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Originally published In Press as doi:10.1074/jbc.M104566200 on August 2, 2001

J. Biol. Chem., Vol. 276, Issue 39, 36652-36663, September 28, 2001
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Molecular Basis for Selectivity of High Affinity Peptide Antagonists for the Gastrin-releasing Peptide Receptor*

Kenji TokitaDagger , Tatsuro KatsunoDagger , Simon J. Hocart§, David H. Coy§, Muriel Llinares, Jean Martinez, and Robert T. JensenDagger ||

From the Dagger  Digestive Diseases Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892-1804, the § Department of Medicine, Peptide Research Laboratories, Tulane University Health Sciences Center, New Orleans, Louisiana 70112, and  Faculté de Pharmacie, Universités de Montpellier, Montpellier 34060, France

Received for publication, May 18, 2001, and in revised form, June 28, 2001

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Few gastrointestinal hormones/neurotransmitters have high affinity peptide receptor antagonists, and little is known about the molecular basis of their selectivity or affinity. The receptor mediating the action of the mammalian bombesin (Bn) peptide, gastrin-releasing peptide receptor (GRPR), is an exception, because numerous classes of peptide antagonists are described. To investigate the molecular basis for their high affinity for the GRPR, two classes of peptide antagonists, a statine analogue, JMV594 ([D-Phe6,Stat13]Bn(6-14)), and a pseudopeptide analogue, JMV641 (D-Phe-Gln-Trp-Ala-Val-Gly-His-Leupsi (CHOH-CH2)-(CH2)2-CH3), were studied. Each had high affinity for the GRPR and >3,000-fold selectivity for GRPR over the closely related neuromedin B receptor (NMBR). To investigate the basis for this, we used a chimeric receptor approach to make both GRPR loss of affinity and NMBR gain of affinity chimeras and a site-directed mutagenesis approach. Chimeric or mutated receptors were transiently expressed in Balb/c 3T3. Only substitution of the fourth extracellular (EC) domain of the GRPR by the comparable NMBR domain markedly decreased the affinity for both antagonists. Substituting the fourth EC domain of NMBR into the GRPR resulted in a 300-fold gain in affinity for JMV594 and an 11-fold gain for JMV641. Each of the 11 amino acid differences between the GRPR and NMBR in this domain were exchanged. The substitutions of Thr297 in GRPR by Pro from the comparable position in NMBR, Phe302 by Met, and Ser305 by Thr decreased the affinity of each antagonist. Simultaneous replacement of Thr297, Phe302, and Ser305 in GRPR by the three comparable NMBR amino acids caused a 500-fold decrease in affinity for both antagonists. Replacing the comparable three amino acids in NMBR by those from GRPR caused a gain in affinity for each antagonist. Receptor modeling showed that each of these three amino acids faced inward and was within 5 Å of the putative binding pocket. These results demonstrate that differences in the fourth EC domain of the mammalian Bn receptors are responsible for the selectivity of these two peptide antagonists. They demonstrate that Thr297, Phe302, and Ser305 of the fourth EC domain of GRPR are the critical residues for determining GRPR selectivity and suggest that both receptor-ligand cation-pi interactions and hydrogen bonding are important for their high affinity interaction.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The gastrin-releasing peptide (GRP)1 receptor, which mediates the diverse actions of the mammalian bombesin (Bn)-related peptide (1, 2), GRP, has numerous high affinity peptide antagonists (3-5). This is in contrast to most other gastrointestinal (GI) hormone/neurotransmitter receptors for which no high affinity peptide antagonists exist (6). These GRP receptor antagonists are now widely used in both in vitro studies (5) and in vivo studies in animals (3, 7-11) and humans (12). Recent studies show that for many nonpeptide antagonists, differences in amino acids in the transmembrane domains between receptor subtypes are frequently particularly important for determining receptor subtype selectivity (13, 14). A recent study (15) shows a similar result with the peptoid antagonist PD168368 for the neuromedin B receptor. However, with peptide antagonists of non-GI hormone/neurotransmitter receptors, interactions with transmembrane regions (16, 17) or extracellular domains (18) are important for high affinity interaction or receptor subtype selectivity. Which if any of these results apply to the different classes of GRPR peptide antagonists, at present, is unclear.

GRP and neuromedin B (NMB), mammalian homologues of the amphibian tetradecapeptide bombesin, have structurally related carboxyl termini (19). These peptides mediate a spectrum of biological activities such as stimulating growth of both normal and neoplastic tissues (20-23), secretion (1), muscle contraction (24), central nervous system effects (including satiety (25), thermoregulation (26), and circadian rhythm (27)), changed developmental (28, 29) and immunologic effects (30). These effects are mediated by binding to two structurally and pharmacologically distinct receptors, the GRPR and NMB receptor (NMBR) (31-33). These two receptors are members of the bombesin receptor family within the G protein-coupled receptor (GPCR) superfamily and share 56% overall amino acid sequence identity (34). Both the GRPR and NMBR are widely distributed in the central nervous system and peripheral tissues including in the GI tract (1, 35). Which of the widespread effects of these peptides (1) are important physiologically or in pathologic processes is largely unknown at present. The availability of potent Bn receptor subtype-specific antagonists and a molecular understanding of their basis of action are important steps in addressing these questions.

In the present study, we have examined the molecular basis for the GRPR selectivity of two different classes of closely related high affinity Bn receptor peptide antagonists: a Bn statine analogue, JMV594 (36), and a Bn pseudopeptide analogue, JMV641 (37). A receptor chimeric and site-directed mutagenesis approach was used to identify critical domain and amino acid(s) responsible for these antagonists' selectivity and high affinity for GRPR. In this study we show that the selectivity of peptide antagonists JMV594 and JMV641 for the GRPR over the NMBR depends primarily on interactions with amino acids in the fourth extracellular region of the GRPR. Site-directed mutagenesis studies demonstrate that Thr297, Phe302, and Ser305 in this region of GRPR are the critical amino acids for high affinity binding and selectivity of these two antagonists. Computer modeling of this region of the GRPR demonstrates that these amino acids all face inward forward the proposed binding pocket and are all within 5 Å of it, suggesting that cation-pi and hydrogen bonding interactions between these antagonists and the above three amino acids are essential for receptor subtype selectivity and high affinity interaction.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- pcDNA3 was from Invitrogen (Carlsbad, CA). Oligonucleotides were from Midland Certified Reagent Company (Midland, TX) and Life Technologies, Inc. SeamlessTM Cloning Kit and QuikChangeTM Site-Directed Mutagenesis Kit were from Stratagene (La Jolla, CA). Restriction endonucleases (HindIII, XbaI, and EcoRI), fetal bovine serum, penicillin-streptomycin, LipofectAMINETM reagent, LipofectAMINETM Plus reagent, and trypsin-EDTA (0.05% trypsin, 0.53 mM EDTA-4Na) were from Life Technologies. Dulbecco's modified Eagle's medium and Dulbecco's phosphate-buffered saline were from Biofluids, Inc. (Rockville, MD). Balb/c 3T3 cells were from the American Type Culture Collection (Manassas, VA). A 100 × 20-mm tissue culture dish (Falcon® 3003) was from Becton Dickinson (Plymouth, United Kingdom). Bn and neuromedin B were from Peninsula Laboratories, Inc. (Belmont, CA). Na125I (2,200 Ci/mmol) was from Amersham Pharmacia Biotech. 1,3,4,6-Tetrachloro-3alpha ,6alpha -diphenylglycouril (IODO-GEN®) and dithiothreitol were from Pierce. Bovine serum albumin fraction V and HEPES were from ICN Pharmaceutical Inc. (Aurora, OH). Soybean trypsin inhibitor type I-S and bacitracin were from Sigma. Nyosil M20 oil (specific gravity 1.0337) was from Nye Lubricants Inc. (New Bedford, MA). All other chemicals were of the highest purity commercially available.

Construction of Chimeric and Mutant Receptors-- The cDNAs of the mouse GRPR and rat NMBR were identical to those described previously (31, 32). The cDNA of the wild-type mouse GRPR was cloned between the HindIII site and XbaI site of pcDNA3, and the wild-type rat NMBR was cloned into the EcoRI site of pcDNA3. The GRPR/NMBR chimeras were constructed using the SeamlessTM Cloning Kit (38) using hydropathy plots for the GRPR and for the NMBR as described previously (15). Mutant receptors were made by using the QuikChangeTM Site-Directed Mutagenesis Kit, following the manufacturer's instructions, except that the annealing temperature was 60 °C and the DpnI digestion was for 2 h. Nucleotide sequence analysis of the entire coding region was performed using an automated DNA sequencer (ABI PRISMTM 377 DNA sequencer; Applied Biosystems Inc., Foster City, CA).

Cell Transfection-- Balb/c 3T3 cells were seeded in a 10-cm diameter tissue culture dish at a density of 106 cells/dish and grown overnight at 37 °C in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal bovine serum, 100 units/ml penicillin, and 100 mg/ml streptomycin. The following morning, cells were transfected with 5 µg of plasmid DNA by the cationic lipid-mediated method (39) using 30 µl of LipofectAMINETM reagent and 20 µl of LipofectAMINETM Plus reagent in serum-free Dulbecco's modified Eagle's medium for 3 h at 37 °C. At the end of the incubation period, the medium was replaced with Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal bovine serum, 100 units/ml penicillin, and 100 mg/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-- 125I-[Tyr4]Bn at a specific activity of 2,200 Ci/mmol was prepared by a modification of the methods described previously (40, 41). Briefly, 0.8 µg of IODO-GEN in chloroform was transferred to a vial, dried under a stream of nitrogen, and washed with 100 µl of KH2PO4 (pH 7.4). To this vial, 20 µl of KH2PO4 (pH 7.4), 8 µg of peptide in 4 µl of water, and 2 mCi (20 µl) of Na125I were added, mixed gently, and incubated at room temperature for 6 min. The incubation was stopped by the addition of 100 µl of distilled water and 300 µl of 1.5 M dithiothreitol. The iodination mixture was incubated at 80 °C for 60 min. The reaction mixture was applied to a Sep-Pak column (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 µl of sequential elutions (× 10) with 60% acetonitrile in 0.1% trifluoroacetic acid. The two or three fractions with the highest radioactivity were combined and purified on a reverse-phase, high performance liquid chromatography with a µBondaPak column (0.46 × 25 cm). The column was eluted with a linear gradient of acetonitrile in 0.1% trifluoroacetic acid (v/v) from 16 to 60% acetonitrile in 60 min. One-ml fractions were collected and checked for radioactivity and receptor binding. The pH values of the fractions were adjusted to 7 using 0.2 M Tris (pH 9.5), and radioligands were stored in aliquots with 0.5% bovine serum albumin at -20 °C.

Whole Cell Radioligand Binding Assays-- Competitive binding assays were performed 48 h post-transfection. Disaggregated transiently transfected cells were incubated for 1 h at room temperature in 250 µl of binding buffer (pH 7.4) with the ligand 50 pM 125I-[Tyr4]Bn (2,200 Ci/mmol) in the presence of the indicated concentration of unlabeled peptides. The binding buffer contained 98 mM NaCl, 6 mM KCl, 11.5 mM glucose, 5 mM fumarate, 5 mM glutamate, 5 mM 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 serum albumin, 0.05% (w/v) bacitracin, and 0.01% (w/v) soybean trypsin inhibitor. The cell concentration was adjusted to 0.2-2.6 × 106 cells/ml to assure that no more than 20% of the total added radioactive ligand bound. Bound tracer was then separated from unbound tracer by layering 100 µl of the binding reaction on top of an oil phase (100 µl of Nyosil M20; Nye Lubricants Inc., New Bedford, MA) in a 0.4-ml microcentrifuge tube (PGC Scientific, Frederick, MD) and pelleting the cells through the oil by centrifugation at 10,000 × g in a Microfuge ETM (Beckman Instruments) for 3 min. The supernatant was aspirated, and the pelleted cells were rinsed twice with distilled water. The amount of radioactivity bound to the cells was measured in a Cobra II gamma  counter (Packard Instrument Co.). A 100-µl aliquot of the incubation mixture were taken in duplicate to determine the total radioactivity. Binding was expressed as the percentage of total radioactivity that was associated with the cell pellet. All binding values represented saturable binding (i.e. total binding minus nonsaturable binding). Nonsaturable binding was <15% of the total binding in all experiments. Each point was measured in duplicate, and each experiment was replicated at least three times. Calculation of IC50 values was performed with a curve-fitting program, KaleidaGraph graphing software (Synergy Software, Reading, PA). Affinity and receptor density were calculated using a least-squares curve-fitting program (LIGAND) (42).

Homology Modeling of Extracellular Loop Region-- The amino acid sequences of mouse GRPR and rat NMBR were retrieved from the G protein-coupled receptor data base (43). The three-dimensional crystal structure of bovine rhodopsin (1f88) was obtained from the Protein Data Bank (Research Collaboratory for Structural Bioinformatics, Rutgers University) (44). Examination of the hydropathy profile of the fourth extracellular domain of bovine rhodopsin using SYBYL6.6 demonstrated that the hydrophobicity of the sequence is markedly less in the region of the extracellular loop as compared with the adjoining helical regions. This was confirmed by protein sequence analysis (45-47). Hydropathy evaluation and protein sequence analysis were used to locate the fourth extracellular domain regions in both mouse GRPR and rat NMBR. An additional eight amino acids were included at each end of the putative regions, and the sequences were imported into Deep View, the Swiss-PdbViewer (48) and aligned to the structure of bovine rhodopsin. The threading energy (49) of the alignment was at a minimum and rose if the sequences were shifted in either direction. The orientation of the side chains was optimized to reduce clashes, and the three-dimensional structure of the extracellular loop region was exported to SYBYL as a pdb file.

Binding Site Model-- The putative, solvent-accessible binding sites of the extracellular loop of the receptor were explored using the SiteID module in SYBYL6.6. Briefly, the loop region was solvated with water molecules, and clusters of solvent molecules adjacent to solvent-accessible residues were defined as a potential binding site. This demonstrated a possible binding site near the extracellular surface bounded by the extracellular loop and potentially extending into the interior of the helical bundle with a volume of 430 Å3. The surface of the solvent cluster adjacent to the extracellular loop was visualized using the SYBYL program Molcad.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Wild-type GRPR and NMBR-- The Bn-related natural occurring agonists Bn and GRP (Fig. 1) had high affinity (IC50 2.7 nM) for the GRPR (Table I), and neuromedin B (Fig. 1) had high affinity for the NMBR (IC50 1.2 nM) (Table I). Bn had a 2-fold and GRP a 12-fold selectivity for the GRPR over the NMBR, whereas neuromedin B had 200-fold selectivity for the NMBR over the GRPR (Table I). Both the Bn statine antagonist, JMV594 (Fig. 1), and the pseudopeptide antagonist, JMV641 (Fig. 1) had a high affinity (IC50 0.5-2.2 nM) for the wild-type GRPR as reported previously (36, 37) (Fig. 2, Table I). However, we found that each antagonist had a low affinity for the NMBR (IC50 1,500-10,000 nM) (Fig. 2, Table I). Therefore, JMV594 and JMV641 had >5,000 and 3,260 times higher selectivity, respectively, for the GRPR over the NMBR (Fig. 2, Table I). To explore the molecular basis for this GRPR selectivity of JMV594 and JMV641, we first made both loss of affinity GRP chimeric receptors (Fig. 3) and gain of affinity NMBR chimeric receptors (Fig. 4). Then to determine the exact amino acids involved we made GRPR loss of affinity point mutations (Figs. 5-8) and gain of affinity point mutants of NMBR (Figs. 9 and 10).


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  		Fig. 1.   Structure of bombesin, GRP-(14-27), NMB, and the GRPR-selective antagonists, JMV594 and JMV641. Sta, statine, which is 4-amino-3-hydroxy-6-methylheptanoic acid. psi , a pseudopeptide bond in which the C=O is replaced by CHOH. Boxes indicate amino acid identities with bombesin.

                              
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Table I
Comparison of binding affinities of Bn-related naturally occurring peptides and the antagonists JMV594 and JMV641 for wild-type GRPR and NMBR
Balb/c 3T3 cells were transfected with either wild type GRPR or NMBR and incubated with 50 pM 125I-[Tyr4]Bn alone or with increasing concentrations of the indicated unlabeled peptides. The concentration causing half-maximal inhibition of binding, IC50, was determined by using the curve-fitting program KaleidaGraph. Values are mean ± S.E. from four different experiments, and in each experiment each value was determined in duplicate. Structures of JMV594 and JMV641 are shown in Fig. 1.


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  		Fig. 2.   Affinities of JMV594 and JMV641 for wild-type GRPR and wild-type NMBR expressed in Balb/c 3T3 cells. Balb/c 3T3 cells were transfected with either wild-type receptor as outlined under "Experimental Procedures" and were incubated with 50 pM 125I-[Tyr4]Bn and the indicated concentrations of JMV594 and JMV641. Each point is the mean of three separate experiments, and in each experiment each point was determined in duplicate. All curves were best fit by a single binding site model (LIGAND) (42).


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  		Fig. 3.   Affinities of JMV594 and JMV641 for wild-type GRPR, extracellular chimeric GRPRs, and wild-type NMBR expressed in Balb/c 3T3 cells (loss of affinity chimeras). The diagrams of the chimeric receptors formed are shown at the top. The chimeric GRPRs were formed by replacing each of the extracellular loops one at a time by the comparable NMBR extracellular loop. The affinity was measured by competitive radioligand displacement of 50 pM 125I-[Tyr4]Bn by JMV594 and JMV641 at the concentrations shown. Each point on a curve is the mean from three separate experiments, and in each experiment each point was measured in duplicate. All curves were best fit by a single binding site model (LIGAND) (42). e1-, e2-, e3-, and e4-NMBR refer to the substitution of this extracellular loop of the NMBR for the comparable extracellular loop in GRPR. The arrows indicate large changes in affinity from the wild-type GRPR.


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  		Fig. 4.   Affinities of JMV594 and JMV641 for wild-type NMBR, extracellular chimeric NMBRs, and wild-type GRPR expressed in Balb/c 3T3 cells (gain of affinity chimeras). Diagrams of the chimeric receptors formed are shown at the top. The chimeric NMBRs were formed by replacing each of the extracellular loops of NMBR by the comparable loop of the GRPR one at a time. The affinity was measured by competitive radioligand displacement of 50 pM 125I-[Tyr4]Bn by JMV594 and JMV641 at the concentrations shown. Each point on a curve is the mean from three separate experiments, and in each experiment each point was measured in duplicate. All curves were best fit by a single binding site model (LIGAND) (42). e1-, e2-, e3- and e4-GRPR refer to substitution of this extracellular loop of the GRPR for the comparable extracellular loop of the NMBR. The arrows indicate large gains of affinity for the antagonists.


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  		Fig. 5.   Alignment of amino acid sequences in the fourth extracellular domain of GRPR and NMBR. The boxes indicate divergent amino acids between these two receptors in the fourth extracellular domain. Shown are the 21 GRPR mutants made to explore the importance of 11 amino acid differences for determining JMV594 and JMV641 selectivity. The arrow indicates that the top set of amino acids in the indicated GRPR position were replaced by the bottom set of amino acids from the comparable position in the NMBR.

Extracellular Chimeric Receptors-- Four loss of affinity GRPR chimeric receptors were made with the extracellular domains of NMBR substituted for the comparable domains in GRPR (Fig. 3), and four potential gain of affinity NMBR chimeras were made with the extracellular domains of GRPR substituted into NMBR (Fig. 4). Substitution of the first, second, and third extracellular domain in the GRPR by the comparable domain from the NMBR did not alter the affinity for JMV594 or JMV641, and each chimeric GRPR had similar affinities to wild-type GRPR (Fig. 3 and Table II). Substitution of the fourth extracellular domain in the GRPR by the comparable domain of the NMBR decreased the affinity 1,400-fold (from 2.2 ± 0.05 to 3,100 ± 120 nM) for the statine antagonist, JMV594, and 1,200-fold (from 0.46 ± 0.03 to 530 ± 22 nM) for the pseudopeptide antagonist, JMV641 (Fig. 3, Table II). When the reverse study was performed by substituting in the NMBR the extracellular domain of the GRPR to attempt to gain affinity, replacement of the fourth extracellular domain increased the affinity for JMV594 by 300-fold (from >10,000 to 29 ± 0.58 nM) and with JMV641 caused an 11-fold gain in affinity (from 1,500 ± 40 to 140 ± 4.2 nM) (Fig. 4, Table II). Replacement of the first, second, or third extracellular domain of the NMBR by the comparable domain of the GRPR had no effect on the affinity of JMV594. However, substitution of the third extracellular domain had a small effect on the affinity for JMV641, increasing affinity less than 1-fold (Fig. 4, Table II). These results from the study of chimeric receptors demonstrated that the fourth extracellular domain was principally involved in determining selectivity for GRPR over NMBR for these two structurally different peptide antagonists.

                              
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Table II
Affinities of JMV594 and JMV641 for wild-type GRPR and NMBR and extracellular chimeric GRPRs and NMBRs
Balb/c 3T3 cells were transiently transfected with either wild-type, chimeric GRPRs or NMBRs. Cell concentration (conc.) was adjusted as indicated so that <20% of the added counts were bound after incubation with 50 pM 125I-[Tyr4]Bn alone (100,000 cpm/ml) or with increasing concentrations of the indicated unlabeled antagonists. The percentage of added counts bound with 50 pM 125I-[Tyr4]Bn alone (Total) and the percentage of added counts bound with 50 pM 125I-[Tyr4]Bn plus 1 µM Bn (nonsaturable) are shown. The concentration causing half-maximal inhibition of binding, IC50, was determined by using the curve-fitting program KaleidaGraph. Values are mean ± S.E. from four different experiments, and in each experiment each value was determined in duplicate.

GRPR Fourth Extracellular Domain Mutants (Loss of Affinity Point Mutants)-- To identify which amino acid(s) in the fourth extracellular domain of GRPR are responsible for the high affinity for JMV594 and JMV641, the amino acid differences and identities were compared between the mouse GRPR and rat NMBR in the fourth extracellular domain (Fig. 5). The rat, mouse, and human NMBR are identical in the fourth extracellular domain (33, 50). The human and mouse GRPR are identical also in this region and differ from the rat by one conservative substitution of isoleucine for valine in position 303 (33). In the fourth extracellular domain, 11 amino acid differences were present, occurring at positions 290-291, 293, 295, 297, 299-300, and 302-305 of GRPR, which are comparable with positions 291-292, 294, 296, 298, 300-301, and 303-306 of NMBR (Fig. 5). To study the 11 amino acid differences in the fourth extracellular domains of these receptors, we first made six GRPR loss of affinity group point mutants (Fig. 5). Two of the six GRPR group point mutants caused a decrease in affinity for both antagonists (Fig. 6). The GRPR mutant with Thr297 of the GRPR replaced by Pro297 from the comparable position of NMBR (Thr297 right-arrow Pro297) and a second mutant with Phe-Val-Thr-Ser302-305 of the GRPR replaced by Met-Ile-Val-Thr302-305, decreased the affinities for JMV594 by 17- and 90-fold, respectively, and for JMV641 by 2- and 48-fold, respectively (Fig. 6 and Table III). The other four GRPR group point mutants, including replacement of Tyr290-His291 of the GRPR by Phe-Asn290,291, Ser293 of the GRPR by Lys293, Val295 of the GRPR by Ile295, and Met-Leu299,300 of the GRPR by Leu-Gly from NMBR had no effect on the affinity for either antagonist (Fig. 6, Table III). However, neither of the two group changes alone (i.e. 2-100-fold decrease) caused a decrease in affinity equal to the >1,100-fold decrease in affinity seen for each antagonist when the entire fourth extracellular GRPR domain was replaced by that from the NMBR (Table III). Therefore, a series of point mutations and combinations were made to identify which other amino acids were important for determining the antagonists' selectivity.


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  		Fig. 6.   Affinities of JMV594 and JMV641 for wild-type GRPR, fourth extracellular domain group point mutants of GRPR, and wild-type NMBR expressed in Balb/c 3T3 cells (loss of affinity group point mutants). The amino acid mutants of GRPR were formed by replacing the amino acid(s) of the fourth extracellular domain of GRPR in the positions indicated by the comparable amino acid(s) of the NMBR as shown in Fig. 5. The affinity was measured by competitive radioligand displacement of 50 pM 125I-[Tyr4]Bn by JMV594 and JMV641 at the concentrations shown. Each point on a curve is the mean from three separate experiments, and in each experiment each point was measured in duplicate. All curves were best fit by a single binding site model (LIGAND) (42). [Phe-Asn290,291]GRPR refers to replacement of the amino acid residues in positions 290 and 291 of the GRPR by phenylalanine and aspartic acid, which exists in the comparable position in NMBR. The arrows indicate large changes in affinity of the GRPR by the mutations.

                              
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Table III
Affinities of GRP, JMV594, and JMV641 for wild-type GRPR, wild-type NMBR, and fourth extracellular domain amino acid mutants of GRPR and NMBR
Balb/c 3T3 cells were transiently transfected with either wild-type, chimeric GRPRs or NMBRs. Cell concentration (conc.) was adjusted as indicated so that <20% of the added counts were bound after incubation with 50 pM 125I-[Tyr4]Bn alone (100,000 cpm/ml) or with increasing concentrations of the indicated unlabeled antagonists. The percentage of added counts bound with 50 pM 125I-[Tyr4]Bn alone (Total) and the percentage of added counts bound with 50 pM 125I-[Tyr4]Bn plus 1 µM Bn (Nonsaturable) are shown. The concentration causing half-maximal inhibition of binding, IC50, was determined by using the curve-fitting program KaleidaGraph. Values are mean ± S.E. from four different experiments, and in each experiment each value was determined in duplicate.

To understand which amino acids were important in [Met-Ile-Val-Thr302-305]GRPR for determining the decrease in affinity from the wild-type GRPR, we made mutant GRPRs with four single amino acid substitutions (Phe302 of the GRPR replaced by Met302 in the comparable position in NMBR, Val303 replaced by Ile303, Thr304 replaced by Val304, and Ser305 replaced by Thr305) and examined the affinities of these four mutants for the two peptide antagonists. Two of these GRPR point mutants (Phe302 right-arrow Met302, Ser305 right-arrow Thr305) significantly (3-9-fold) decreased the affinities for each of the two antagonists (Fig. 7, Table III). However, each of the single GRPR amino acid mutations that altered GRPR affinity for the two antagonists (Thr297 right-arrow Pro297, Phe302 right-arrow Met302, and Ser305 right-arrow Thr305) had only a small change (<10-fold decrease in affinity) compared with the 1,200-1,400-fold decrease caused by replacement of the entire fourth extracellular domain. Therefore, we next made seven GRPR combination point mutants in which combinations of amino acids were replaced by the comparable different amino acids of the fourth extracellular domain of NMBR (Fig. 5). GRPR mutants with three combinations of fourth extracellular domain NMBR amino acid replacements (Thr297 right-arrow Pro297, Phe302 right-arrow Met302, and Ser305 right-arrow Thr305) had the largest decrease in affinity for both antagonists, demonstrating that differences in these three amino acids were the most important for the selectivity of the two antagonists for GRPR over NMBR (Fig. 8, Table III). However, even the [Pro297,Met302,Thr305]GRPR mutant did not cause the full decrease in affinity seen with the complete replacement of the fourth extracellular GRPR domain. Specifically, [Pro297,Met302,Thr305]GRPR decreased affinity for JMV594 and JMV641 500- and 460-fold compared with a 1,400- and 1,200-fold decrease with replacement of the entire fourth extracellular domain. GRPR mutants with combinations of at least eight amino acids of the 11 different amino acids in the fourth extracellular domain between GRPR and NMBR were required to reproduce the magnitude of decrease in affinity for both antagonists seen with the entire fourth extracellular domain (Fig. 8, Table III). The only different amino acids in the fourth extracellular domain of the GRPR from the NMBR that did not contribute to the selectivity of either antagonist were Met299, Leu300, Val303, and Thr304 (Table III).


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  		Fig. 7.   Affinities of JMV594 and JMV641 for wild-type GRPR, fourth extracellular domain point mutants of GRPR, and wild-type NMBR expressed in Balb/c 3T3 cells (loss of affinity point mutants). The point mutants of GRPR were formed by replacing the amino acids in positions 302-305 of GRPR by the comparable amino acids of the NMBR as shown in Fig. 5. The affinity was measured by competitive radioligand displacement of 50 pM 125I-[Tyr4]Bn by JMV594 and JMV641 at the concentrations shown. Each point on a curve is the mean from three separate experiments, and in each experiment each point was measured in duplicate. All curves were best fit by a single binding site model (LIGAND) (42). [Met302]GRPR refers to replacement of the amino acid residue in position 302 of the GRPR by methionine, which exists in the comparable position in NMBR. The arrows indicate large changes in affinity of the GRPR by the mutations.


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  		Fig. 8.   Affinities of JMV594 and JMV641 for wild-type GRPR, fourth extracellular domain combination amino acid(s) mutants of GRPR, and wild-type NMBR expressed in Balb/c 3T3 cells (loss of affinity point mutant combinations). The mutants of GRPR were formed by replacing the amino acid(s) of the fourth extracellular domain of GRPR by the comparable amino acid(s) of the NMBR as shown in Fig. 5. The affinity was measured by competitive radioligand displacement of 50 pM 125I-[Tyr4]Bn by JMV594 and JMV641 at the concentrations shown. Each point on a curve is the mean from three separate experiments, and in each experiment each point was measured in duplicate. All curves were best fit by a single binding site model (LIGAND) (42). [Pro297,Met302]GRPR refers to replacement of the amino acid residues in positions 297 and 302 of the GRPR by proline and methionine, which exist in the comparable positions in NMBR. The arrows indicate large changes in affinity of the GRPR by the mutations.

NMBR Fourth Extracellular Domain Mutants (Gain of Affinity Point Mutants)-- To provide additional insight into the importance of the different fourth extracellular domain amino acids in determining antagonist selectivity, we constructed and assessed the possible gain in affinity of four NMBR mutants made by replacing the three most important amino acids identified from the study of GRPR point mutants either alone or in combination (i.e. Pro298 right-arrow Thr298; Met303 right-arrow Phe303; Thr306 right-arrow Ser306; and Pro298, Met303, Thr306 right-arrow Thr298, Phe303, Ser306) (Fig. 9). These three amino acids (i.e. Pro298, Met303, and Thr306) in NMBR are in the position comparable with that of the amino acids in GRPR that were the key amino acids for high selectivity of GRPR for the antagonists identified in the loss of affinity studies (Fig. 9). For the statine antagonist JMV594, two single amino acid mutants (Pro298 right-arrow Thr298 and Thr306 right-arrow Ser306) caused ~3-fold gain in affinity, and the combination mutant (Pro298, Met303, Thr306 right-arrow Thr298, Phe303, Ser306) demonstrated a >130-fold gain in affinity (Fig. 10, Table III). This increase in affinity was only 2.5-fold less than the gain of affinity seen when the entire fourth extracellular domain of the GRPR was substituted into the NMBR (Fig. 10 and Table III). With the pseudopeptide JMV641, the substitutions of these three amino acids singularly or together into NMBR showed a different result. The substitution of Thr298 for Pro298 alone was the most critical amino acid for the gain in affinity for JMV641, causing a 5-fold increase in affinity (Fig. 10 and Table III). In contrast to the statine analogue JMV594, with the pseudopeptide JMV641 the replacement of Thr306 in the NMBR by Ser306 from the comparable position in the GRPR had no effect. Furthermore, the combination replacement of all three of these key fourth extracellular domain amino acids of the NMBR (Pro298, Met303, Phe306) resulted in no greater gain in affinity than the effect of replacing the Pro298 in NMBR alone (Fig. 10 and Table III), whereas in the case of the statine antagonist JMV594 it caused a 40-fold gain in affinity over any single replacement (Fig. 10 and Table III).


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  		Fig. 9.   Alignment of amino acid sequences in the fourth extracellular domain of NMBR compared with GRPR. Boxes indicate divergent amino acids between these two receptors in the fourth extracellular domain. Shown are the four NMBR gain of affinity point mutants made to explore the importance of amino acid differences for determining JMV594 and JMV641 selectivity.


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  		Fig. 10.   Affinities of JMV594 and JMV641 for wild-type NMBR, fourth extracellular domain amino acid mutants of NMBR, and wild-type GRPR expressed in Balb/c 3T3 cells (gain of affinity mutants). The point mutants of NMBR were formed by replacing the amino acid(s) of the fourth extracellular domain of NMBR by the comparable amino acid(s) of the GRPR as shown in Fig. 9. The affinity was measured by competitive radioligand displacement of 50 pM 125I-[Tyr4]Bn by JMV594 and JMV641 at the concentrations shown. Each point on a curve is the mean from three separate experiments, and in each experiment each point was measured in duplicate. All curves were best fit by a single binding site model (LIGAND) (42). [Thr298]NMBR refers to replacement of the amino acid residue in position 298 of the GRPR by threonine, which exists in the comparable position in GRPR. The arrows indicate large changes in affinity of the NMBR by the mutations.

Binding Site Model-- To attempt to gain additional insight into why certain amino acids within the fourth extracellular domain of GRPR were important for determining selectivity for these two antagonists, the proposed binding sites of the antagonists to the GRPR in the fourth extracellular domain of GRPR were analyzed using modeling programs (Fig. 11). The modeling results based on the crystal structure of bovine rhodopsin (44) revealed that Arg288, Ser289, Tyr290, Tyr292, Ser293, Glu294, Val295, Asp296, Thr297, His301, Phe302, and Ser305 in the fourth extracellular domain of GRPR all contained atoms that were within 5 Å of the putative binding site (Fig. 11). Six of these 12 amino acids in the GRPR (i.e. Tyr290, Ser293, Val295, Thr297, Phe302, and Ser305) are different from the comparable amino acids in the NMBR (Fig. 5). The three key amino acids (i.e. Thr297, Phe302, and Ser305) that were found to be the primary determinants for the specificity of these two antagonists for the GRPR all projected into the interior of the putative binding pocket, with most atoms within a 5-Å distance of the projected binding pocket (Fig. 11).


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  		Fig. 11.   Modeling of the putative binding site of the fourth extracellular domain of GRPR. The structure of the fourth extracellular domain of GRPR, from homology modeling determined as described under "Experimental Procedures," based on the crystal structure of bovine rhodopsin (Protein Data Bank code 1f88), is illustrated. The solvent-accessible amino acids with any atoms within 5 Å of a possible binding site are colored yellow, with the atoms of these amino acids within 5 Å colored red. The blue residues reside farther from the solvent-accessible site, which is illustrated as a transparent shape, defined as an envelope enclosing a cluster of water molecules surrounding the solvent-accessible residues. Val303 and Thr304 are at the rear (lipid-facing side) of the helical region and are therefore not accessible in this model. The critical amino acids Thr297, Phe302, and Ser305, which differ between GRPR and NMBR are all within 5 Å of the possible binding site.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In general, the roles of GPCRs mediating the action of most GI hormones/neurotransmitters in physiological or in pathological processes are still unclear. This is in large part because, for many, specific receptor antagonists do not exist, and for others, only recently have high affinity antagonists been developed (51). The antagonists for these receptors generally fall into one of three types: nonpeptide antagonists (the largest group); peptide antagonists; or, in a few cases, peptoid antagonists, which have features of both peptides and nonpeptides (52, 53). There are a number of studies of the molecular basis of action of nonpeptide antagonists for various GI hormone receptors (14, 54-57); however, there are only a few studies for peptide antagonists (14, 16, 17, 55). This has occurred in large part, because potent peptide antagonists have been described for only a few GI hormone/neurotransmitter receptors (51). One of these exceptions is GRPR, which mediates the actions of the mammalian bombesin-related peptide, GRP, in the central nervous system and peripheral tissues (1-3, 58). Six different classes of peptide receptor antagonists are described for the GRPR (3, 4, 36, 37, 59-62), some with sufficient potency and stability to be used recently for in vivo studies to determine GRP's action in humans (12). At present, for each of these peptide GRPR antagonists, their molecular basis of action (the molecular determinants of their receptor selectivity or for their high affinity receptor interaction) is unknown. In this study, we examined the molecular basis of action of two of the most potent GRPR peptide antagonists, the statine analogue, JMV594, and the pseudopeptide analogue, JMV641 (36, 37).

In the present study, we not only found that each of these peptide antagonists had an affinity as high as GRP or bombesin for the GRPR as reported previously (36, 37) but also found that they were highly selective for the GRPR over the other mammalian Bn receptor, the NMBR. Each of the antagonists had >3,000-fold higher affinity for GRPR than NMBR, despite the fact that these receptors share an ~50% overall amino acid identity (2, 33). The analyses of both the loss of affinity GRPR chimera and gain of affinity NMBR chimera support the conclusion that differences in the fourth extracellular domains of these two receptors play the major role in determining selectivity of these two different classes of peptide antagonists.

This result has both similarities with and differences from studies on the interaction of peptide antagonists and agonists with other GPCRs. Studies of several GPCRs demonstrate that the receptor extracellular domains can be an important receptor region for determining high affinity ligand binding (63). However, only a few studies have explored whether the determinants of high affinity interaction or selectivity for a peptide antagonist are due to interactions with the receptor extracellular domains. Such an interaction is not important for determining high affinity interaction of the peptide antagonist D-Arg-[Hyp3,D-Phe7]bradykinin (NPC567) with the B2 bradykinin receptor (16) or BQ-123 with the endothelin A receptor (14). However, it is important for the high affinity interaction of the peptide antagonists JMV179 with the human CCK-A receptor (55) and [Sar1,Ile8]angiotensin II with the AT1 receptor (18). For a number of peptide GI receptor neurotransmitter/hormone agonists, high affinity receptor binding or receptor subtype selectivity depends on interaction with receptor extracellular domains, including CCK-8 with the CCK-A or CCK-B receptor (64), bradykinin with B2 bradykinin receptor (65), and substance P with the neurokinin-1 receptor (66). However, for other peptide agonists such as the high affinity interaction of NMB with the NMBR (67), endothelin (ET) with the ETA receptor (57), and neuropeptide Y with neuropeptide Y Y1 receptor (68), the high affinity interaction or selectivity is primarily determined by amino acids in the transmembrane regions. Similarly, with many nonpeptide antagonists such as the interaction of L365,260 with the CCK-B receptor (54), losartan with the AT1B angiotensin II receptor (69), bosentan with the ETA receptor (57), and L161,664 with the neurokinin-1 receptor (70), the extracellular domains do not contain determinants for high affinity interactions or receptor subtype selectivity.

To determine which amino acids in the fourth extracellular domain of the GRPR account for the high affinity of these two antagonists for the GRPR and its selectivity for this receptor over the NMBR, we performed a comparative alignment of the amino acids in this region between the GRPR and the NMBR. Our results support the conclusion that the threonine residue in position 297 of GRPR instead of a proline in the NMBR, a phenylalanine in position 302 of GRPR instead of a methionine in NMBR, and a serine in position 305 of GRPR rather than a threonine in NMBR are the key amino acid differences responsible for the high affinity of the two peptides for the GRPR and their high selectivity for the GRPR over the NMBR. Whereas the individual replacement of these three amino acids caused only a 10-fold change in affinity for each antagonist, a combined substitution of these three residues by the comparable NMBR amino acids led to potentiated effect with a marked decrease in affinity (~500-fold) for each of the antagonists. This result demonstrates that amino acid differences at the both the amino and carboxyl terminus of the fourth extracellular domain are involved in determining the high affinity and selectivity for the GRPR of both antagonists.

This result has both similarities and differences from studies of binding of other peptide ligands to GI hormone/transmitter GPCRs. It is similar to results with interaction of the peptide antagonist [Sar1,Ile8]angiotensin II (18) with the AT1 receptor, in which a cooperative interaction between His24, Tyr26, and Ile27 in the first extracellular domain is required for high affinity ligand interaction or for the peptide antagonist D-Arg-[Hyp3,D-Phe7]bradykinin's interaction with the B2 bradykinin receptor, in which the ligand's interaction with multiple amino acids in the sixth transmembrane domain is required for high affinity interaction and selectivity. Similarly, with almost all studies on peptide agonists, such as bradykinin's interaction with the bradykinin 1 or 2 receptor (65); CCK-8 interaction with the CCK-B receptor (64); and substance P, neurokinin A, or neurokinin B interaction with the neurokinin-1 receptor (66), cooperative interactions with a number of residues in the same extracellular domain are required for high affinity interaction and selectivity for the one receptor subtype. In contrast to these cooperative interactions, with some peptide antagonists such as the interaction of the pentapeptide BQ123 with the ETA receptor (14) or JMV179 with the CCK-A receptor (55) and with many nonpeptide antagonists such as L365,260 interaction with the CCK-B receptor (54), BMS-182874 with the ETA receptor (14) or SB209670, and Ro 46-2005 with the ETB receptor (56), a single amino acid difference between receptor subtypes accounts primarily for the high affinity or selectivity of the antagonist.

The threonine, serine, and phenylalanine residues found to be important in determining GRPR selectivity for both classes of antagonists in the present study have been reported in several studies in other GPCRs to play a critical role in determining high affinity interaction and selectivity of the ligand for a GPCR subtype (16, 17, 57, 64, 69). A threonine in the sixth transmembrane domain of the bradykinin 2 receptor is required for high affinity interaction with the peptide antagonist D-Arg-[Hyp3,D-Phe7]BK (NPC567) (16), two threonine residues in the fifth transmembrane domain of the m3 muscarinic receptor are critical amino acids for high affinity interaction with agonists (acetylcholine, carbachol) (71), and a serine in the third transmembrane domain of the AT1A AII receptor is one of the critical amino acids required for high affinity interaction with the peptide antagonist [Sar1, Ile8]AII (72). In the former two studies (16, 71), it was proposed that the mechanism of the enhanced affinity due to the threonine or serine was by enhancing hydrogen bonding with the ligand. The presence of a phenylalanine residue in either a transmembrane domain or extracellular domain in other GPCRs also has been shown to play an important role in high affinity ligand-receptor interaction and ligand GPCR selectivity (16, 17, 57, 64, 69). The presence of a phenylalanine in the sixth transmembrane domain of the bradykinin 2 receptor (Phe261) is necessary for high affinity and selectivity of the peptide antagonist NPC567, and this effect was proposed to be mediated by an amino-aromatic or aromatic-aromatic interaction (16). A phenylalanine residue in the second transmembrane domain of the CCK-A receptor (Phe107) or two phenylalanine residues in the second extracellular domain of the CCK-B receptor are critical amino acids for high affinity interaction with peptide agonist CCK-8 (64). Similarly, with substance P interaction with the neurokinin-1 receptor (66), endothelin with the ETA receptor (57), and the peptide antagonist [des-Arg10-Leu9]kallidin with the bradykinin 1 receptor (17), phenylalanines in either extracellular domains or transmembrane regions are essential for high affinity interaction and selectivity. Phenylalanine, similar to other aromatic amino acids, characteristically interacts with ligands by functioning as a strong locus of cation-pi binding (73). The cation-pi binding occurs through the side chains from the aromatic amino acids such as phenylalanine, tyrosine, or tryptophan (74). In our study, the substitution of methionine from the comparable position of NMBR for phenylalanine in the GRPR resulted in a marked decrease in affinity for JMV594 and JMV641, supporting the conclusion that cation-pi interactions are important for the high affinity and selectivity of both of the peptide antagonists.

At present, it is not possible to compare the receptor structural determinants of high affinity interaction and GRPR selectivity of these two different classes of peptide antagonists with that for either the agonist bombesin or GRP, peptide agonists of which both antagonists are close structural analogues. There are no studies on the molecular determinants of either bombesin or GRP's high affinity interaction with the GRPR or of the selectivity of GRP for GRPR over the NMBR. However, our study does demonstrate that these two different antagonists have both similarities and differences in their molecular determinants of high affinity GRPR interaction and selectivity. They are similar in that with both the statine antagonist JMV594 and the pseudopeptide antagonist JMV641, the amino acid differences in the fourth extracellular domains of NMBR and GRPR are primarily responsible for their high affinity and selectivity for GRPR. They are also similar in that of the 11 amino acid differences in this domain between GRPR and NMBR, the three most important for each antagonist for high affinity interaction are Thr297, Phe302, and Ser305 in the GRPR. Furthermore, with each antagonist, these three amino acids had a potentiating effect on enhancing affinity, with the combination having a much greater effect than any of the three substitutions alone. These results suggest that for both antagonists cation-pi interactions and hydrogen bonding through serine and threonine receptor residues are of primary important in determining high affinity interaction. However, some results suggest that these two different classes of antagonists have some important differences in the molecular determinants of their GRPR selectivity. First, for the pseudopeptide antagonist, JMV641, the GRPR loss of affinity and NMBR gain of affinity chimeric studies showed that differences in second and third extracellular domains were of some importance in determining its GRPR selectivity, whereas for the statine analogue, JMV594, they were not important. Second, within the fourth extracellular domain, which was the most important domain for GRPR selectivity for both antagonists, the presence of Thr297, Phe302, and Ser305 in the GRPR was relatively more important for the statine antagonist JMV594's selectivity than for the pseudopeptide antagonist, JMV641. With both NMBR gain of affinity chimeric receptors made by substituting the extracellular domain of NMBR with that from GRPR and with the gain of affinity combination point mutant, [Thr298,Phe303,Ser306]NMBR, the gain in affinity was much greater for JMV594 than JMV641. Specifically, with the replacement of the fourth extracellular domain of NMBR by that of GRPR ([e4-GRPR]NMBR) there was a 30-fold greater gain in affinity for JMV594 than JMV641 (i.e. 345- versus 11-fold increase, respectively). Similarly, with insertion of Thr298, Phe303, and Ser306 in NMBR, there was a >20-fold greater increase in affinity for JMV594 than JMV641 (>133- versus 6.5-fold increase, respectively). These results demonstrate that while the molecular determinants of receptor subtype selectivity for these two closely related classes of GRPR antagonists is generally similar, there are also some important differences.

To attempt to gain additional insight into why these three amino acids of the fourth extracellular domain are important in determining the high GRPR selectivity of these two antagonists, three-dimensional modeling of this region of the GRPR was undertaken. Modeling of the GRPR was performed based on the results with bovine rhodopsin, which has recently been crystallized and characterized by x-ray diffraction (75). In rhodopsin, the fourth extracellular domain is well defined and solvent-accessible (75). The hydropathy profile of the sequence is markedly more hydrophilic in the loop region than in the adjoining transmembrane helical regions. This profiling was used to determine the ends of the loop region in GRPR, and the sequence of the extracellular loop was threaded onto the three-dimensional structure of rhodopsin. When the sequences were aligned, the loop regions coincided, and any shift in the alignment resulted in a higher threading energy, indicating a less favorable environment for the sequence. In this model, the critical Thr297, Phe302, and Ser305 residues in the fourth extracellular domain of the GRPR were found to face the interior of a binding pocket formed primarily by the loop of the fourth extracellular domain (Fig. 11). Our receptor model indicates that Arg288, Ser289, Tyr290, Tyr292, Ser293, Glu294, Val295, Asp296, Thr297, His301, Phe302, and Ser305 in the fourth extracellular domain of GRPR are within the 5 Å of the putative binding site and could interact with the antagonists (Fig. 11). However, Arg288, Ser289, Tyr292, Glu294, Asp296, and His301 are in a comparable position in the NMBR and therefore are unlikely to be important in the selectivity of the JMV594 and JMV641 for the GRPR. Of the other six amino acids (Tyr290, Ser293, Val295, Thr297, Phe302, and Ser305), our mutagenesis studies show that only Thr297, Phe302, and Ser305 are important in determining the selectivity of JMV594 and JMV641 for the GRPR over the NMBR. Substitution of the other three amino acids (Tyr290, Ser293, and Val295) by the comparable amino acids from the NMBR (Phe291, Lys294, and Ile296) caused no change in the affinities of JMV594 and JMV641, suggesting that either the backbone substitutions of these amino acids were not involved in ligand interaction or the comparable amino acid from NMBR was sufficiently similar to effectively replace the GRPR amino acid. Therefore, we conclude that Thr297, Phe302, and Ser305, which are the critical amino acids in determining the selectivity of JMV594 and JMV641, each are facing and within 5 Å of the proposal binding pocket.

In conclusion, our receptor chimeric gain and loss of affinity studies showed that the fourth extracellular domain of the GRPR was the principal receptor region responsible for the high affinity and selectivity of the statine antagonist JMV594 and the pseudopeptide antagonist JMV641 for the GRPR over the NMBR. Our mutagenesis studies show that Thr297, Phe302, and Ser305 in the fourth extracellular domain of the GRPR were the key amino acid differences in determining the GRPR selectivity and high affinity interaction of these two antagonists. To our knowledge, this is the first study that reveals the molecular basis of action between the GRPR and any of its classes of high affinity peptide antagonists. The results from these studies allowed us to obtain a more detailed picture of the receptor-ligand interaction and propose a model that could account for important receptor-ligand interactions. The availability of this model could be of use for studying the molecular basis of the interaction of this class of receptor not only with other peptide antagonists but also with natural agonists and peptoid antagonists.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom correspondence and reprint requests should be addressed: Dr. Robert T. Jensen, 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; E-mail: robertj@bdg10.niddk.nih.gov.

Published, JBC Papers in Press, July 19, 2001, DOI 10.1074/jbc.M104566200

    ABBREVIATIONS

The abbreviations used are: GRP, gastrin-releasing peptide; GRPR, GRP receptor; Bn, bombesin; GI, gastrointestinal; NMB, neuromedin B; NMBR, NMB receptor; GPCR, G protein-coupled receptor; ET, endothelin.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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