The orphan receptor, bombesin (Bn) receptor subtype 3 (BRS-3), shares high homology with bombesin receptors (neuromedin B receptor (NMB-R) and gastrin-releasing peptide receptor (GRP-R)). This receptor is widely distributed in the central nervous system and gastrointestinal tract; target disruption leads to obesity, diabetes, and hypertension, however, its role in physiological and pathological processes remain unknown due to lack of selective ligands or identification of its natural ligand. We have recently discovered (Mantey, S. A., Weber, H. C., Sainz, E., Akeson, M., Ryan, R. R. Pradhan, T. K., Searles, R. P., Spindel, E. R., Battey, J. The 399-amino acid orphan receptor, bombesin receptor subtype 3 (BRS-3), 1 shares 51 and 47% amino acid sequence homology with the mammalian bombesin (Bn) receptors (gastrinreleasing peptide receptor (GRP-R) and the neuromedin B receptor (NMB-R), respectively) (1, 2). Studies of the distribution of this orphan receptor show that the BRS-3 receptor is present in the central nervous system and peripheral tissues although the distribution is more limited than the GRP-R and NMB-R (3-6). The BRS-3 receptor has been found on such diverse structures as secondary spermatocytes, pregnant uterus, a number of brain regions, and some human lung, breast, and epidermal cancer cell lines (1, 2)The role of BRS-3 in physiological or pathological processes is unknown even though BRS-3-deficient mice, produced by targeted disruption, develop obesity, diabetes, and hypertension (7). These results (7) suggest that the BRS-3 receptor may be required for the regulation of glucose metabolism, energy balance, and maintenance of blood pressure. This proposition is yet to be confirmed because the natural ligand of the BRS-3 receptor is still unknown. Results from previous studies (8 -10) have demonstrated that the hBRS-3 receptor has a unique pharmacology compared with that of any of the closely related Bn receptor family.

Vasoactive intestinal peptide (VIP) is a neurotransmitter involved in a number of pathological and physiological processes. VIP is rapidly degraded and simplified stable analogs are needed. VIP's action was extensively studied in rat and guinea pig. However, it is largely unknown whether its pharmacophore in these species resembles human. To address this issue we investigated the VIP pharmacophore for VPAC 1 (the predominant receptor subtype in cancers and widely distributed in normal tissues) by using alanine and D-amino acid scanning. Interaction with rat, guinea pig, and human VPAC 1 was assessed using transfected Chinese hamster ovary (CHO) and PANC1 cells and cells possessing native VPAC 1 . Important species differences existed in the VIP pharmacophore. The human VPAC 1 expressed in CHO cells, which were used almost exclusively in previous studies, differed markedly from the na-

The search for synthetic analogues of somatostatin (SRIF) which exhibit selective affinities for the five known receptor subtypes (sst1-5) has generated a large number of potent agonist analogues. Many of these agonists display good subtype selectivities and affinities for the subtypes 2, 3, and 5, with very few selective for sst1 or sst4. Until the recent report by Bass and co-workers (Mol. Pharmacol. 1996, 50, 709-715; erratum Mol. Pharmacol. 1997, 51, 170), no true antagonists of somatostatin had been discovered, let alone any displaying differential receptor subtype selectivity. In this present study, we further explore the effect of this putative L,5D6 antagonist motif on somatostatin octapeptide analogues with a cyclic hexapeptide core. The most potent antagonist found to date is H-Cpa-cyclo[DCys-Tyr-DTrp-Lys-Thr-Cys]-Nal-NH2, PRL-2970 (21), which has an IC50 of 1.1 nM in a rat pituitary growth hormone in vitro antagonist assay versus SRIF (1 nM). This analogue bound to cloned human somatostatin subtype 2 receptors with a Ki of 26 nM. The highest hsst2 affinity analogue was H-Cpa-cyclo[DCys-Pal-DTrp-Lys-Tle-Cys]-Nal-NH2, PRL-2915 (15), with a Ki of 12 nM (IC50 = 1.8 nM). This analogue was also selective for hsst2 over hsst3 and hsst5 by factors of 8 and 40, respectively, and had no agonist activity when tested alone at concentrations up to 10 microM. Regression analysis of the binding affinities versus the observed antagonist potencies revealed high correlations for hsst2 (r = 0.65) and hsst3 (r = 0.52) with a less significant correlation to hsst5 (r = 0.40). This is quite different from the somatostatin agonist analogues which show a highly significant correlation to hsst2 (r > 0.9). Receptor-selective somatostatin antagonists should provide valuable tools for characterizing the many important physiological functions of this neuropeptide.

]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 search for synthetic analogues of somatostatin (SRIF) which exhibit selective affinities for the five known receptor subtypes (sst1-5) has generated a large number of potent agonist analogues. Many of these agonists display good subtype selectivities and affinities for the subtypes 2, 3, and 5, with very few selective for sst1 or sst4. Until the recent report by Bass and co-workers (Mol. Pharmacol. 1996, 50, 709-715; erratum, Mol. Pharmacol. 1997, 51, 170), no true antagonists had been discovered, let alone any displaying differential receptor subtype selectivity. In this present study, we explore the effect of this putative L5,D6 antagonist motif on various series of somatostatin agonist analogues, both linear and cyclic. It was found that many D5,L6 agonists could be converted into competitive antagonists by applying this motif, the most potent of which was H-Nal-cyclo[DCys-Pal-DTrp-Lys-Val-Cys]-Nal-NH2 (32). This antagonist was selective for hsst2 with an affinity of 75 nM and an IC50 of 15.1 nM against SRIF-14 in a rat in vitro antagonist bioassay. Receptor-selective somatostatin antagonists should provide valuable tools for characterizing the many important physiological functions of this neuropeptide.