]Bn(6 -14)), and a pseudopeptide analogue, JMV641 (D-Phe-Gln-Trp-Ala-Val-GlyHis-Leu(CHOH-CH 2 )-(CH 2 ) 2 -CH 3 ), 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. 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 Thr 297 , Phe 302 , and Ser 305 of the fourth EC domain of GRPR are the critical residues for determining GRPR selectivity and suggest that both receptor-ligand cation-interactions and hydrogen bonding are important for their high affinity interaction.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 activi...

The mammalian bombesin peptides [gastrin-releasing peptide (GRP) and neuromedin B (NMB)] are important in numerous biological and pathological processes. These effects are mediated by the heptahelical GRP receptor (GRPR) and NMB receptor (NMBR). GRP has high affinity for GRPR and lower affinity for NMBR. Almost nothing is known about the molecular basis for the selectivity of GRP. To address this question, we first studied four loss-of-affinity GRPR chimeric receptors formed by exchanging the four extracellular (EC) domains of GRPR with the corresponding NMBR EC domains. Receptors were transiently expressed, and affinities were determined by binding studies. Only substitution of the third EC domain (EC3) of GRPR markedly decreased GRP affinity. In the reverse study using gain-of-affinity NMBR chimeras, only replacement of EC3 of NMBR markedly increased GRP affinity. Replacing each of the 20 comparable EC3 amino acids that differed in the NMBR in GRPR showed that two separate NMBR substitutions in the GRPR, Ile for Phe 185 or Ile for Ala 198 , markedly decreased GRP affinity. Additional point mutants demonstrated that an amino acid with an aromatic ring in position 185 of GRPR and the size of the backbone substitution in position 198 of GRPR were important for GRP selectivity. These results demonstrate that selectivity of GRP for GRPR over NMBR is primarily determined by two amino acid differences in the EC3 domains of the receptor. Our results suggest that an interaction between the aromatic ring of Phe 185 of the GRPR with GRP is the most important for GRP selectivity.In contrast to adrenergic and muscarinic cholinergic receptors, with many receptors for gastrointestinal (GI) hormones/ neurotransmitters, little is known about the molecular basis of their agonist selectivity. Some of the most important GI hormone/neurotransmitter receptors are those mediating the actions of the mammalian bombesin (Bn)-related peptides, gastrin-releasing peptide (GRP), and neuromedin B (NMB). These peptides mediate a wide spectrum of biological activities, including stimulating the growth of both normal and neoplastic tissues (Willey et al

Peptoid antagonists are increasingly being described for G protein-coupled receptors; however, little is known about the molecular basis of their binding. Recently, the peptoid PD168368 was found to be a potent selective neuromedin B receptor (NMBR) antagonist. To investigate the molecular basis for its selectivity for the NMBR over the closely related receptor for gastrin-releasing peptide (GRPR), we used a chimeric receptor approach and a site-directed mutagenesis approach. Mutated receptors were transiently expressed in Balb 3T3. The extracellular domains of the NMBR were not important for the selectivity of PD168368. However, substitution of the 5th upper transmembrane domain (uTM5) of the NMBR by the comparable GRPR domains decreased the affinity 16-fold. When the reverse study was performed by substituting the uTM5 of NMBR into the GRPR, a 9-fold increase in affinity occurred. Each of the 4 amino acids that differed between NMBR and GRPR in the uTM5 region were exchanged, but only the substitution of Phe 220 for Tyr in the NMBR caused a decrease in affinity. When the reverse study was performed to attempt to demonstrate a gain of affinity in the GRPR, the substitution of Tyr 219 for Phe caused an increase in affinity. These results suggest that the hydroxyl group of Tyr 220 in uTM5 of NMBR plays a critical role for high selectivity of PD168368 for NMBR over GRPR. Receptor and ligand modeling suggests that the hydroxyl of the Tyr 220 interacts with nitrophenyl group of PD168368 likely primarily by hydrogen bonding. This result shows the selectivity of the peptoid PD168368, similar to that reported for numerous non-peptide analogues with other G protein-coupled receptors, is primarily dependent on interaction with transmembrane amino acids.