (GRPR) (19, 20). These peptides have impor-tant effects in the central nervous system including thermoreg-ulation (21), satiety (22), control of circadian rhythm (23), andin peripheral tissues causing stimulation of gastrointestinalhormone release (18, 24, 25), activation of macrophages (26),and effects on development (27, 28). These peptides also havepotent growth effects causing proliferation of normal cells (18,29) and various tumor cell lines (18, 30). The NMBR and GRPRare members of the bombesin receptor family within the GPCRsuperfamily and share ;50% overall amino acid sequence iden-tity (19, 20). Both the NMBR and GRPR are widely distributedin the central nervous system and alimentary tract (18). Al-though the potential physiological role of GRP and its receptorhas been a major focus of research (18, 20), the role of NMB inphysiological or pathophysiological processes has receivedmuch less attention. Some studies suggest that NMB may playan important role in a number of biological processes, includingcausing growth of some tumor cells (31), a modulatory role insuppression of feeding behavior or gastric emptying (32), con-* The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby marked“advertisement” in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.

¶ To whom correspondence and reprint requests should be addressed:Digestive Diseases Branch, NIDDK, National Institutes of Health,Bldg. 10, Rm. 9C-103, 10 Center Dr., MSC 1804, Bethesda, MD 20892-1804. Tel.: 301-496-4201; Fax: 301-402-0600; E-mail: robertj@bdg10.niddk.nih.gov.

1 The abbreviations used are: GI, gastrointestinal; NMB, neuromedinB; NMBR, neuromedin B receptor; GRP, gastrin-releasing peptide;GRPR, gastrin-releasing peptide receptor; GPCR, G protein-coupledreceptor; FBS, fetal bovine serum; DMEM, Dulbecco’s modified Eagle’smedium; Bn, bombesin; CCK, cholecystokinin; ET, endothelin; NK1,neurokinin 1.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 1, Issue of January 5, pp. 495–504, 2001Printed in U.S.A.

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trol of the hypothalamic-pituitary-adrenocortical axis and thy-rotropin release (33), sensory transmission in the spinal cord(34), excitation of serotonin neurons in the dorsal raphe nu-cleus (35), control of potassium secretion by the blood-brainbarrier (36), and smooth muscle contractility (37). However,which of these are physiological or the principal roles of NMBRactivation in pathological processes remains unclear. The de-velopment of selective, high affinity NMBR agonists and an-tagonists would enable a more precise definition of the role ofNMB in these processes.

In contrast to GRPR antagonists for which numerous classesof high affinity antagonists have been described (38), the dis-covery process for NMBR antagonists has been slower. None ofthe strategies successfully used previously yielded potent an-tagonists when applied to the NMBR (38, 39). However, apeptoid based on a 3-amino acid template, PD165929, wasrecently characterized as a high affinity NMBR antagonist(10). In a previous study, we evaluated the pharmacology of asecond generation peptoid from this series, PD168368 (40, 41).The results confirmed that PD168368 was a potent and selec-tive antagonist for NMBR over GRPR regardless of speciesorigin, which could prove generally useful in understanding therole of NMBR in physiological and pathological processes.

To attempt to provide insight into the molecular basis of thespecificity of the peptoid PD168368 for the NMBR receptor, inthe present study we have investigated in detail the highaffinity of PD168368 for the NMBR, and its selectivity for theNMBR over the GRPR using a chimeric and mutagenesis ap-proach. To identify which domains of the NMBR are importantfor high affinity binding of PD168368, we used a chimericreceptor approach, which has proven useful in elucidating thestructural basis of GPCR interaction with ligands (42). A site-directed mutagenesis approach was then used to identify crit-ical amino acid(s) within these domains. Here we report thepeptoid antagonist PD168368’s selectivity for the NMBR overthe GRPR depends primarily on an interaction with aminoacids in the 5th upper transmembrane region of the NMBR.Detailed site-directed mutagenesis studies demonstrate thatTyr220 in this region is a key amino acid for high affinityPD168368 binding. Computer modeling of the receptor andanalysis of the ligand support the conclusion that PD168368interacts with the hydroxyl group of Tyr220 principally throughhydrogen bonding.

EXPERIMENTAL PROCEDURES

Materials—pcDNA3 was from Invitrogen (Carlsbad, CA). Oligonu-cleotides were from Midland Certified Reagent Company (Midland, TX)and Life Technologies, Inc. Seamlessy cloning kit and QuikChangeysite-directed mutagenesis kit were from Stratagene (La Jolla, CA).Restriction endonucleases (HindIII, XbaI, and EcoRI), fetal bovine se-rum (FBS), penicillin-streptomycin, LipofectAMINEy reagent, Lipo-fectAMINEy Plus reagent, and trypsin-EDTA (0.05% trypsin, 0.53 mM

EDTA-4Na) were from Life Technologies, Inc. Dulbecco’s modified Ea-

gle’s medium (DMEM) and Dulbecco’s phosphate-buffered saline werefrom Biofluids, Inc. (Rockville, MD). Balb 3T3 cells were from AmericanType Culture Collection (Rockville, MD). A 100 3 20-mm tissue culturedish (Falcon® 3003) was from Becton Dickinson (Plymouth, UnitedKingdom). Bombesin (Bn) was from Peninsula Laboratories, Inc. (Bel-mont, CA). Na125I (2,200 Ci/mmol) was from Amersham PharmaciaBiotech. 1,3,4,6-Tetrachloro-3a,6a-diphenylglycouril (IODO-GEN®)and dithiothreitol were from Pierce. Bovine serum albumin fraction Vand HEPES were from ICN Pharmaceutical Inc. (Aurora, OH). Soybeantrypsin inhibitor type I-S and bacitracin were from Sigma. Nyosil M20oil (specific gravity 1.0337) was from Nye Lubricants Inc. (New Bedford,MA). PD168368 was a gift from Robert Pinnock (Medicinal ChemistryDepartment, Pfizer Global Research, Cambridge, United Kingdom). Allother chemicals were of the highest purity commercially available.

Construction of Chimeric and Mutant Receptors—The cDNAs of themouse GRPR and rat NMBR were identical to those described previ-ously (42). The receptor extracellular domains or upper transmembranedomains used to make chimeric receptors were those identified usinghydropathy plots for the GRPR and for the NMBR. The amino acids inthese regions of the two receptors were aligned using the WisconsinPackage (Version 9.1; Genetics Computer Group, Madison, WI) forcomparisons. The cDNA of the wild-type mouse GRPR was insertedbetween the HindIII site and XbaI site of pcDNA3, and the wild-type ratNMBR was inserted into the EcoR-I site of pcDNA3. Both the GRPR/NMBR extracellular domain chimeras and upper transmembranechimeras were constructed using the Seamlessy cloning kit. Mutantreceptors were made by using the QuikChangey site-directed mu-tagenesis kit, following the manufacturer’s instructions except that theannealing temperature was 60 °C and the DpnI digestion was for 2 h.Nucleotide sequence analysis of the entire coding region was performedusing an automated DNA sequencer (ABI Prismy 377 DNA sequencer;Applied Biosystems Inc., Foster City, CA).

Cell Transfection—Balb 3T3 cells were seeded in a 10-cm diametertissue culture dish at a density of 106 cells/dish and grown overnight at37 °C in DMEM supplemented with 10% (v/v) FBS, 100 units/ml peni-cillin, and 100 mg/ml streptomycin. The following morning cells weretransfected with 5 mg of plasmid DNA by cationic lipid-mediatedmethod using 30 ml of LipofectAMINEy reagent and 20 ml of Lipo-fectAMINEy Plus reagent in serum-free DMEM for 3 h at 37 °C. At theend of the incubation period, the medium was replaced with DMEMsupplemented with 10% (v/v) FBS, 100 units/ml penicillin, and 100mg/ml streptomycin. Cells were maintained at 37 °C, with a 5% CO2

atmosphere and were used 48 h later for binding assays.Preparation of 125I-[Tyr4]Bn and 125I-[D-Tyr6,b-Ala11,Phe13,Nle14]Bn-

(6–14)—125I-[Tyr4]Bn and 125I-[D-Tyr6,b-Ala11,Phe13,Nle14]Bn-(6–14) ata specific activity of 2,200 Ci/mmol were prepared by a modification ofthe methods described previously (39, 40, 43). Briefly, 0.8 mg of IODO-GEN in chloroform was transferred to a vial, dried under a stream ofnitrogen and washed with 100 ml of KH2PO4 (pH 7.4). To this vial, 20 mlof 0.5 M KH2PO4 (pH 7.4), 8 mg of peptide in 4 ml of water, and 2 mCi (20ml) of Na125I were added, mixed gently and incubated at room temper-ature for 6 min. The incubation was stopped by the addition of 100 ml ofdistilled water and in the case of 125I-[Tyr4]Bn, 300 ml of 1.5 M dithio-threitol was also added to reduce the oxidized methionines. The iodi-nation mixture was re-incubated at 80 °C for 60 min. The reactionmixtures were applied to a Sep-Pak (Waters Associates, Milford, MA),and free 125I was eluted with 5 ml of water followed by 5 ml of 0.1% (v/v)trifluoroacetic acid. The radiolabeled peptides were eluted with 200 mlof sequential elutions (310) with 60% acetonitrile in 0.1% trifluoroace-tic acid. The two or three fractions with the highest radioactivity were

FIG. 1. Structure of bombesin, gas-trin-releasing peptide (GRP-(14–27)),NMB, and the peptoid antagonist,PD168368.

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combined and purified on a reverse-phase, high performance liquidchromatography with a mBondaPak column (0.46 3 25 cm). The columnwas eluted with a linear gradient of acetonitrile in 0.1% trifluoroaceticacid (v/v) from 16–60% acetonitrile in 60 min, and 1-ml fractions werecollected and checked for radioactivity and receptor binding. The pH ofthe pooled fractions were adjusted to 7 using 0.2 M Tris (pH 9.5), andradioligands were stored in aliquots with 0.5% bovine serum albumin at220 °C.

Whole Cell Radioligand Binding Assays—Competitive binding as-says were performed 48 h after transfection as described previously (43,44). Briefly, disaggregated transiently transfected cells were incubatedfor 1 h at room temperature in 250 ml of binding buffer (pH 7.4) with theligands 125I-[Tyr4]Bn (2,200 Ci/mmol) or 125I-[D-Tyr6,b-Ala11,Phe13,Nle14]Bn-(6–14) (2,200 Ci/mmol) in the presence of the indicated con-centration of unlabeled peptides. The binding buffer contained 98 mM

NaCl, 6 mM KCl, 11.5 mM glucose, 5 mM fumarate, 5 mM glutamate, 5mM pyruvate, 24.5 mM HEPES, 0.2% (v/v) essential amino acid solution,2.5 mM KH2PO4, 1 mM MgCl2, 0.5 mM CaCl2, 0.2% (w/v) bovine serumalbumin, 0.05% (w/v) bacitracin, and 0.01% (w/v) soybean trypsin in-hibitor. The cell concentration was adjusted to 0.15–9 3 106 cells/ml toassure that no more than 20% of the total added radioactive ligandbound. Bound tracer was then separated from unbound tracer by lay-ering 100 ml of the binding reaction on top of an oil phase (100 ml ofNyosil M20, Nye Lubricants Inc., New Bedford, MA) in a 0.4-ml micro-centrifuge tube (PGC Scientific, Frederick, MD), and pelleting the cellsthrough the oil by centrifugation at 10,000 3 g in a Microfuge Ey(Beckman, Palo Alto, CA) for 3 min. The supernatant was aspirated,and the pelleted cells were rinsed twice with distilled water. Theamount of radioactivity bound to the cells was measured in a Cobra IIg counter (Packard, Downers, IL). Aliquots (100 ml) of the incubation

mixture were taken in duplicate to determine the total radioactivity.Binding was expressed as the percentage of total radioactivity that wasassociated with the cell pellet. All binding values represented saturablebinding (i.e. total binding minus nonsaturable binding). Nonsaturablebinding was ,15% of the total binding in all experiments. Each pointwas measured in duplicate, and each experiment was replicated at leastthree times. Calculation of IC50 values was performed with a curve-fitting program, KaleidaGraph graphing software (Synergy Software,Reading, PA).

Visualization of PD168368—In an attempt to visualize the hydrogenbonding potential and likely hydrogen bonding donor and acceptor sitesof PD168368, we generated a model for the ligand in the molecularmodeling suite Sybyl 6.6 (Tripos Inc., St. Louis, MO). The model wasinitially generated in a reasonable low energy conformation using Con-cord (45, 46), a program widely used to generate three-dimensionalstructures. This model was then optimized by energy minimizationusing the Sybyl 6.6 Tripos force field with atomic charges calculated bythe method of Gasteiger-Huckel (47–49). The molecular surface wasvisualized as a Connolly dot surface. The molecular volume was 558 Å2.

Binding Site Model—The coordinates of the NMB transmembranereceptor model (NMBR_Vriend) were retrieved from the GPCR database server on the World Wide Web (50). In a attempt to better definethe NMBR binding pocket of PD168368, the receptor was modeled usingthe a-carbon template from bacteriorhodopsin, a seven-transmembranereceptor characterized by electron cryomicroscopy (51), the WHAT IFprogram (52), and Sybyl 6.6 (Tripos, Inc.). Following optimization of themodel receptor transmembrane bundle by extensive energy minimiza-tion using the Kollman all force field (51) in Sybyl 6.6, putative solventaccessible binding sites were explored using the SiteID module in Sybyl6.6. The surface of the solvent cluster filling the binding site wasvisualized using the Molcad module of Sybyl.

RESULTS

Wild-type NMBR and GRPR—The Bn-related natural occur-ring agonists Bn and GRP (Fig. 1, Table I) had high affinities(IC50 values of 0.5 and 2 nM, respectively) for the GRPR, andNMB (Fig. 1, Table I) for the NMBR (IC50 5 1.0 nM). Bn had a3-fold and GRP an 11-fold selectivity for the GRPR, whereasNMB had 100-fold selectivity for the NMBR (Table I).PD168368 (Fig. 1) had 96-fold lower affinity for the NMBR(IC50 5 96 6 8 nM) than NMB (Table I). However, PD168368also had a very low affinity for the GRPR (IC50 5 3,500 6 110nM) with the result that PD168368 had a 36-fold selectivity forthe NMBR over the GRPR (Table I).

Extracellular Chimeric Receptors—To begin to explore themolecular basis for this difference in affinity of PD168368 forNMBR and GRPR, four chimeric receptors with the extracel-lular domains of GRPR substituted for the comparable domains

FIG. 2. Importance of extracellularreceptor domains for determiningPD168368 selectivity for NMBR overGRPR: loss of affinity NMBR chime-ras. The chimeric NMBRs were formed byreplacing each of the extracellular loopsone at a time by the comparable GRPRloop as shown in the diagram at the top.The affinity was measured by competitiveradioligand displacement of 50 pM 125I-[Tyr4]Bn by PD168368 at the concentra-tions shown. Each point on each dose-inhibition curve is the mean from threeseparate experiments, and in each exper-iment each point was measured in dupli-cate. e1-, e2-, e3-, and e4-GRPR refer tosubstitution of this extracellular loop ofthe GRPR for the comparable loop in theNMBR.

TABLE IComparison of binding affinities of Bn-related naturally occurring

peptides and the peptoid antagonist, PD168368, for wild-type GRPRand NMBR

Balb 3T3 cells were transfected with either wild-type GRPR or NMBRand incubated with 50 pM 125I-[Tyr4]Bn alone or with increasing con-centrations of the indicated unlabeled peptides/peptoid. The concentra-tion causing half-maximal inhibition of binding, IC50, was determinedby using the curve-fitting program KaleidaGraph. Values are mean 6S.E. from four different experiments and in each experiment each valuewas determined in duplicate.

Peptide/peptoid

IC50

GRPR NMBR

nM

Bn 0.49 6 0.01 1.5 6 0.1GRP 1.9 6 0.1 21 6 1NMB 100 6 2 1.0 6 0.1PD168368 3,500 6 110 96 6 8

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in NMBR (loss of affinity chimeras, see Fig. 2) and four chime-ras with the extracellular domains of NMBR substituted intoGRPR (gain of affinity chimeras, see Fig. 3) were made. Theaffinities of PD168368 for the NMBR chimeras in which the1st, 2nd, and 4th extracellular domain in the NMBR was sub-stituted by the comparable domain of GRPR were similar towild-type NMBR (Fig. 2, Table II). Substitution of the 3rdextracellular domain in the NMBR by the comparable domainof the GRPR decreased the affinity almost 2-fold from 96 6 7.8nM to 180 6 17 nM (Fig. 2, Table II). However, this representedonly a small change compared with the 36-fold lower affinityPD168368 had for the wild type GRPR compared with the wildtype NMBR (Fig. 2, Tables I and II). To confirm these results,gain of affinity chimeras were made by substituting in theGRPR, the comparable extracellular domains of the NMBR(Fig. 3). The 3rd extracellular domain substitution into theGRPR increased potency, causing a 3-fold gain in affinity from3,500 6 110 nM to 1,100 6 49 nM (Fig. 3, Table II). However,chimeras formed by the insertions of the 1st, 2nd, or 4th extra-cellular loop of NMBR into GRPR caused no change in affinity(Fig. 3, Table II).

Upper Transmembrane Chimeric Receptors—Because substi-tutions of the extracellular domains caused only a small changein affinity of PD168368, suggesting they were playing only aminor role in the selectivity of PD168368 for the NMBR, weapplied a similar chimeric approach to the receptor’s uppertransmembrane regions, which are the transmembranes areasmost likely to interact with the ligand. The affinities of poten-tial loss of affinity NMBR chimeras in which 1st, 2nd, 3rd, 4th,and 7th upper transmembrane domain in the NMBR was sub-stituted by the comparable domain from GRPR showed nochange in affinity for PD168368 compared with the wild typeNMBR (Fig. 4, Table III). However, substitution of the 5th orthe 6th upper transmembrane domain of the NMBR by thecomparable GRPR domains decreased the affinity 16-fold (i.e.96 6 7.8 nM to 1,540 6 150 nM) and 2-fold (96 6 7.8 nM to 190 69.4 nM), respectively (Fig. 4, Table III). When the reverse studywas performed to make potential gain of affinity chimeras bysubstituting into the GRPR the 5th upper transmembranedomain of NMBR, a 9-fold increase in affinity (3,500 6 110 nM

to 390 6 36 nM) occurred (Fig. 5, Table III). In contrast, sub-stitutions of the other upper transmembrane domains ofNMBR into the GRPR, including the upper 6th transmembrane

region of NMBR, caused no gain of affinity (Fig. 5, Table III). Asimultaneous substitution into the GRPR of the 3rd extracel-lular domain and the 5th upper transmembrane domain ofNMBR resulted in a 36-fold increase in affinity for PD168368and the resultant receptor had similar affinity for PD168368 asthe wild type NMBR (Fig. 5, Table III).

Point Mutants in the 5th Upper Transmembrane Do-main—To identify which amino acid(s) in the 5th upper trans-membrane domain of the NMBR and GRPR were responsiblefor the selectivity of PD168368 for the NMBR, the amino acidsequence of this region of the GRPR and NMBR was compared(Fig. 6). The two receptors differed in 4 amino acids in the 5thtransmembrane region (Fig. 6). Specifically, the isoleucine inposition 216 in the NMBR was replaced by a serine in theGRPR, tyrosine 220 by phenylalanine, phenylalanine 221 bytyrosine, and leucine 222 in NMBR by valine in the comparableposition of GRPR (Fig. 6). To determine the importance of thesefour amino acid differences, potential loss of affinity NMBRpoint mutations were made by substituting into the NMBR thecomparable amino acid from the GRPR (Figs. 6 and 7) and fourpotential gain of affinity GRPR point mutations were made bysubstituting into the GRPR the comparable amino acid fromthe NMBR (Figs. 6 and 8). For the potential loss of affinityNMBR point mutations, three of the substitutions (Ser216 for

TABLE IIAffinities of PD168368 for wild-type NMBR, loss of affinity NMBR,and gain of affinity GRPR extracellular loop chimeric receptors and

wild-type GRPRAffinities were calculated by competitive displacement of 50 pM 125I-

[Tyr4]Bn by PD168368. Values represent the mean 6 S.E. from at leastthree independent experiments.

Receptor IC50 for PD168368

nM

Wild-type NMBR 96 6 8[e1-GRPR]NMBR 87 6 10[e2-GRPR]NMBR 96 6 10[e3-GRPR]NMBR 180 6 17[e4-GRPR]NMBR 98 6 4

Wild-type GRPR 3,500 6 110[e1-NMBR]GRPR 4,600 6 260[e2-NMBR]GRPR 2,100 6 80[e3-NMBR]GRPR 1,100 6 49[e4-NMBR]GRPR 3,000 6 92

FIG. 3. Importance of extracellularreceptor domains for determiningPD168368 selectivity for NMBR overGRPR: gain of affinity GRPR chime-ras. The chimeric GRPR’s were formed byreplacing each of the extracellular loops ofGRPR by the comparable loop of theNMBR one at a time as shown in thediagram. The affinity was measured bycompetitive radioligand displacement of50 pM 125I-[Tyr4]Bn by PD168368 at theconcentrations shown. Each point on thedose-inhibition curve is the mean fromthree separate experiments, and in eachexperiment each point was measured induplicate. e1-, e2-, e3-, and e4-NMBR re-fer to substitution of this extracellularloop of the NMBR for the comparable loopin the GRPR.

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Ile, Tyr221 for Phe, and Val222 for Leu) did not significantlyalter the high affinity for PD168368 compared with the wildtype NMBR (Table IV, Fig. 7). In contrast, the substitution ofPhe220 for Tyr in the NMBR caused a 8.6-fold decrease inaffinity (96 6 7.8 nM to 830 6 63 nM) (Table IV, Fig. 7). Whenthe reverse study was performed to attempt to demonstrate again of affinity in the GRPR, the substitution of Tyr219 for Phecaused 6.4-fold increase in affinity (Table IV, Fig. 8). The otherthree potential GRPR gain of affinity point mutants showed nogain in affinity for PD168368 (Table IV, Fig. 8).

Visualization of PD168368—To attempt to identify using thecomputer modeling suite Sybyl 6.6 and calculating charges bythe method of Gasteiger-Huckel with the molecular energyminimized, the hydrogen bonding potential of PD168368 wasvisualized (Fig. 9). This generated a Connolly surface of themolecule, colored blue in hydrogen bond accepting regions andred in hydrogen bond donating regions (Fig. 9). The molecularvolume was 558 Å2. PD168368 had a large hydrogen bondaccepting region around the nitrophenyl group and smaller

hydrogen bond acceptor regions associated with the pyridylgroup and amide bonds (Fig. 9).

Binding Site Model—To attempt to better define the bindingpocket of PD168368 to NMBR, the NMBR (Fig. 10) was ana-lyzed using modeling programs. The NMBR was analyzed us-ing the a-carbon template from the bacteriorhodopsin receptorderived from electron microscopy (51), the WHAT IF program(52), and Sybyl 6.6. This indicated a possible binding site nearthe extracellular surface, bounded by the extracellular surfaceand Pro120, Gln123, Leu124, and Val127 of helix 3; Trp168, Ser171,and Glu178 of helix 4; Ile216 and Tyr220 of helix 5; Trp279,Asn282, His283, and Tyr286 of helix 6; and Ser313 of helix 7, witha volume of .700 Å2 (Fig. 10). Amino acid side chains facingthe interior of the transmembrane bundle within 3.5 Å of thesolvent cluster were highlighted to indicate the putative bind-ing site of PD168368 (Fig. 10). The critical Tyr220 residue of the5th transmembrane domain of NMBR was found to face theinterior of the putative binding pocket formed by transmem-brane domains 3–7 (Fig. 10).

DISCUSSION

There are now more than 30 GPCRs mediating the action ofGI hormones or neurotransmitters such as bradykinin, GRP, orcholecystokinin (CCK) (53). In most cases their roles in physi-ological processes or in pathological conditions are still unclearbecause only recently have high affinity antagonists been de-veloped for some of these receptors. The antagonists for thesereceptors generally fall into one of three types: peptide antag-onists, nonpeptide antagonists, or peptoid antagonists, whichhave features of both peptides and nonpeptides (1, 2). In gen-eral the molecular basis of action of these antagonists is poorlyunderstood, and it is unclear whether it is similar to the wellstudied adrenergic or muscarinic cholinergic receptor antago-nists (54–56). Whereas there have been a number of studies ofthe molecular basis of action of nonpeptide antagonists forvarious GI hormone receptors (11–15), there are only a fewstudies of peptide antagonists (12, 57, 58) and almost none forpeptoid antagonists (59). In this study we examined the molec-ular basis of action of the novel peptoid receptor antagonistPD168368, which is reported to be selective for the NMBR inthe bombesin family of receptors (10, 40, 41).

A number of our results support the conclusion that theNMBR receptor extracellular domains do not play a prominentrole in determining the selectivity of PD168368 for NMBR overthe structurally closely related receptor, the GRPR. First, when

FIG. 4. Importance of the uTM re-gions in determining selectivity ofPD168368 for NMBR over GRPR: lossof affinity NMBR transmembranechimeras. The chimeric uTM NMBRswere formed by replacing each of thetransmembrane domains of NMBR by thecomparable domain of the GRPR one at atime as shown in the diagram at the top ofthe figure. Affinities were measured bycompetitive radioligand displacement of50 pM 125I-[Tyr4]Bn or 125I-[D-Tyr6,b-Ala11,Phe13,Nle14]Bn-(6–14) by PD168368at the concentrations shown. Each pointon the dose-inhibition curve is the meanfrom three separate experiments, and ineach experiment each point was meas-ured in duplicate. uTM1-, uTM2-, uTM3-,uTM4-, uTM5-, uTM6-, and uTM7-GRPR-NMBR refer to replacement bythis transmembrane domain of theNMBR by that from the GRPR.

TABLE IIIAffinities of PD168368 for wild-type NMBR, loss of affinity NMBR,and gain of affinity GRPR upper transmembrane chimeric receptors

and wild-type GRPRAffinities were calculated by competitive displacement of 50 pM 125I-

[Tyr4]Bn or 125I-[D-Tyr6,b-Ala11,Phe13,Nle14]Bn-(6–14) (*) byPD168368. Values represent the mean 6 S.E. from at least threeindependent experiments.

Receptor IC50 forPD168368

nM

Wild-type NMBR 96 6 8[uTM1-GRPR]NMBR 63 6 3[uTM2-GRPR]NMBR 91 6 4[uTM3-GRPR]NMBR 42 6 2[uTM4-GRPR]NMBR 79 6 7[uTM5-GRPR]NMBR 1,540 6 145*[uTM6-GRPR]NMBR 194 6 9[uTM7-GRPR]NMBR 47 6 3

Wild-type GRPR 3,500 6 110[uTM1-NMBR]GRPR 6,900 6 270[uTM2-NMBR]GRPR 4,700 6 420[uTM3-NMBR]GRPR 5,200 6 370[uTM4-NMBR]GRPR 4,400 6 210[uTM5-NMBR]GRPR 390 6 36[e3,uTM5-NMBR]GRPR 67 6 3[uTM6-NMBR]GRPR 3,600 6 130[uTM7-NMBR]GRPR 3,700 6 190

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extracellular domains of the GRPR were substituted into theNMBR to assess loss of affinity for PD168368, only the substi-tution of the 3rd extracellular domain in the NMBR by thecomparable domain of the GRPR altered affinity and the de-crease in affinity was less than 2-fold. Second, when the reversestudy was performed by substituting into the GRPR the extra-cellular domain of the NMBR to produce gain of affinity chi-meras, only the substitution of the 3rd extracellular domain inthe GRPR by the comparable domain of the NMBR alteredaffinity causing an almost 3-fold increase for PD168368. Thecontribution of the 3rd extracellular domain for high selectivityof PD168368 was small compared with the almost 40-foldhigher affinity PD168368 had for the native NMBR over GRPR.These results therefore suggest that differences in the 3rdextracellular domain of these two receptors play only a smallrole in the selectivity of PD168368. This result has both simi-larities and differences from studies on the interaction of pep-tide and nonpeptide agonists and antagonists with otherGPCRs. Mutagenesis and biophysical analyses of severalGPCRs indicate that the receptor extracellular domain can bean important receptor region for determining high affinity li-gand binding (16). Only a few studies have explored whetherpeptide antagonists interacting with receptor extracellular do-mains. Such an interaction is important for determining high

affinity of the peptide antagonist JMV179 for the humanCCK-A receptor (12); however, for other peptide antagonistssuch as the interaction of D-Arg-[Hyp3,D-Phe7]bradykinin(NPC567) with the B2 bradykinin receptor (57) and BQ-123with the endothelin A (ET-A) receptor (13), the receptor extra-cellular domains are not important for high affinity interaction.Numerous studies show that with most nonpeptide antagonistssuch as the interaction of losartan with the AT1b angiotensin IIreceptor, L365,260 with the CCK-B/gastrin receptor, or BMS-182874 with the ET-A receptor, the extracellular region doesnot contain important determinants for high affinity interac-tion (11, 13, 60). However, interaction of nonpeptide substanceP antagonist CP96345 with the 3rd extracellular domain of theneurokinin 1 (NK1) receptor is important for determining se-lectivity for this receptor (61). For a number of peptide GIreceptor neurotransmitter/hormones that function as agonistshigh affinity receptor binding has been shown to dependent oninteraction with receptor extracellular domain, includingCCK-8 with the CCK-A or CCK-B receptor (62), bradykininwith B2 bradykinin receptor (63), and substance P with theNK1 receptor (64). On the other hand, with many nonpeptide

FIG. 5. Importance of the uTM re-gions in determining selectivity ofPD168368 for NMBR over GRPR: gainof affinity GRPR transmembrane chi-meras. The chimeric uTM GRPRs wereformed by replacing each of the trans-membrane domains of GRPR by the com-parable domain of the NMBR one at atime as shown in the diagrams at the topof the figure. Affinities were measured bycompetitive radioligand displacement of50 pM 125I-[Tyr4]Bn by PD168368 at theconcentrations shown. Each point on thedose-inhibition curve is the mean fromthree separate experiments, and in eachexperiment each point was measured induplicate. uTM1-, uTM2-, uTM3-, uTM4-,uTM5-, uTM6-, and uTM7-NMBR-GRPRrefer to replacement by this transmem-brane domain of the GRPR by that fromthe NMBR. Arrows indicate large changesin affinity of the GRPR.

FIG. 6. Alignment of amino acid sequences in the 5th uppertransmembrane domain of NMBR and GRPR. Boxes indicate di-vergent amino acids between these two receptors in the 5th uppertransmembrane region. Shown are the four NMBR and four GRPRpoint mutants made to explore the importance of each of the four aminoacid differences for determining PD168368 selectivity.

TABLE IVAffinities of PD168368 for the wild-type, receptor uTM5 loss and gain

of affinity point mutants of NMBR and GRPRAffinities were calculated by competitive displacement of 50 pM 125I-

[Tyr4]Bn or 125I-[D-Tyr6,b-Ala11,Phe13,Nle14]Bn-(6–14) (*) byPD168368. Values represent the mean 6 S.E. from at least threeindependent experiments. The numbers refer to the position in eitherthe NMBR or GRPR of the amino acid substitutions as shown in Fig. 6.[I216S]NMBR refers to replacement of the isoleucine in position 216 ofthe NMBR by serine, which exists in the comparable position in GRPR(see Fig. 6).

Receptor IC50 for PD168368

nM

Wild-type NMBR 96 6 8[uTM5-GRPR]NMBR 1,500 6 150*[I216S]NMBR 110 6 6*[Y220F]NMBR 830 6 63[F221Y]NMBR 150 6 3[L222V]NMBR 81 6 3

Wild-type GRPR 3,500 6 110[uTM5-NMBR]GRPR 390 6 36[S215I]GRPR 3,600 6 290[F219Y]GRPR 550 6 14[Y220F]GRPR 3,600 6 170[V221L]GRPR 4,900 6 320

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agonists such as a- and b-adrenergic agents, nucleosides, eico-sanoids, muscarinic cholinergic agents, and lipid moieties, theextracellular receptor domains are not important generally indetermining receptor selectivity and high affinity interaction(17). However, with small molecular nonpeptide agonists forthe G protein-coupled Ca21-sensing receptor; Ca21 and Gd31

interaction with the large NH2-terminal extracellular segmentof the receptor is important for high affinity interaction (65).

Because the extracellular domains play only a minor role inthe selectivity of PD168368 for the NMBR over the GRPR, weapplied a similar chimeric approach to the receptor’s uppertransmembrane region, which is also likely to interact withsmall ligands (16, 17). Our results support the conclusion thatamino acids in the 5th upper transmembrane region play amajor role, and in the 6th upper transmembrane region aminor role in determining the high selectivity of PD168368 forthe NMBR over the GRPR. In contrast, the amino acids in theother five upper transmembrane regions did not play a signif-icant role in this ligand’s selectivity. The importance of the 5thupper transmembrane region for PD168368 selectivity is con-sistent with the interaction of some small ligands (a- andb-adrenergic agents, nucleosides, eicosanoids, or muscariniccholinergic agents) (16, 17) but not others (Ca21, glutamate, org-aminobutyric acid) with their GPCRs, which provide evi-dence that upper transmembrane domains can be importantregions for determining the affinity of some small molecular

ligands to interact with receptors (17). High affinity receptorbinding of some peptide agonists have been shown to primarilydepend on interactions with the receptor upper transmem-brane domains. This includes interaction of various peptideagonists with angiotensin II receptor (66), the endothelin-Areceptor (ETA-R) (15), neuropeptide Y1 receptor (67), and thethyrotropin-releasing hormone receptor (68). However, withpeptide agonists for the B2 bradykinin receptor (63), the corti-cotropin-releasing factor receptor (69), or m- and d-opioid recep-tors (70), the transmembrane regions are not important. Insimilar fashion, several nonpeptide agonists have been shownto primarily interact with GPCR upper transmembrane regionsincluding nonpeptide agonists for adrenergic receptors (54, 55),muscarinic cholinergic receptors (56, 71, 72), or eicosanoid (73,74). High affinity receptor binding of some peptide antagonistsalso have been shown to depend primarily on interaction withthe upper transmembrane domains of receptors such as theinteraction of NPC567 and Hoe140 with the B2 bradykininreceptor (57, 58) or BQ-123 with the ETA-R (13), whereas withother GPCRs such as interaction of CCK-JMV179 with theCCK-A receptor (12), the upper transmembrane regions are notimportant. A number of studies show that for most nonpeptideantagonists the interaction with the GPCRs’ transmembranedomains are the primary determinants of high affinity receptorinteraction (13, 14, 60, 75–77). For example, with the AT1b

angiotensin II receptor, the nonpeptide antagonist losartan

FIG. 7. Importance of specific aminoacids in the receptors 5th uppertransmembrane region in determin-ing selectivity of PD168368 for theNMBR over the GRPR: loss of affinityNMBR point mutations. The point mu-tants in the NMBR were formed by re-placing each of the amino acid(s) of the5th upper transmembrane region ofNMBR by the comparable amino acid(s) ofthe GRPR one at a time, as shown in Fig.6. Affinities were measured by competi-tive radioligand displacement of 50 pM125I-[Tyr4]Bn or 125I-[D-Tyr6,b-Ala11,Phe13,Nle14]Bn-(6–14) by PD168368 at the con-centrations shown. Each point on thedose-inhibition curve is the mean fromthree separate experiments, and in eachexperiment each point was measured induplicate. I216S refers to replacement ofthe isoleucine in position 216 of theNMBR by serine, which exists in the com-parable position in GRPR (see Fig. 6).

FIG. 8. Importance of specific aminoacids in the receptors 5th uppertransmembrane region in determin-ing selectivity of PD168368 for theNMBR over the GRPR: gain of affin-ity GRPR point mutations. The pointmutants in the GRPR were formed by re-placing each of the amino acid(s) of the5th upper transmembrane region ofGRPR by the comparable amino acid(s) ofthe NMBR one at a time, as shown in Fig.6. Affinities were measured by competi-tive radioligand displacement of 50 pM125I-[Tyr4]Bn by PD168368 at the concen-trations shown. Each point on the dose-inhibition curve is the mean from threeseparate experiments, and in each exper-iment each point was measured in dupli-cate. [S215I] refers to replacement of theserine in position 215 of the GRPR byisoleucine, which exists in the comparableposition in NMBR (see Fig. 6). The arrowsindicate mutants showing a large changein affinity from the native GRPR.

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requires specific amino acid residues within the 3rd to 7thtransmembrane regions for high affinity binding (60). Similarlythe high affinity binding of the nonpeptide antagonist L365,260with CCK-B/gastrin receptor (75), BMS-182874 with ETA-R(13), SB209670 and Ro 46-2005 with ETB-R (14), or CP96345

and L-161,664 with NK1 receptor (76, 77) are all dependentprimarily on interaction with receptor upper transmembraneregions. Our results show that the peptoid antagonistPD168368 primarily resembles nonpeptide receptor antago-nist’s interaction with GPCRs in that its high affinity andselectivity for the NMBR depends primarily on interactionswith the receptor upper transmembrane areas.

For a few other ligands, the 5th transmembrane region isalso a critical region to interact with a GPCR as we found in thepresent study for PD168368. For example, the critical bindingsite for the 8-amino acid peptide agonist angiotensin II is in the5th transmembrane region of AT1a angiotensin II receptor (66).The high affinity interaction of the nonpeptide agonists acetyl-choline and carbachol depends on interaction with the 5thtransmembrane region of the m3 muscarinic receptor (72).Furthermore, high affinity receptor binding and selectivity forseveral nonpeptide antagonists depend on interact with spe-cific amino acids in the 5th transmembrane region, includingthe binding of the nonpeptide corticotropin releasing factorreceptor 1 antagonist NBI27914 (69) to the human cortico-tropin releasing factor receptor 1 and the binding of the NK1receptor antagonist L161,664 to the NK1 receptor (77).

To determine which amino acids in the 5th upper transmem-brane region of the NMBR account for the PD168368’s selec-tivity, we performed a comparative alignment of the aminoacids in this region between the NMBR and the GRPR. Withinthe 5th upper transmembrane domain, there were four aminoacid residues that differ between NMBR and GRPR (Fig. 6).These differences included a Ser for Ile change at position 216of the NMBR and Phe, Tyr, and Val at positions 219–221 of theGRPR for Tyr, Phe, and Leu at positions 220–222 of theNMBR. Our results support the conclusion that the tyrosineresidue in position 220 of NMBR instead of a phenylalanine inthis position in the GRPR is the key amino acid residue indetermining the selectivity of the antagonist PD168368 causedby the differences in the receptors’ 5th transmembrane region.

FIG. 9. An energy-minimized structure for PD168368. PD168368is shown as a ball and stick model (hydrogen, cyan; carbon, white;nitrogen, blue; oxygen, red). It is surrounded by a Connolly molecularsurface that has been colored to indicate hydrogen bond acceptor (blue)and donor (red) regions. Hydrophobic regions are colored gray.

FIG. 10. The NMB transmembrane receptor putative binding site surrounding the critical residue Tyr220. Helices 1–7 are coloredlight cyan, orange, red, purple, blue, green, and yellow, respectively. The putative binding site, established by computer modeling using SiteID, isdepicted as a gray opaque space-filling model. Residues surrounding the binding site, including Tyr220 (green), are shown as stick models. Aminoacid side chains not facing the center of the transmembrane region are omitted for clarity.

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In a previous study (42), we showed that the 5th transmem-brane domain of NMBR was critical also for high affinity NMBbinding. However, the isoleucine in position 216 was the keyamino acid for high affinity interaction of NMB with the NMBRover the GRPR. These results demonstrate that the key recep-tor determinants for the selectivity of the agonist NMB andpeptoid antagonist PD168368 for the NMBR over the GRPRare in very close proximity in the NMBR. This result is con-sistent with the strategy used to design PD168368, which de-pended strongly on incorporating critical structural featuresfor determining high affinity NMB binding to NMBR into thePD168368 (10). However, despite these considerations the pep-toid antagonist PD168368 and the peptide agonist NMB showa significant difference in their determinants of high affinityinteraction with the NMBR. Specifically, the key amino acid inthe 5th transmembrane domain (Tyr220) for high affinityNMBR interaction of PD168368 is different from the key aminoacid for the high affinity interaction of NMB (Ile216) with theNMBR, supporting the conclusion that significantly differentmolecular interactions are likely responsible for their highaffinities for the NMBR.

In several studies, a tyrosine residue in the GPCRs plays acritical role in ligand-receptor interaction (13, 66, 68, 73, 77).The tyrosine of the 3rd transmembrane domain of the thyro-tropin-releasing hormone receptor binds the pyroglutamyl moi-ety of thyrotropin-releasing hormone through hydrogen bond-ing (68). The tyrosine of the 6th transmembrane domain of them2 muscarinic receptor is a critical amino acid for the interactionwith nonpeptide agonists (acetylcholine, carbachol, oxotremorineM, and pilocarpine) (71), likely by a hydrogen bonding mecha-nism. The tyrosine hydroxyl moiety characteristically interactswith ligands by functioning as a hydrogen bond donor or as astrong locus of cation-p binding (73). The cation-p bindingoccurs through the side-chains of aromatic amino acids such asphenylalanine, tyrosine, or tryptophan (78). In our study thesubstitution of phenylalanine for tyrosine in the NMBR re-sulted in a marked decrease in affinity for PD168368. There-fore, it might be argued that the interaction of the tyrosinehydroxyl with PD168368 through hydrogen bonding is likelymore important than cation-p binding. The calculated D(DG°)value of 0.5 kcal/mol for the peptoid antagonist PD168368obtained from the difference in affinities by replacing Tyr220 ofthe NMBR by the comparable Phe219 of the GRPR is consistentwith the elimination of a hydrogen bond (79). However, tyro-sine is predicted to have a higher cation-p binding potentialthan phenylalanine secondary to the negative electrostatic po-tential of the oxygen (80). Therefore, whether the tyrosinehydroxyl is involved in increasing negative electrostatic poten-tial due to cation-p binding site interaction or to hydrogenbonding cannot be resolved by our studies.

To further explore the putative binding site for PD168368,three-dimensional modeling of the NMBR based on the struc-ture of bacteriorhodopsin (51) was employed. In this model, thecritical Tyr220 residue on the 5th transmembrane domain of theNMBR was found to face the interior of a large binding pocketformed primarily by transmembrane domains 3–7 (Fig. 10).Examination of a minimum energy conformation of the ligandshowed that it is dominated by a large hydrogen bond acceptingregion around the nitrophenyl group, and smaller acceptorregions associated with the pyridyl moiety and amide bonds(Fig. 9). The ligand also has smaller hydrogen bond donorregions associated with the Trp moiety and the amide linkages.The rest of the molecule consists of uncharged hydrocarbonstructures that prefer a lipid or hydrophobic environment (Fig.9). Thus, it is feasible for the ligand PD168368 to be able toenter the intratransmembrane binding pocket to interact with

Tyr220 while simultaneously being capable of interacting withextracellular loops. If this interaction is primarily dependenton hydrogen bonding as our data suggest, it is most likely thatthe hydroxyl of the Tyr220 interacts with nitrophenyl group ofPD168368 or perhaps one of the other hydrogen acceptorgroups on PD168368. This proposal is supported by the factthat, in PD168368, the largest hydrogen bond accepting groupis the nitro group on the phenyl ring (Fig. 9). This is also themost accessible of the hydrogen bond acceptors in the ligand.The other hydrogen bond acceptors, the pyridyl group, and thebackbone carbonyls are much less accessible. Our receptormodel indicates that Pro120, Gln123, Leu124, and Val127 of TMhelix 3; Trp168, Ser171, and Glu178 of TM helix 4; Ile216 andTyr220 of TM helix 5; Trp279, Asn282, His283, and Tyr286 of TMhelix 6; and Ser313 of TM helix 7 are all facing the putativebinding pocket and could interact with a small ligand such asPD168368 (Figs. 9 and 10). Pro120, Gln123, Leu124, and Val127 ofTM helix 3; Trp168, Ser171, and Glu178 of TM helix 4; Trp279,Asn282, His283, and Tyr286 of TM helix 6; and Ser313 of TM helix7 in the NMBR are the same as the comparable amino acids inthe GRPR and therefore are unlikely to be important in theselectivity of PD168368 for the NMBR over the GRPR. Of theother two amino acids, Ile216 and Tyr220, our mutagenesisstudies show only Tyr220 is important in determining the se-lectivity of PD168368 for the NMBR over the GRPR. Therefore,we conclude that Tyr220 is the important amino acid in deter-mining the selectivity of PD168368 of the transmembraneamino acids facing the binding pocket. Our studies suggest thatamino acids within the 3rd extracellular domain have a smalleffect on determining the affinity of PD168368. Although the3rd extracellular domain of the NMBR is in close proximity tothe putative binding pocket of PD168368 (Fig. 10) in the pres-ent study because of its relative small effect on PD168368affinity, we did not investigate in detail which amino acidswithin this domain are responsible for the slight differences inaffinity. We therefore cannot speculate on the type of ligandreceptor interaction responsible for this small effect of the 3rdextracellular domain.

In conclusion, our receptor chimeric studies showed that the5th transmembrane domain of the NMBR was a responsibleregion for the high affinity and selectivity of the peptoid antag-onist PD168368 for the NMBR over the GRPR. Our mutagen-esis studies show Tyr220 in the 5th upper transmembranedomain of the NMBR was the key amino acid interacting withthis antagonist. To our knowledge, this is the first study thatreveals the molecular basis of action between a peptoid antag-onist and receptor. The described data from these studies al-lowed us to obtain a more detailed picture of the receptor-ligand interaction and propose a model that could account forimportant receptor-ligand interactions. The availability of thismodel could be of use not only for studying the molecular basisof the interaction of natural agonists, but also for peptoidantagonists or peptide antagonists for this important family ofreceptors.

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Basis of NMBR Selectivity of PD168368504

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Robert T. JensenKenji Tokita, Simon J. Hocart, Tatsuro Katsuno, Samuel A. Mantey, David H. Coy and

Critical for the High Selectivity of the Peptoid Antagonist PD168368Tyrosine 220 in the 5th Transmembrane Domain of the Neuromedin B Receptor Is

doi: 10.1074/jbc.M006059200 originally published online September 29, 20002001, 276:495-504.J. Biol. Chem. 

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