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.

Bombesin (Bn) receptor subtype 3 (BRS-3) is an orphan receptor that is a predicted member of the heptahelical G-protein receptor family and so named because it shares a 50% amino acid homology with receptors for the mammalian bombesin-like peptides neuromedin B (NMB) and gastrin-releasing peptide. In a recent targeted disruption study, in which BRS-3-deficient mice were generated, the mice developed obesity, diabetes, and hypertension. To date, BRS-3's natural ligand remains unknown, its pharmacology unclear, and cellular basis of action undetermined. Furthermore, there are few tissues or cell lines found that express sufficient levels of BRS-3 protein for study. The mammalian bombesin (Bn) 1 -like peptides gastrin-releasing peptide (GRP) and neuromedin B (NMB) contribute to diverse biological functions in the central nervous system (1, 2) and peripheral tissues (1, 2), which include thermoregulation (3), satiety (4), control of circadian rhythm (5), stimulation of pancreatic secretion (6), stimulation of gastrointestinal hormone release (7-9), and macrophage activation (10). These peptides also have important developmental effects (11,12) and potent growth effects (13-15), causing proliferation of normal cells (13,14,16,17) and various tumor cell lines (15, 16, 18 -20). To date, two mammalian receptor subtypes and their ligands have been identified, each of which has an architecture suggesting they are members of the heptahelical G-protein coupled receptor superfamily (21-23). One subtype, the GRP receptor, exhibits selectivity for GRP (21, 22, 24 -26), whereas the other, the NMB receptor, has selectivity for NMB (23,26,27). The intracellular signaling pathways of these two receptors have been characterized, with ligand binding resulting in stimulation of phospholipase C (14, 28 -30), protein kinase C activation (14), [Ca 2ϩ ] i mobilization (14,29,30), and tyrosine phosphorylation of various intracellular proteins (31-34).Recently, it has been proposed that an orphan receptor may represent a third type of mammalian bombesin receptor (35,36). This 399-amino acid protein, which was later identified in human tissues (35), was named bombesin receptor subtype-3 (BRS-3), due to its 51% and 47% amino acid sequence homology

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-

Pancreatic acini from most species possess vasoactive intestinal peptide (VIP) receptors. Recently, two subtypes of VIP receptors, VIP(1)-R and VIP(2)-R, were cloned. Which subtype exists on pancreatic acini or mediates secretion is unclear. To address this, we examined pancreatic acini from both rat and guinea pig. VIP(1)-R and VIP(2)-R mRNA were identified in dispersed acini from both species by Northern blot analysis and in rat by Southern blot analysis. With the use of the VIP(2)-R-selective ligand Ro-25-1553 in both species, inhibition of binding of (125)I-labeled VIP to acini showed a biphasic pattern with a high-affinity component (10%) and a second representing 90%. The VIP(1)-R-selective ligand, [Lys(15),Arg(16),Leu(27)]VIP-(1-7)-GRF-(8-27), gave a monophasic pattern. Binding of Ro-25-1553 was better fit by a two-site model. In both rat and guinea pig acini, the dose-response curve of Ro-25-1553 for stimulation of enzyme secretion was biphasic, with a high-affinity component of 10-15% of the maximal secretion and a low-affinity component accounting for 85-90%. At low concentrations (10 nM) of Ro-25-1553 and [Lys(15),Arg(16), Leu(27)]VIP-(1-7)-GRF(8-27), which only occupy VIP receptors, a 4-fold and a 56-fold increase in cAMP occurred, respectively. These results show that both VIP(1)-R and VIP(2)-R subtypes exist on pancreatic acini of rat and guinea pig, their activation stimulates enzyme secretion by a cAMP-mediated mechanism, and the effects of VIP are mediated 90% by activation of VIP(1)-R and 10% by VIP(2)-R. Because VIP has a high affinity for both VIP-R subtypes, its effect on pancreatic acini is mediated by two receptor subtypes, which will need to be considered in future studies of the action of VIP in the pancreas.