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Bombesin (BN) interacts with two mammalian receptor subtypes termed gastrin-releasing peptide (GRP)-preferring (GRP-R) and neuromedin B (NMB)-preferring (NMB-R) that may mediate the satiety action of BN. We examined the feeding behavior of mice that were deficient in the GRP-R (GRP-R KO) to assess the overall contribution of this receptor subtype in the feeding actions of BN-related peptides. GRP-R KO mice failed to suppress glucose intake in response to systemically administered BN and GRP(18-27), whereas both peptides elicited a potent reduction of intake in wild-type (WT) mice. Neither GRP-R KO nor WT mice suppressed glucose intake following NMB administration. Unlike the impaired responses to BN-like peptides, the feeding inhibitory action of cholecystokinin was enhanced in GRP-R KO mice. Consistent with behavioral results, GRP-R KO mice also exhibited a reduction in c-Fos immunoreactivity in the nucleus of the solitary tract (NTS) and paraventricular nucleus (PVN) following peripheral administration of BN. An evaluation of meal patterns showed that GRP-R KO mice ate significantly more at each meal than WT mice, although total 24 h food consumption was equivalent. A long-term analysis of body weight revealed a significant elevation in GRP-R KO mice compared with WT littermates beginning at 45 weeks of age. These data suggest that the GRP-R mediates the feeding effects of BN-like peptides and participates in the termination of meals in mice.

Three mammalian bombesin receptor subtypes have been characterized: the gastrin-releasing peptide receptor (GRP-R), the neuromedin B receptor (NMB-R), and bombesin receptor subtype 3 (BRS-3). In a previous report we identified four amino acids that are critical for high affinity binding of bombesin and gastrin-releasing peptide (GRP) to the GRP-R. These four amino acids are conserved in all species variants of the GRP-R and NMB-R which bind bombesin with high affinity, but they are diverged in BRS-3, the bombesin receptor subtype that binds bombesin with much lower affinity. Substituting these four divergent amino acids in BRS-3 for the conserved amino acids in either GRP-R or NMB-R increased the affinity of the mutated BRS-3 (4DeltaBRS-3) for bombesin compared with wild-type BRS-3. We hypothesized that the same four amino acids might be critical for high affinity NMB binding to the NMB-R. In this study we confirm this hypothesis by showing that the affinity of NMB is increased in a mutant BRS-3 receptor (4DeltaBRS-3) that contains these four substitutions resulting in an affinity that is close to the affinity of wild-type NMB-R for NMB. In contrast, these four amino acid substitutions in BRS-3 did not result in the formation of a high affinity binding site for the recently described non-peptide NMB-R antagonist PD168368.

The receptor for neuromedin B (NMB-R), a mammalian bombesin-related peptide, is widely distributed in the central nervous system and gastrointestinal tract. While it is known that this receptor is coupled to phospholipase C, like many other phospholipase C-activating receptors, little is known about regulation of the NMB-R subsequent to agonist stimulation. We studied both native NMB-R on C-6 rat glioblastoma cells and wild type NMB-R cloned from rat esophageal muscle which was stably transfected into Balb/3T3 fibroblasts. Both cell types rapidly increased [3H]inositol phosphates and [Ca2+]i in response to 1 microM NMB, whereas preincubation with 3 nM NMB for 3 h markedly attenuated the ability of 1 microM NMB, but not 1 microM endothelin-1, to alter either cell type's biological activity. Prolonged exposure to 3 nM NMB caused a rapid decrease in the number of NMB-R, with the maximal receptor down-regulation seen at 24 h due to NMB-R internalization. After maximal down-regulation, removal of agonist resulted in a rapid restoration of NMB-R to the cell surface of both cell types. NMB-R recovery at 6 h was blocked by monensin, an inhibitor of receptor recycling, but was not affected by cycloheximide, a protein synthesis inhibitor. Resensitization to agonist paralleled the recovery of NMB-R in both cell types, and resensitization likewise was blocked by monensin. Our data demonstrate that the NMB-R undergoes rapid homologous desensitization consequent to agonist stimulation, which is mediated by receptor down-regulation and which, in turn, is regulated by internalization. During resensitization, NMB-R reappearance on the cell surface membrane is independent of protein synthesis and is due to a recycling from an intracellular site.

The two bombesin receptor subtypes, neuromedin B (NMB-R) and gastrin releasing peptide (GRP-R) receptors, bind their respective ligands with high affinity. To identify molecular components mediating high affinity NMB binding, four mutant receptors were constructed, in which different parts of the NMB-R were replaced with the corresponding regions of the GRP-R. When stably expressed in Balb 3T3 fibroblasts, all four NMB-R/GRP-R chimeras were functional and showed NMB-induced stimulation of inositol phosphate (IP) formation. Results of 125I-[D-Tyr0]NMB displacement assays using unlabeled NMB for competition indicated that high affinity NMB binding was determined by amino acid sequences in transmembrane domain V (TM-V) of the NMB-R. To identify which amino acid(s) in TM-V of NMB-R contributed to high affinity NMB binding, four additional NMB-R mutants were constructed where non-conserved amino acids in TM-V of NMB-R were replaced by the corresponding GRP-R amino acids. Three of the mutations, TyrPheLeu220-222-->PheTyrVal, Ile230-->Val, and His234-->Phe, did not affect high affinity NMB binding. The Ile216-->Ser substitution, however, abolished high affinity NMB binding and severely impaired the ability of the mutant receptor to stimulate NMB-dependent inositol phosphate formation. These results suggest that ILe216 in TM-V of NMB-R may be critical for high affinity NMB binding.

Recent cloning studies confirm the presence of two subtypes of bombesin (Bn) receptors. In contrast to the gastrin-releasing peptide (GRP)-preferring subtype, which has been widely studied, nothing is known about the cellular mechanisms of the neuromedin B (NMB)-preferring subtype, which occurs widely in the central nervous system and gastrointestinal tissues, partially because of the lack of a cell line with functional receptors. In the present study we have investigated Bn receptors on the rat glioblastoma cell line C-6, reported to contain mRNA of the NMB receptor subtype. Binding of 125I-[D-Tyr0]NMB to these cells was time- and temperature-dependent, saturable, reversible, and only inhibited by Bn receptor agonists or antagonists. For Bn receptor agonists the relative potencies were: NMB (1.7 nM) approximately equal to litorin (3 nM) greater than ranatensin (8 nM) greater than Bn (19 nM) greater than neuromedin C (NMC) (210 nM) greater than GRP (500 nM). These relative affinities were almost identical to those for the NMB receptor subtype on rat oesophageal tissue and for Balb 3T3 cells stably transfected with the NMB receptor subtype. These potencies differed from those for the GRP receptor subtype on rat pancreatic acini [Bn approximately equal to litorin (4 nM) greater than ranatensin, NMC, GRP (15-20 nM) much greater than NMB (351 nM)]. The relative potencies of four different classes of Bn receptor antagonists were compared. Results from C-6 tumour cells agreed closely with those for binding to the NMB receptor subtype on rat oesophageal tissue and in Balb 3T3 cells stably transfected with this receptor, and differed markedly from those for binding to the GRP receptor subtype on rat pancreatic acini. Four Bn receptor antagonists had a higher affinity for the GRP subtype ([D-Phe6]Bn-(6-13)ethyl ester (500 x), [D-Phe6][psi 13-14,Cpa14]Bn- (6-14) (70 x) (where psi 13-14 refers to the replacement of the -CONH- peptide bond between Leu13 and Met14 by -CH2NH2) [psi 13-14,Leu14]Bn, [D-Phe6]Bn-(6-13) propylamide (30 x)] and two had a higher affinity for the NMB subtype on C-6 cells and transfected cells ([D-Pro4,D-Trp7,9,10] substance P-(4-11) (9 x) and [Tyr4,D-Phe12]Bn (18 x)]. In C-6 tumour cells, Bn receptor agonists caused an increase in cytosolic Ca2+ and the generation of inositol phosphates. For both responses, NMB was more than 50-fold more potent than GRP. Neither NMB nor GRP increased cyclic AMP. These results demonstrate that the rat glioblastoma cell line C-6 possesses functional NMB-preferring Bn receptors, and agonist occupation activates phospholipase C, thus increasing cytosolic Ca2+ and inositol phosphate formation. Because the interaction of Bn-related peptides with C-6 cell receptors is identical with that reported in other tissues containing the mRNA for the NMB subtype, this cell line should prove useful in exploring further the cellular basis of action of the peptides that interact with this receptor in the central nervous system and various other tissues.

The bombesin-like peptides are a family of structurally related amidated peptide ligands that are known to have a variety of potent pharmacological actions on various cells, including neurons in the rat brain. Two mammalian representatives of the bombesin family of peptides have been identified, gastrin-releasing peptide (GRP) and neuromedin B (NMB). Previously, we cloned the rat preproGRP gene and determined the locations of neurons expressing this gene using in situ hybridization. In this study, we describe the structure and sequence of the rat preproNMB gene, and the first detailed cellular localization of preproNMB mRNA in rat brain using in situ hybridization. Nucleotide sequence analysis of cDNA and genomic clones reveals a 117 amino acid precursor whose overall structure is similar to that described for human preproNMB. Sequence similarity between the rat NMB and GRP genes is observed only over a limited 10 amino acid sequence encoding the carboxy termini of the GRP and NMB peptides, the region shown to be necessary and sufficient for high-affinity receptor binding. In situ hybridization studies performed with cRNA probes specific for NMB or GRP mRNA show that the distribution of cells expressing either mRNA in brain is very distinct. NMB mRNA is found most prominently in the olfactory bulb, dentate gyrus, and dorsal root ganglion. In contrast, the highest levels of GRP mRNA are observed in the forebrain (isocortex and hippocampal formation). This heterogeneity of mRNA distribution for these peptides suggests that these 2 structurally related peptides may have very distinct functions as neuropeptides in the rat nervous system.