International Union of Pharmacology. LXVIII. Mammalian Bombesin Receptors: Nomenclature, Distribution, Pharmacology, Signaling, and Functions in Normal and Disease States

A. Early Studies of the BB₁ Receptor

Before the identification of the BB₁ in 1989 in rat esophageal muscle tissue sections by direct binding studies using ¹²⁵I-Bolton-Hunter-labeled NMB and subsequent esophageal muscle strip contraction studies (von Schrenck et al., 1989), there were no early studies that unequivocally established the existence of BB₁. Numerous previous studies had demonstrated that the frog peptides ranatensin and litorin, which closely resembled NMB (Minamino et al., 1983), had potent effects on various tissues and especially on smooth muscle contraction, which in some classes had differences from bombesin (Falconieri Erspamer et al., 1988; Regoli et al., 1988). However, these differences were not significant enough to clearly establish the existence of a separate class of BB₁ receptors (Minamino et al., 1983; Falconieri Erspamer et al., 1988; Regoli et al., 1988). Although there had been many binding studies to numerous tissues from the late 1970s, in almost all cases ¹²⁵I-[Tyr⁴] bombesin or another radiolabeled bombesin analog was used (Moody et al., 1978; Ladenheim et al., 1993b; Shapira et al., 1993). Unfortunately, bombesin has high affinity for both BB₁ and BB₂, making it more difficult to distinguish subtypes. Numerous classes of selective BB₂ receptor antagonists were developed before the cloning of the BB₁, and these also confirmed the presence of the BB₁ on esophageal smooth muscle (von Schrenck et al., 1990). After the pharmacologic description of BB₁ on esophageal muscle and before its cloning in 1991, by use of selective BB₂ receptor antagonists or binding studies with radiolabeled NMB and selective agonists or BB₂ receptor antagonists, BB₁ receptors were demonstrated in the CNS (Ladenheim et al., 1990) and on gastric smooth muscle cells (Severi et al., 1991).

B. Cloned BB₁ Receptor and Receptor Structure

The human BB₁ receptor is a 390-amino acid protein, and it shows an 89% amino acid identity with the rat BB₁ (Corjay et al., 1991). The human BB₁ receptor has 55% amino acid identities with the human BB₂ (Corjay et al., 1991) and 47% with the human BB₃ receptor (Fathi et al., 1993b). The human BB₁ receptor has two consensus sites for potential PKC phosphorylation and three potential N-linked glycosylation sites (Corjay et al., 1991). Hydropathy plots yielded results consistent with a seven-transmembrane structure typical for a G protein-coupled receptor (Corjay et al., 1991). The BB₁ receptor has been cloned from rat (Wada et al., 1991) (Fig. 2), mouse (Ohki-Hamazaki et al., 1997a), and the frog, B. orientalis (Nagalla et al., 1995). Cross-linking studies demonstrate that the mature human BB₁ receptor had a molecular mass of 72 ± 1 kDa and when deglycosylated 43 ± 1 kDa (Benya et al., 1995b). Detailed cross-linking and serial deglycosylation studies using enzymatic digestion in the rat BB₁ receptor demonstrated a molecular mass of 63 kDa in the membrane and showed that there were no O-linked carbohydrates, but that the mature BB₁ receptor was a sialoprotein (Kusui et al., 1994). However, each of the potential N-linked glycosylation sites was, in fact, glycosylated, with tri-antennary and/or tetra-antennary complex oligosaccharide chains (Kusui et al., 1994).

C. BB₁ Receptor Genomic Organization

The human BB₁ receptor gene is localized at human chromosome 6p21-qter and in the mouse on chromosome 10 (Table 1). Both the human, rat, and mouse genes contained three exons with two introns (Corjay et al., 1991; Wada et al., 1991; Ohki-Hamazaki et al., 1997a; Ohki-Hamazaki, 2000). In the mouse the gene for the BB₁ receptor spanned more than 10 kb with exon 1 of the BB₁ gene separated from exon 2 by 6 kb, and this in turn is separated from exon 3 by 3 kb (Ohki-Hamazaki et al., 1997a). In human and mouse the first intron of the BB₁ gene was located between transmembrane domains 3 and 4 and the second between transmembrane domains 5 and 6 (Corjay et al., 1991; Ohki-Hamazaki et al., 1997a). The first intron interrupted a codon for arginine located immediately COOH terminal to the transmembrane domain 3, and the second intron was located between glutamine and methionine codons in both the mouse and human BB₁ gene (Corjay et al., 1991; Ohki-Hamazaki et al., 1997a). The positions of the first and second introns were identical in the mouse and human BB₁ receptor gene (Corjay et al., 1991; Ohki-Hamazaki et al., 1997a).

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Fig. 2.

Ability of the mammalian peptides, GRP and NMB, the amphibian peptide, bombesin, and the synthetic bombesin analog [d-Phe⁶,β-Ala¹¹,Phe¹³,Nle¹⁴]bombesin_6–14 to interact with the three human classes of bombesin receptors. Data are partially from Benya et al. (1995b) and Ryan et al. (1998b).

D. BB₁ Receptor Expression

Expression levels of BB₁ receptor mRNA have been reported in human, mouse, rat, and monkey (Corjay et al., 1991; Wada et al., 1991; Ohki-Hamazaki et al., 1997a; Sano et al., 2004). In the monkey, in which it was studied in detail, the highest levels of BB₁ mRNA are found in the CNS and in the testis (Sano et al., 2004). In the CNS the BB₁ receptor was expressed widely in different brain regions including the amygdala, caudate nucleus, hippocampus, hypothalamus, thalamus, brain-stem, spinal cord, and peripheral tissues in addition to the testis and the stomach, which is a similar distribution to that found in rats and mice (Wada et al., 1991; Ohki-Hamazaki et al., 1997a; Ohki-Hamazaki, 2000; Sano et al., 2004). In the rat and mouse, BB₁ mRNA is present in high amounts in the olfactory region and esophagus (Wada et al., 1991; Ohki-Hamazaki et al., 1997a). Binding studies and studies of biological activity provide evidence for BB₁ on both gastrointestinal and urogenital smooth muscle cells (von Schrenck et al., 1989; Severi et al., 1991; Bitar and Coy, 1992; Kim et al., 1993). Binding studies have confirmed the widespread distribution of BB₁ in the brain showing especially high levels in the olfactory tract of the rat (Ladenheim et al., 1990, 1992, 1993a).

Using binding studies and/or assessment of BB₁ mRNA, BB₁ receptors have been shown to exist on a large number of different tumors (Reubi et al., 2002; Jensen and Moody, 2006) including CNS tumors (glioblastomas) (Wada et al., 1991; Wang et al., 1992), small cell and non-small cell lung cancers (Corjay et al., 1991; Moody et al., 1992, 2000; Toi-Scott et al., 1996; Siegfried et al., 1997; Jensen and Moody, 2006), carcinoids (intestinal, thymic, and bronchial) (Reubi et al., 2002), human ovarian epithelial cancers (Sun et al., 2000b), and pancreatic cancer cell lines (Jensen and Moody, 2006).

E. BB₁ Receptor Pharmacology

F. BB₁ Receptor Structural Basis of Receptor Binding/Activation

1. BB₁ Receptor Agonist Binding/Activation.

Structure-function studies of NMB demonstrated that the COOH-terminal octapeptide is the minimal peptide length required for BB₁ receptor activation and the full decapeptide was required for full affinity for the BB₁ receptor (Lin et al., 1996). NMB differs from GRP in the COOH octapeptide, which is the biologically active end (Broccardo et al., 1975; Lin et al., 1996), at three residues: substitution of a leucine in NMB for a histidine in GRP at position 3, a threonine for valine at position 6, and a phenylalanine for leucine at position 9 of NMB from the amino terminus (Minamino et al., 1983; Lin et al., 1996) (Fig. 1). Structure-function studies of all naturally occurring bombesin-related peptides for BB₁ and BB₂ receptors suggested the presence of the phenylalanine instead of leucine, as the penultimate amino acid from the COOH terminus in NMB was not important for selectivity for the BB₁ receptor, Single amino acid substitutions in NMB demonstrated the Leu for His substitution in position 3 was the most important for determining high affinity and selectivity for the BB₁ receptor (Lin et al., 1996) (Fig. 1).

A chimeric receptor approach (Fathi et al., 1993a) and homology screening after computer alignment of bombesin receptor family members (Sainz et al., 1998), followed by site-directed mutagenesis studies, have been used to explore the molecular basis of NMB high affinity and selectivity for the BB₁ receptor over the BB₂ receptor (Fig. 3). A study of BB₁/BB₂ chimeric receptors (Fathi et al., 1993a) demonstrated that differences in the amino terminus of the two receptors were of minimal importance for high-affinity NMB interaction. High affinity and selectivity for the BB₁ receptor were primarily determined by differences in transmembrane (TM) domain 5 (Fathi et al., 1993a) (Fig. 3). Site-directed mutagenesis of the amino acid differences between the BB₁ receptor and the BB₂ receptor in this region demonstrated that the substitution of an Ile²¹⁶ instead of Ser in the comparable position of the TM5 of the BB₂ receptor was the critical difference accounting for high-affinity NMB interaction with the BB₁ and not the BB₂ receptor (Fathi et al., 1993a). A second study (Sainz et al., 1998) used a different approach to select potentially important amino acids for NMB selectivity for the BB₁ receptor and further study. Using amino acid sequence alignment of bombesin receptor family members and identifying conserved amino acids in members with similar peptide affinities (Akeson et al., 1997), four amino acids were identified that could be important for high-affinity bombesin binding to either the BB₁ or BB₂ receptor (Akeson et al., 1997) (i.e., in the BB₁ receptor, Gln¹²³, Pro²⁰⁰, Arg²⁹⁰, and Ala³¹⁰, and in the BB₂ receptor, Gln¹²¹, Pro¹⁹⁹, Arg²⁸⁸, and Ala ³⁰⁸). Possible gain-of-affinity mutants were made in the BB₃ receptor, which has a low affinity for NMB (Mantey et al., 1997; Ryan et al., 1998a,b), by substituting alone or in combination each of these four BB₁ receptor amino acids for the comparable amino acid(s) of the BB₃ receptor (Arg¹²⁷, Ser²⁰⁵, His²⁹⁴, and Ser³¹⁵) (Fig. 3). It was found that each of these four amino acids is important for determining NMB affinity because the affinities for NMB of the BB₃ mutants with these BB₁ receptor amino acids substituted one at a time were increased (Sainz et al., 1998). The substitution of all four amino acids for the comparable amino acids in the BB₃ receptor, which has a very low affinity for NMB (i.e., K_i 3450 nM), increased the affinity and the potency for NMB, almost up to that seen with the native BB₁ receptor (Sainz et al., 1998). This study helped to define the binding pocket for NMB by identifying four amino acids needed for high-affinity NMB interaction in markedly different BB₁ regions [transmembrane domain 2 (Gln¹²³), extracellular domain 2 (Pro²⁰⁰), extracellular domain 3 (Arg²⁹⁰), and transmembrane region 7 (Ala³¹⁰)] (Fig. 3) (Sainz et al., 1998).

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Fig. 3.

Schematic representation of the rat BB₁ receptor showing the postulated transmembrane topology, sites for NH₂-linked glycosylation, possible palmitoylated cysteines in the cytoplasmic tail, and the key amino acids for high-affinity NMB interaction (dark circles) or interaction with the BB₁ receptor specific peptoid antagonist PD 168368 (shaded circles). Amino acid data are from Wada et al. (1991), data for NMB high-affinity sites are from Fathi et al. (1993a) and Sainz et al. (1998), and data for PD 168368 are from Tokita et al. (2001a).

G. BB₁ Receptor Signaling, Activation, and Modulatory Processes (Internalization, Down-Regulation, and Desensitization)

The human BB₁ receptor (Moody et al., 1986, 1992, 1995a; Corjay et al., 1991; Benya et al., 1995b), as well as the rat BB₁ receptor (Wada et al., 1991; Jones et al., 1992; Wang et al., 1992; Dobrzanski et al., 1993; Lach et al., 1995; Akeson et al., 1997; Tsuda et al., 1997b; Vigne et al., 1997; Hou et al., 1998) is coupled to phospholipase C, resulting in breakdown of phosphoinositides, mobilization of cellular calcium, and activation of protein kinase C. BB₁ receptor activation also results in the stimulation phospholipase A₂ (Moody et al., 1995a) and phospholipase D by a PKC-dependent and -independent mechanism (Tsuda et al., 1997b) but does not activate adenylate cyclase (Benya et al., 1992). BB₁ receptor stimulation also results in activation of tyrosine kinases (Lach et al., 1995; Tsuda et al., 1997b) stimulating tyrosine phosphorylation of p125^FAK by a phospholipase C-independent mechanism that requires p21^rho and the integrity of the actin cytoskeleton (Tsuda et al., 1997b). BB₁ receptor activation also stimulated tyrosine phosphorylation of paxillin and MAP kinase activation (Lach et al., 1995). The native and transfected rat BB₁ receptor in BALB 3T3 cells have been shown to behave in a similar manner in their binding and signaling cascades (Benya et al., 1992), demonstrating the usefulness of this cell line for studying BB₁ receptor interaction and signaling.

The BB₁ receptor is coupled to heterotrimeric guanine-nucleotide binding proteins in both native and BALB 3T3-transfected cells (Benya et al., 1992; Wang et al., 1993). In an Xenopus oocyte assay with the injection of antisense oligonucleotides, G_αq was identified as a mediator of the BB₁ receptor response (Shapira et al., 1994). With an in situ reconstitution assay with purified G protein α subunits, it was found that cells expressing the BB₁ receptor activated G_αq, but not G_αt or G_αi/o (Jian et al., 1999). This activation was enhanced by βγ dimers with a relative potency of βγ > β1γ 2 >> β1γ1. In this study (Jian et al., 1999), these results were contrasted with those for the BB₂ receptor, and differences were found in their kinetics of activation and preference for G_αq proteins from different sources and for βγ dimers, demonstrating distinct coupling mechanisms for these two closely related receptors (Jian et al., 1999).

In contrast with the BB₂ receptor there have been few studies of BB₁ receptor modulatory processes (internalization, down-regulation, or desensitization). Both the human (Benya et al., 1995b) and rat BB₁ receptors (Benya et al., 1992, 1994c; Wang et al., 1993) are rapidly internalized with receptor activation of the BB₁ receptor. The rat BB₁ receptor internalized 60 to 80% of the bound ligand, and human BB₁ receptors internalized 70% of the bound ligand. In addition to being rapidly internalized by BB₁ receptor-bearing cells, the ligand is rapidly degraded by these cells (Benya et al., 1992; Wang et al., 1993). Protease inhibitors markedly decreased ligand degradation by either rat native or rat BB₁ receptor-transfected BALB 3T3 cells (Benya et al., 1992; Wang et al., 1993) with the acid proteinase inhibitor, leupeptin being the most potent followed by bacitracin > chymostatin > phosphoramidon >> bestatin and amastatin. The BB₁ receptor also undergoes desensitization, which is mediated by receptor down-regulation and internalization (Benya et al., 1994c). Preincubation for 3 h with 3 nM NMB markedly attenuated the ability of a maximally effective concentration of NMB (1 μM) to subsequently stimulate either native or BB₁-transfected BALB 3T3 cells but did not alter the response to other stimulants (Benya et al., 1994c). This desensitization was associated with a rapid decrease in BB₁ receptors due to internalization of the receptors. Restoration of receptor number and response recovered over a 6-h period, and it was not dependent on new protein synthesis but was due to receptor recycling, because it was inhibited by the recycling inhibitor, monesin, a monocarboxylic acid cation ionophore (Benya et al., 1994c).

H. BB₁ Receptor Function in Various Tissues and in Vivo

One of the main difficulties in assessing the effects of BB₁ receptor activation in the CNS as well as in peripheral tissues, especially in older studies, is that bombesin was frequently used as the agonist, and it interacts with both BB₁ and BB₂ receptor with relatively high affinity. Furthermore, many tissues possess both BB₁ and BB₂ receptors, and therefore it was difficult to assess whether a particular response was due to activation of the BB₁ or BB₂ receptors present.

Numerous effects of NMB in both in vivo and in vitro studies have been reported, but it is not clear in many cases which are physiological and which are pharmacological. Studies comparing the potencies of NMB to GRP as well as binding studies or antagonist studies provide evidence that the BB₁ receptor can stimulate contraction of urogenital and gastrointestinal smooth muscle (esophageal, gastric, colon, and gallbladder) (Regoli et al., 1988; von Schrenck et al., 1989, 1990; Severi et al., 1991; Kilgore et al., 1993; Parkman et al., 1994; Milusheva et al., 1998), potently inhibit thyrotropin release from the pituitary gland by acting as an autocrine and paracrine regulator (Rettori et al., 1992; Pazos-Moura et al., 1996; Ortiga-Carvalho et al., 2003), and have potent CNS effects including inhibiting food intake independent of BB₂ stimulation (Ladenheim et al., 1994, 1996b, 1997b; Merali et al., 1999; Ladenheim and Knipp, 2007) and mediating aspects of the stress and fear responses as well as various behaviors such as spontaneous activity (Merali et al., 2002, 2006).

BB₁ receptor knockout mice are now available and have undergone a limited number of investigations for actions of NMB (Ohki-Hamazaki et al., 1999; Oeffner et al., 2000; Yamada et al., 2002b, 2003; Yamano et al., 2002) (Table 1). In these mice the hypothermic effect of NMB was reduced by 50% without a change in the GRP response, supporting a possible BB₁ receptor-mediated role in thermoregulation: NMB-mediated gastric smooth muscle contraction was not affected, suggesting this is mediated not through BB₁ receptors, and no effect on feeding could be confirmed, although NMB did not have an effect in the control animals (Ohki-Hamazaki et al., 1999). The satiety effects of the BB₁ receptor are mediated through peripheral neural pathways different from those mediating the satiety effects of the BB₂ receptor, because only the satiety effects of BB₁ receptors are inhibited by capsaicin treatment, suggesting the involvement of primary sensory afferent neurons (Ladenheim and Knipp, 2007). Recently, NMB has found to be expressed in human and rodent adipose tissue and to be regulated by changes in energy balance. It was proposed that because of the known anorectic effects of NMB centrally, it may form part of a new adipose tissue-hypothalamic regulating system for food intake (Hoggard et al., 2007). In BB₁ receptor knockout mice dysregulation of the thyroid occurred, suggesting that BB₁ receptor pathways are significantly involved in both TSH gene regulation and function (Oliveira et al., 2006), dysfunction in response to stress was seen (Yamada et al., 2002b; Yamano et al., 2002), impairment in the modulation of the CNS 5-HT system in response to stress occurred (Yamano et al., 2002), and an impairment of learning and memory was seen (Yamada et al., 2003). The alterations in the CNS 5-HT and stress in these animals is particularly interesting, because the dorsal raphe nucleus is one of the brain regions that has a preponderance of BB₁ receptors (Wada et al., 1990; Ladenheim et al., 1992; Pinnock et al., 1994; Merali et al., 2006), which are located on 5-HT neurons, and stimulation of this nucleus by NMB stimulates release of 5-HT, resulting in anxiogenesis (Merali et al., 2006). In a study in rats using BB₁ and BB₂ receptor agonists and antagonists (Bédard et al., 2007), data were provided to show that both GRP and NMB affect the stress response. NMB affected both anxiety and fear responses, whereas GRP affected only fear responses (Bédard et al., 2007).

Whereas the growth effects of the BB₂ receptor in normal and especially in neoplastic tissues have received the most attention, stimulation of the BB₁ receptor and/or administration of NMB has been shown to have growth-promoting effects in a number of neoplastic tissues. NMB is an autocrine growth factor for non-small cell lung cancer with 14 of 14 such cell lines possessing BB₁ receptors in one study (Siegfried et al., 1997), and in four non-small cell lung cancer cell lines examined in detail NMB was synthesized and released into the media by the tumor cell in 7 to 15 times greater amounts than was GRP (Siegfried et al., 1997). Blockade of the BB₂ receptor only partially blocked the proliferative effect of NMB on these cells, demonstrating the importance of BB₁ receptor activation for the proliferative effects in these tumor cells (Siegfried et al., 1997). Furthermore, in human colon cancers NMB and the BB₁ receptor are coexpressed, and they act in an autocrine growth fashion (Matusiak et al., 2005). Activation of BB₁ receptors causes proliferation of rat C6 glioblastoma cells (Moody et al., 1995a), BB₁ receptor transfected RAT-1 cells (Lach et al., 1995), small cell lung cancers (Moody et al., 1992), and adrenal zona fasciculata cells (Malendowicz et al., 1996).

I. BB₁ Receptor in Diseases

At present, no disease has been shown to be caused specifically by alterations in the BB₁ receptor. Activation of the BB₁ receptor in various human cancers (particularly human small cell lung cancers, non-small cell lung cancers, colon, cancer, and various carcinoid tumors) due to an autocrine growth pathway may have an important effect on their growth (Moody et al., 1992; Moody and Jensen, 1996; Siegfried et al., 1999; Matusiak et al., 2005; Jensen and Moody, 2006). In various studies BB₁ receptors were overexpressed by 55% of small cell lung cancers, 67% of non-small cell lung cancers, 46% of intestinal carcinoids, and a proportion of colon cancers, prostate cancers, and CNS tumors such as glioblastomas (Moody et al., 1995a; Reubi et al., 2002; Matusiak et al., 2005; Jensen and Moody, 2006).

Numerous studies (Rettori et al., 1992; Pazos-Moura et al., 1996; Ortiga-Carvalho et al., 2003) including BB₁ receptor knockout studies (Oliveira et al., 2006) support the conclusion that NMB plays an important physiological role in the regulation of thyrotropin release, having primarily an inhibitory effect. NMB is produced in the pituitary (Jones et al., 1992), and it is proposed that NMB functions as a tonic inhibitor of TSH secretion, acting as an autocrine/paracrine regulator (Rettori et al., 1992; Oliveira et al., 2006) (Table 1). Conditions with increased TSH release such as hypothyroidism are associated with decreased pituitary NMB levels (Jones et al., 1992; Ortiga-Carvalho et al., 2003), whereas in hyperthyroidism in which the TSH levels are suppressed; there is an increased pituitary NMB level (Jones et al., 1992; Ortiga-Carvalho et al., 1997). These results suggest NMB could play an important role in human thyroid disorders causing hyperor hypofunction.

The role of NMB in human feeding disorders is unclear at present. Two genetic studies have suggested that the NMB gene is a possible candidate for eating disorders and predisposition to obesity (Oeffner et al., 2000; Bouchard et al., 2004).

A. Early Studies of the BB₂ Receptor

Many of the early studies provided limited information on the BB₂ receptor, as discussed in section III.A. for the BB₁ receptor. This occurred because many of the tissues studied are now known to possess both BB₂ and BB₁ receptors and in most studies bombesin analogs were used, which have high affinity for both subclasses of receptors. This situation continued after the isolation of GRP in 1978 (McDonald et al., 1979), even though it had greater selectivity than bombesin analogs for the BB₂ over the BB₁ (von Schrenck et al., 1989; Lin et al., 1995; Benya et al., 1995b; Reubi et al., 2002), because of its limited availability. In vivo studies were even more difficult to interpret because numerous studies demonstrated that GRP-related peptides can have both a direct action on tissues as well as indirect action as they are potent for stimulating the release of many hormones (gastrin, insulin, somatostatin, CCK, pancreatic polypeptide, enteroglucagon, pancreatic glucagon, and gastric inhibitory peptide) (Greeley et al., 1986; McDonald et al., 1979, 1983; Modlin et al., 1981; Ghatei et al., 1982; Knuhtsen et al., 1987; Pettersson and Ahren, 1987; Kawai et al., 1988; Hermansen and Ahren, 1990). With the development of selective BB₂ receptor antagonists (von Schrenck et al., 1990; Jensen and Coy, 1991; Benya et al., 1995b) and the increased use of BB₂ selective ligands such as GRP, it became clear that a separate GRP-preferring receptor existed, even before the cloning of the mouse and human BB₂ receptor in the early 1990s (Spindel et al., 1990; Battey et al., 1991; Corjay et al., 1991) (Table 2). It subsequently became clear that a number of the tissues that had been extensively used to characterize bombesin receptors/responses such as pancreatic acinar cells (Jensen et al., 1978; Jensen, 1994) and Swiss 3T3 cells (Rozengurt, 1988) possessed only BB₂ receptors, whereas other tissues such as the CNS (Battey and Wada, 1991; Ladenheim et al., 1992) and smooth muscle preparations possessed both BB₁ and BB₂ receptors (Severi et al., 1991).

B. Cloned BB₂ Receptor and Receptor Structure

The human BB₂ receptor has 384 amino acids and shows high homology (90% amino acid identities) with the mouse BB₂ receptor (Corjay et al., 1991) (Fig. 4). The human BB₂ receptor has 55% amino acid identities with the human BB₁ receptor (Corjay et al., 1991) and 51% with human BB₃ receptor (Fathi et al., 1993b). Hydropathy analysis of the predicted human BB₂ structure revealed seven regions of hydrophobic amino acids consistent with a seven-transmembrane structure typical for G protein-coupled receptors (Corjay et al., 1991). There were two consensus sites of potential PKC phosphorylation and two potential sites for N-linked glycosylation in the human BB₂ receptor (Corjay et al., 1991). The BB₂ receptor has been completely or partially cloned from 21 species (Baldwin et al., 2007) and the most highly conserved regions are in the transmembrane domains and the third intracellular domain (Baldwin et al., 2007). The presence of a likely disulfide bond between cysteines at the end of the extracellular domain 1 and middle of extracellular domain 2 (Cys¹¹³ and Cys¹⁹⁶ in human BB₂) is preserved in all noninsect species (Baldwin et al., 2007) (Fig. 4). Solubilization studies as well as cross-linking studies demonstrate that the mature human BB₂ receptor has a molecular weight greater than that predicted from the structure (Kris et al., 1987; Rozengurt, 1988; Feldman et al., 1990; Huang et al., 1990; Staley et al., 1993; Benya et al., 1994b; Kusui et al., 1994; Williams and Schonbrunn, 1994; Benya et al., 1995b). Cross-linking studies demonstrate that the mature human BB₂ receptor has a molecular mass of 60 ± 1 kDa and the mouse BB₂ receptor has a molecular mass of 82 ± 2 kDa and when each is deglycosylated the molecular mass is 43 ± 1 kDa (Kris et al., 1987; Rozengurt, 1988; Huang et al., 1990; Benya et al., 1994b; Kusui et al., 1994; Williams and Schonbrunn, 1994; Benya et al., 1995b). These results demonstrate that 35% of the molecular mass of the mature human BB₂ receptor is due to glycosylation, whereas in the mouse BB₂ receptor it is 47%. This difference is probably due to the existence of two potential sites of N-linked glycosylation in the human BB₂ receptor compared with four potential sites in the mouse BB₂ receptor (Spindel et al., 1990; Battey et al., 1991; Corjay et al., 1991; Benya et al., 1995b) (Fig. 4). Using cross-linking studies with serial deglycosylation by enzymatic digestion (Kusui et al., 1994, 1995), and a molecular approach involving mutating the four potential N-linked glycosylation sites either alone or in combination in the murine BB₂ receptor followed by receptor expression and cross-linking analysis (Benya et al., 1994d), the murine BB₂ receptor was shown to be glycosylated at all four potential N-linked sites (Asn⁵, Asn²⁰, Asn²⁴, and Asn¹⁹¹) (Benya et al., 1994d; Kusui et al., 1994, 1995). The extent of glycosylation varied, however, with carbohydrate residues of 12 kDa on Asn⁵, 10 kDa on Asn²⁰, 5 kDa on Asn²⁴, and 9 kDa on Asn¹⁹¹ (Benya et al., 1994d). The presence of the glycosylation on Asn²⁴ and Asn¹⁹¹ was especially important for sorting and expression of the murine BB₂ receptor on the plasma membrane (Benya et al., 1994d). Digestion of the cross-linked receptor with different enzymes demonstrated that the murine BB₂ receptor was not a sialoprotein, contained no O-linked glycosylation, and had four tri-antennary and or tetra-antennary complex oligosaccharide chains (Kusui et al., 1994). Studies using baculovirus expression of the BB₂ receptor (Kusui et al., 1995) demonstrated that neither full glycosylation was needed for receptor expression on the cell surface nor did the glycosylation have to be tri- or tetra-antennary for expression, because in the baculovirus only 11 kDa of glycosylation was seen on different sites, and the glycosylation was entirely bi-antennary complex oligosaccharide chains (Kusui et al., 1995).

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Fig. 4.

Schematic representation of the murine BB₂ receptor showing the postulated transmembrane topology, sites for NH₂-linked glycosylation, possible palmitoylated cysteines in the cytoplasmic tail, and the key amino acids for high-affinity GRP interaction or signaling (dark circles) or interaction with the BB₂ selective antagonist statin analog JMV594 or the pseudopeptide analog JMV641 (shaded circles). Amino acid data are from Spindel et al. (1990) and Battey et al. (1991); GRP high-affinity sites are from Akeson et al. (1997), Donohue et al. (1999), Carroll et al. (2000b), Lin et al. (2000), Tokita et al. (2002), Glover et al. (2003), and Nakagawa et al. (2005); and data for JMV594 and JMV641 are from Tokita et al. (2001b).

C. BB₂ Receptor Genomic Organization

The human BB₂ receptor gene was localized to Xp22 (Maslen and Boyd, 1993; Xiao et al., 2001) and the murine BB₂ receptor gene to X chromosome between the Pdha-1 and Amg loci (Maslen and Boyd, 1993). Both the human (Xiao et al., 2001) and murine (Weber et al., 2000) BB₂ receptor gene organizations have been studied in detail. The human BB₂ receptor gene has three exons (Corjay et al., 1991; Xiao et al., 2001) spanning more than 27 kb with intron 1 and intron 2 being 23 and 1.6 kb (Xiao et al., 2001). Exon one encodes the first three membrane-spanning domains of the BB₂ receptor, and the splice site is located in the proximal second intracellular loop (residue 137). Exon 2 encodes for the transmembrane regions 4 and 5 and most of the third intracellular loop with the splice site located at residue 254. Exon 3 encodes for transmembrane domains 5 as well as the cytoplasmic carboxyl terminus of the BB₂ receptor (Xiao et al., 2001). Two major transcription start sites for the human BB₂ receptor gene were found in gastrointestinal and breast cancer cells located 43 and 36 bp downstream of a TTTAAA motif, which is identified 407 to 402 bp upstream of the ATG start codon (Xiao et al., 2001). Truncation studies of the transfected promoter region suggested that a cyclic AMP response element motif located 112 bp upstream of the major transcription start site is required to confer basal BB₂ receptor promoter activity in duodenal cancer cells (Xiao et al., 2001).

D. BB₂ Receptor Expression

Expression levels of BB₂ receptor mRNA have been reported in human, mouse, and monkey (Spindel et al., 1990; Battey et al., 1991; Corjay et al., 1991; Ohki-Hamazaki et al., 1997a; Sano et al., 2004). BB₂ receptor mRNA distribution was studied in detail in the monkey, in which it is found in the greatest amount in the pancreas and in lesser amounts in the stomach, prostate, skeletal muscle, and CNS (Sano et al., 2004). This result generally agrees with studies of location of the human BB₂ receptor gene, in which a very strong signal was found in the normal pancreas with four specific transcripts of 9, 4.6, 3.1, and 2.1 kb sizes, a weaker signal in the stomach with two transcripts of 9 and 3.1 kb, and a very weak 9-kb transcript signal in whole brain and adrenal gland (Xiao et al., 2001). In the monkey CNS BB₂ receptor mRNA was widely expressed with the highest amounts in hippocampus, hypothalamus, amygdala, and pons (Sano et al., 2004). In the mouse BB₂ receptor mRNA was present in high amounts in the digestive tract and in the colon, but not in the stomach or small intestine (Battey et al., 1991). Detailing mapping in the rat brain was reported, which showed BB₂ receptor expression in all brain regions, with the highest amounts of BB₂ receptor mRNA in the hypothalamus, particularly the suprachiasmatic and supraoptic nuclei, and in the magnocellular preoptic nucleus in the basal ganglia and the nucleus of the lateral olfactory tract (Battey and Wada, 1991).

Detailed CNS location of the murine BB₂ receptor has been reported using a specific BB₂ receptor antibody (Kamichi et al., 2005). The BB₂ receptor was widely distributed in the mouse brain in the isocortex, hippocampal formation, pyriform cortex, amygdala, hypothalamus, and brain stem (Kamichi et al., 2005). Strong BB₂ immunoreactivity was observed in many nuclei of the amygdala and in the nucleus tractus solitarius (Kamichi et al., 2005). Double-labeling studies in the amygdala demonstrated subpopulations of BB₂ receptors present in the GABAergic neurons, providing support for a possible role of BB₂ receptors mediating memory by modulating neurotransmitter release in the local GABAergic network (Kamichi et al., 2005).

Binding studies have confirmed the widespread distribution of BB₂ receptors in the brain, showing high levels in the cortex as well as the suprachiasmatic and supraoptic nuclei of the rat (Ladenheim et al., 1990, 1992, 1993a; Moody and Merali, 2004). Binding studies and studies of biological activity provide evidence for BB₂ receptors on both gastrointestinal and urogenital smooth muscle cells (Severi et al., 1991; Kilgore et al., 1993; Ladenheim et al., 1997a; Milusheva et al., 1998; ter Beek et al., 2004; Fleischmann et al., 2005). BB₂ receptors in the gastrointestinal tract are also found in gastric antral G cells (Giraud et al., 1987), other gastric mucosa cells (D cell, mucus cell, and parietal cell) (Nakamura et al., 1988), and pancreatic acinar cells (Jensen et al., 1978, 1988a; Jensen, 1994). In the epithelial cells lining the normal human gastrointestinal tract, BB₂ receptor mRNA was only found in the antrum with the esophagus, jejunum, and ileum and not in the descending colon (Ferris et al., 1997).

BB₂ receptors are present on a large number of different tumors using binding studies and immunohistochemical localization with specific receptor antibodies and/or assessment of BB₂ receptor mRNA. BB₂ receptors have been widely studied in prostate cancer (Reubi et al., 2002; Jensen and Moody, 2006; Patel et al., 2006), small cell lung cancer (Corjay et al., 1991; Toi-Scott et al., 1996; Jensen and Moody, 2006; Patel et al., 2006), non small cell lung cancer (Corjay et al., 1991; Toi-Scott et al., 1996; Siegfried et al., 1997; Jensen and Moody, 2006), breast cancer (Gugger and Reubi, 1999; Reubi et al., 2002; Jensen and Moody, 2006; Patel et al., 2006), head and neck squamous cell cancer (Lango et al., 2002; Jensen and Moody, 2006), colon cancer (Carroll et al., 1999b, 2000a; Jensen et al., 2001; Glover et al., 2003; Patel et al., 2006), uterine cancer (Fleischmann et al., 2005), various CNS/neural tumors (glioblastomas, neuroblastomas) (Jensen and Moody, 2006), ovarian cancer (Sun et al., 2000b), gastrointestinal carcinoid tumors (Reubi et al., 2002; Scott et al., 2004), and renal cell cancers (Reubi et al., 2002; Heuser et al., 2005).

E. BB₂ Receptor Pharmacology

F. BB₂ Receptor Structural Basis of Receptor Binding/Activation

G. BB₂ Receptor Signaling, Activation, and Modulatory Processes (Internalization, Down-Regulation, and Desensitization)

The human BB₂ receptor (Moody et al., 1986, 1996b; Corjay et al., 1991; Williams and Schonbrunn, 1994; Benya et al., 1995b), as well as the rat (Deschodt-Lanckman et al., 1976; Matozaki et al., 1991; Garcia et al., 1997; Tapia et al., 2006), mouse (Huang et al., 1990; Garcia et al., 1997), guinea pig (Jensen et al., 1978, 1988a,b; Jensen, 1994; Garcia et al., 1997), and canine BB₂ receptors (Seensalu et al., 1997) are coupled to phospholipase C, resulting in breakdown of phosphoinositides, generation of diacylglycerol, stimulation of the mobilization of cellular calcium, and PKC activation (Klein et al., 1979; Rozengurt, 1988, 1998a; Jensen, 1994). BB₂ receptor stimulation activates both phospholipase β₁ and β₃, and this is dependent on Gα_q (MacKinnon et al., 2001). Activation of the BB₂ receptor also results in activation of phospholipase D (Cook et al., 1991; Briscoe et al., 1994) and phospholipase A₂ (Currie et al., 1992; Nishino et al., 1998) and is reported to stimulate increased cAMP in some tissues (Rozengurt and Sinnett-Smith, 1983; Bjøro et al., 1987; Millar and Rozengurt, 1988; Garcia et al., 1997). The increase in cAMP in Swiss 3T3 cells was reduced by PKC down-regulation and inhibition of cyclooxygenase, suggesting that these pathways were involved (Rozengurt et al., 1987). However, a systematic study demonstrated the activation of BB₂ receptors in normal pancreas from three species (rat, mouse, and guinea pig) (Garcia et al., 1997) and the transfected human or mouse BB₂ receptor did not stimulate an increase in cAMP (Benya et al., 1994b, 1995b). These results compared with those in a number of studies in the literature led the authors (Garcia et al., 1997) to propose that the BB₂ receptor may be coupled differentially to different adenylate cyclases in different tissues in the same species. Downstream diacylglycerol leads to the activation of both classic and novel PKCs, which catalyze the phosphorylation of a number of membrane-bound and cytosolic proteins. Furthermore, specific protein kinase cascades are triggered, including the Raf/MEK/ERK kinase cascade, activation of protein kinase D, and rapamycin-sensitive p70s6k, which result in increased expression of immediate early response genes (i.e., c-myc, c-jun, and c-fos), leading to the regulation of the cell cycle and cell proliferation (Rozengurt, 1998a).

BB₂ receptor stimulation also results in the activation of tyrosine kinases and tyrosine phosphorylation of a number of proteins including p125 focal adhesion kinase and PYK2, paxillin, ERK kinase, and P130^CAS (Rozengurt, 1998a,b). Paxillin and P130^CAS function as important adaptors with paxillin promoting protein-protein interactions and P130^CAS interacting with Src and c-Crk and with numerous proteins that have SH2 and SH3 binding domains (Turner, 1994; Harte et al., 1996). BB₂ receptor stimulation of P125^FAK tyrosine phosphorylation occurs largely independent of PKC activation (Sinnett-Smith et al., 1993) but is dependent on the small GTP-binding protein Rho and the integrity of the actin cytoskeleton and focal adhesion plaques (Rozengurt, 1998a). In addition to BB₂ receptor activation stimulating the formation of focal adhesion plaques via a Rho-dependent mechanism, it also stimulates actin proliferation, resulting in membrane ruffling via rac proteins (Nobes et al., 1995). In contrast with P125^FAK and paxillin tyrosine phosphorylation due to BB₂ receptor stimulation, stimulation of ERK activation and tyrosine phosphorylation is not dependent on Rho or the other factors listed above (Seufferlein et al., 1996a). GRP-induced activation of ERK is dependent on PKC (Rozengurt, 1998b) and transactivation of the EGF receptor (MacKinnon et al., 2001; Lui et al., 2003; Thomas et al., 2005) which may be mediated by G_i proteins (MacKinnon et al., 2001). Recent studies provide evidence that BB₂ receptor stimulation of tyrosine phosphorylation of P125^FAK, paxillin, and P130^CAS occurs via an interaction with Gα_12/13 and Rho (Rozengurt, 1998a). BB₂ receptor stimulation also leads to coupling to Gα₁₂ to elicit c-Jun N-terminal kinase activation (MacKinnon et al., 2001; Chan and Wong, 2005).

BB₂ receptor stimulation results in a rapid activation of Src kinase family members (Rodríguez-Fernández and Rozengurt, 1996; Vincent et al., 1999; Pace et al., 2006), which is not dependent on either PKC or mobilization of calcium, nor is it dependent on Rho or the integrity of the cytoskeleton (Rodriguez-Fernandez and Rozengurt, 1996). Blockade of Src family kinases decreases BB₂ receptor-stimulated transactivation of the EGFR as well as MAP kinase stimulation (Vincent et al., 1999). The EGFR transactivation by BB₂ receptor activation in head and neck squamous cancers is dependent on Src-mediated cleavage and release of transforming growth factor-α and amphiregulin and is essential for invasion and growth of these cancers (Vincent et al., 1999).

Acute and chronic BB₂ receptor stimulation results in an activation of a number of receptor modulatory processes (internalization, down-regulation, or desensitization) (Lee et al., 1980; Pandol et al., 1982; Millar and Rozengurt, 1990; Walsh et al., 1993; Benya et al., 1994b; Briscoe et al., 1994; Kroog et al., 1995a), and a number of studies have investigated the cell signaling processes involved. In cells containing human (Benya et al., 1995b), mouse (Zachary and Rozengurt, 1987; Brown et al., 1988; Benya et al., 1993, 1994d; Wang et al., 1993; Tsuda et al., 1997a; Acs et al., 2000), or rat BB₂ receptors (Zhu et al., 1991), with agonist exposure the receptor-ligand complex is rapidly internalized (t_0.5 5 min) with 80 to 85%, 70 to 90%, and 50%, respectively, of the bound ligand internalized. In epithelial cells transfected with the murine BB₂ receptor, agonist ligand and receptor were internalized by 5 min into early endosomes, after 10 min both were in perinuclear vesicles, and after 60 min the BB₂ receptor had recycled back to the surface (Grady et al., 1995). In this study (Grady et al., 1995) and in others (Benya et al., 1994b, 1995a) there was a rapid down-regulation of cell surface receptors, and the recovery was decreased by acidotropic agents but not by inhibitors of new protein synthesis. The internalization of the BB₂ receptor is partially dependent on phospholipase C activation (Benya et al., 1994a; Williams et al., 1998; Schumann et al., 2003) and requires clathrin-coated pits because it is inhibited by hyperosmolar sucrose as well as phenylarsine oxide (Grady et al., 1995). Acute desensitization of the BB₂ receptor occurs within seconds to minutes of agonist exposure (Walsh et al., 1993; Briscoe et al., 1994) and is reported to occur with stimulated phospholipase D activity as well as with stimulation of phosphoinositide breakdown (Briscoe et al., 1994; Williams et al., 1998) and for stimulation of changes in cytosolic calcium, with the latter shown to be homologous in nature (Walsh et al., 1993). In some tissues acute desensitization and down-regulation of the BB₂ receptor are caused by hormones/neurotransmitters activating phospholipase C such as carbachol and cholecystokinin (Younes et al., 1989; Vinayek et al., 1990). Chronic BB₂ receptor desensitization occurs after prolonged incubation with agonist (1–2 h) and is homologous in nature (Lee et al., 1980; Benya et al., 1995a). The receptor structure-function studies reviewed above provide strong support for the conclusion that down-regulation and chronic desensitization are coupled processes being affected by similar receptor structural alterations and cellular signaling cascades and have a mechanism distinct from that causing internalization (Benya et al., 1994d, 1995a; Tsuda et al., 1997a; Schumann et al., 2003). The results of these studies provided no evidence for second messenger-independent processes in mediation of down-regulation or desensitization, whereas internalization is equally stimulated by second messenger-dependent and -independent processes and the presence of the COOH-terminal serines and threonines was essential for mediating these effects. In HIT-T15 cells BB₂ receptor-mediated desensitization was closely coupled to down-regulation (Swope and Schonbrunn, 1990).

Studies in the β-adrenergic receptor and a number of GPCRs demonstrate that receptor phosphorylation, primarily by G protein-coupled receptor kinases (GRKs) and subsequent binding of arrestins are critical for receptor internalization and deactivation during acute desensitization (Krupnick and Benovic, 1998; Ferguson, 2001; Premont and Gainetdinov, 2007). Studies demonstrate that BB₂ receptor activation results in rapid phosphorylation of the receptor (Kroog et al., 1995b, 1999; Williams et al., 1996; Ally et al., 2003) as does stimulation of the BB₂ receptor-containing cells by the phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (Kroog et al., 1995b; Williams et al., 1996; Ally et al., 2003). However, agonist and 12-O-tetradecanoyl-phorbol-13-acetate-induced BB₂ receptor phosphorylation occur at different receptor sites (Williams et al., 1998). GRKs are serine-threonine kinases that preferentially phosphorylate agonist occupied, active conformation GPCRs and lead to uncoupling from G protein and endocytosis (Szekeres et al., 1998; Premont and Gainetdinov, 2007). Bn/GRP stimulates BB₂ receptor phosphorylation at serine/threonine residues in the COOH terminus but does not stimulate tyrosine phosphorylation in the BB₂ receptor (Williams et al., 1996; Ally et al., 2003). With BB₂ receptor activation arrestin translocation occurs to the plasma membrane (Ally et al., 2003) and requires an intact DRY sequence in the second intracellular domain of the BB₂ receptor (Ally et al., 2003). BB₂ receptor internalization has been proposed to play a key role in acute BB₂ receptor desensitization (Swope and Schonbrunn, 1990) because the kinetics of each is identical. Furthermore, the kinetics of BB₂ receptor phosphorylation correlate closely with both internalization and acute desensitization (Kroog et al., 1995b; Williams et al., 1996, 1998). Phosphorylation of the BB₂ receptor after GRP/Bn stimulation is reported in one study (Williams et al., 1996) but not another (Kroog et al., 1995b) to be mediated by both a PKC-dependent and a PKC-independent process (probably a GRK family member).

Studies demonstrate that radiolabeled GRP/Bn is rapidly degraded by the BB₂ receptor (Swope and Schonbrunn, 1987; Zachary and Rozengurt, 1987; Brown et al., 1988; Zhu et al., 1991; Wang et al., 1993; Williams et al., 1998). This degradation is best inhibited by the general inhibitor bacitracin or the thermolysin-like metalloproteinase inhibitor, phosphoramidon, and to a less degree by leupeptin and bestatin > chymostatin > amastatin (Wang et al., 1993). The lysosomal proteinase inhibitor, choroquine, also inhibits degradation (Swope and Schonbrunn, 1987; Williams et al., 1998).

Activation of the BB₂ receptor results in growth of both normal and neoplastic tissues (Moody et al., 2003a; Jensen and Moody, 2006). The cell signaling cascades involved have been studied extensively in both Swiss 3T3 cells and in numerous tumors cells. In 3T3 cells and a number of tumor cells (prostate, head and neck squamous cell cancer, and non-small cell lung cancer cells) activation of the BB₂ receptor results in stimulation of phosphorylation of Akt (Liu et al., 2007) and ERK phosphorylation (Sakamoto et al., 1988; Rozengurt, 1998b; Koh et al., 1999; Vincent et al., 1999; Lui et al., 2003; Thomas et al., 2005), which has been shown in some cells to be dependent on the transactivation of the EGF receptor, which in turn depends on Src and changes in cytosolic calcium in some cases. Mitogenesis in 3T3 cells is dependent on BB₂ receptor-stimulated changes in cytosolic calcium, activation of PKC, PKD, and ERK, and release of arachidonic acid (Rozengurt, 1998b). BB₂ receptor stimulation of ERK phosphorylation is dependent on Ras but not Rap1 in prostate tumor cells (Sakamoto et al., 1988). The transactivation of the EGF receptor by BB₂ receptor activation is dependent on PKC and PKD activation in some cells (Seufferlein et al., 1996b; Rozengurt, 1998b; Sinnett-Smith et al., 2004, 2007). EGF receptor transactivation upon BB₂ receptor stimulation as well as by a number of other GPCRs occurs via metallo-proteinase-dependent cleavage and release of EGF-related peptides that then activate the receptor (Sakamoto et al., 1988; Vincent et al., 1999; Lui et al., 2003). The inhibition of either EGF receptor transactivation or ERK activation inhibited BB₂ receptor-stimulated DNA synthesis in these tumor cells (Sakamoto et al., 1988). BB₂ receptor activation stimulates the invasion and cell migration of tumor cells (Vincent et al., 1999; Thomas et al., 2005; Zheng et al., 2006). This stimulation occurs via G_α13, leading to activation of RhoA and Rho-associated coiled-coil forming protein kinase (Zheng et al., 2006). BB₂ receptor activation promotes progression from the G₁ to the S phase of the cell cycle by increasing the expression of cyclin D₁ and E through the early growth response protein Egr-1, down-regulating the cyclin-dependent kinase inhibitor p27^kip1 and hyperphosphorylating the retinoblastoma protein (Mann et al., 1997; Rozengurt, 1998b; Xiao et al., 2005).

H. BB₂ Receptor Function in Various Tissues and in Vivo

A major difficulty in assessing the effects of BB₂ receptor activation in vivo and in a number of tissues in vitro is the fact that they frequently possess both classes of bombesin receptors, and bombesin, the agonist frequently used, has high affinity for both receptor subtypes. Recently a number of developments have contributed to solving this problem. Selective receptor antagonists for the BB₂ receptor are described, studies on BB₂ receptor knockout animals are being increasing performed, more selective BB₂ receptor agonists such as GRP are being used, and with the cloning of the mammalian bombesin receptors, it has become clear that some widely studied tissues such as Swiss 3T3 cells and pancreatic acinar cells only possess BB₂ receptors.

Many effects of GRP are observed both in vivo and in vitro, but it remains unclear in many cases which are pharmacological or which are physiological. Studies support a role for the BB₂ receptor in numerous gastrointestinal functions, including regulation of gastric acid secretion via both stimulation of gastrin release from antral G cells and somatostatin release from D cells and stimulation of acid secretion (Schubert et al., 1991; Hildebrand et al., 2001; Schubert, 2002); regulation of gastrointestinal motility, especially gastric emptying, small intestinal transit, and gallbladder emptying (Degen et al., 2001; Yeğen, 2003); stimulation of pancreatic secretion (Niebergall-Roth and Singer, 2001; Nathan and Liddle, 2002), insulin release (Persson et al., 2002), and colonic ion transport (Traynor and O'Grady, 1996); and stimulation of the secretion of a variety of hormones (gastrin, somatostatin, CCK, pancreatic polypeptide, enteroglucagon, pancreatic glucagon, and gastric inhibitory polypeptide) (Modlin et al., 1981; Ghatei et al., 1982; Pettersson and Ahren, 1987; Bunnett, 1994). BB₂ receptor activation has number of immunologic effects including functioning as a chemoattractant in peritoneal macrophages, monocytes, and lymphocytes (Ruff et al., 1985; Del Rio and De la Fuente, 1994), stimulating lymphocyte proliferation (Del Rio et al., 1994), and stimulating natural killer and antibody-dependent cellular cytotoxicity in leukocytes (De la Fuente et al., 1993). BB₂ receptors are reported to be important for fetal lung development including lung branching, cell proliferation, and differentiation (Subramaniam et al., 2003) as well as in a number of lung diseases, which will be discussed in section IV.I. BB₂ receptors are widely expressed in the CNS and in the spinal cord, and numerous central effects have been described with their activation including effects on satiety, regulation of circadian rhythm, thermoregulation, grooming behaviors, modulation of stress, fear, and anxiety response, memory, and gastrointestinal function such as acid secretion (Martinez and Tache, 2000; Yeğen, 2003; Moody and Merali, 2004; Karatsoreos et al., 2006; Roesler et al., 2006a,b; Kallingal and Mintz, 2007; Presti-Torres et al., 2007). The satiety effect of BB₂ receptors has been extensively studied (Gibbs et al., 1979; Gibbs and Smith, 1988; Flynn, 1997; Fekete et al., 2007; Ladenheim and Knipp, 2007). A recent study (Ladenheim and Knipp, 2007) showed that the satiety effect of peripherally administered NMB, but not that of GRP, is inhibited by capsaicin pretreatment, suggesting that the neural pathways involved in BB₂ receptor-mediated satiety are either capsaicin-insensitive neurons or involve direct activation of BB₂ receptors in the CNS (Ladenheim and Knipp, 2007). The importance of BB₂ receptors in mediating the satiety effects of GRP was demonstrated by the ability of a specific BB₂ receptor antagonist administered in the hindbrain of rats to inhibit the satiety effects of peripherally administered GRP (Ladenheim et al., 1996a).

BB₂ receptor knockout mice have been described and limited study results are available (Wada et al., 1997; Hampton et al., 1998). In the initial study performed with these mice (Hampton et al., 1998), no developmental abnormalities were seen; however, bombesin failed to suppress glucose intake, whereas it caused a dose-dependent decrease in normal mice (Hampton et al., 1998). In a second study (Wada et al., 1997) the intracerebroventricular administration of GRP failed to cause hypothermia in the BB₂ receptor knockout mice as observed in the wild-type mice. Furthermore, the BB₂ receptor knockout mice demonstrated abnormal behaviors and altered spontaneous activity during darkness (Wada et al., 1997). In a more detailed study of feeding behavior in these mice, neither GRP, NMB, nor bombesin altered satiety in the knockout mice; however, the satiety response to cholecystokinin was present and in fact enhanced (Ladenheim et al., 2002). In a long-term study (Ladenheim et al., 2002) BB₂ receptor knockout mice ate more food than normal mice because of a defect in terminating meals and had greater weight gain, supporting the conclusion that the BB₂ receptor has important roles in satiety. These mice were used to study the effects of BB₂ receptor activation on islet function (Persson et al., 2000, 2002). BB₂ receptor knockout mice had impaired glucose tolerance, a defect in early insulin release (Persson et al., 2000), and their plasma glucagon-like peptide-1 response to gastric glucose administration was significantly reduced, suggesting that the BB₂ receptor had an important role in normal glucagon-like peptide-1 release and insulin and glucose responses after a glucose meal. In a second study (Persson et al., 2002) GRP was found to potentiate glucose-stimulated insulin release in wild-type but not BB₂ receptor knockout mice. This study (Persson et al., 2002) demonstrated that BB₂ receptor activation contributes to insulin secretion induced by activation of autonomic nerves and that the deletion of the BB₂ receptor is compensated for by increased cholinergic sensitivity (Persson et al., 2002). These results are consistent with earlier studies, which demonstrated that GRP potentiated glucose-induced insulin release by both a ganglionic and direct effect but did not alter glucagon or pancreatic somatostatin release (Hermansen and Ahren, 1990; Gregersen and Ahren, 1996; Karlsson et al., 1998). BB₂ receptor knockout mice were used to study possible behavioral effects of GRP (Shumyatsky et al., 2002). In one study the BB₂ receptor was found in wild-type but not knockout mice to be highly expressed in the lateral nucleus of the amygdala, which is important in mediating fear responses. BB₂ receptor knockout mice showed more persistent long-term fear responses (Shumyatsky et al., 2002), supporting other study results, which suggest that the BB2 receptor has an important role in memory and fear responses (Roesler et al., 2006a). Other behavior changes seen in BB₂ receptor knockout mice include increased social investigatory behavior (Yamada et al., 2000b), preference for conspecific odors (Yamada et al., 2000b), and altered social preferences in females (Yamada et al., 2001). BB₂ receptor knockout mice have also been used to investigate the role of this receptor in specific diseases, which will be discussed in the next section.

BB₂ receptor activation has important growth effects on normal and neoplastic tissues (Moody et al., 1992; Jensen and Moody, 2006). BB₂ receptor activation stimulates growth of normal endometrial stomal cells (Endo et al., 1991), bronchial epithelial cells (Willey et al., 1984; Siegfried et al., 1993), melanocytes (Terashi et al., 1998), chondrocytes (Hill and McDonald, 1992), and normal enterocyte growth and turnover after small bowel resection (Chu et al., 1995; Sukhotnik et al., 2007) as well as normal development of the intestinal villus (Carroll et al., 2002) and normal fetal lung development (Emanuel et al., 1999; Shan et al., 2004). The effects of BB₂ receptor activation on neoplastic growth have been extensively studied (Moody et al., 1992; Jensen et al., 2001; Patel et al., 2006). This widespread interest occurred after human small cell lung cancers were shown to possess high-affinity BB₂ receptors (Moody et al., 1985), and bombesin was shown to have an autocrine growth effect on these cells (Cuttitta et al., 1985). Subsequent studies demonstrated such an autocrine growth effect, for which the tumor cells not only possessed BB₂ receptors but also secreted bombesin-like peptides, resulting in a growth stimulatory effect (Moody et al., 2003a; Jensen and Moody, 2006; Patel et al., 2006) in a large number of cells from various types of cancer including neuroblastomas (Kim et al., 2002), squamous head and neck tumors (Lango et al., 2002; Lui et al., 2003), pancreatic cancer (Wang et al., 1996; Murphy et al., 2001), colon cancer (Chave et al., 2000), prostate cancer (Plonowski et al., 2000), human glioblastomas (Sharif et al., 1997), and non-small cell lung cancer (Siegfried et al., 1999). Furthermore, many human cancers or the blood vessels in the cancers either overexpress or ectopically express BB₂ receptors, and the stimulation or inhibition of these receptors is reported to affect growth/differentiation (Jensen et al., 2001; Moody et al., 2003a; Heuser et al., 2005; Jensen and Moody, 2006; Patel et al., 2006; Fleischmann et al., 2007). The potential clinical importance of ectopic expression and overexpression will be discussed further in the next section. The role of the ectopic expression or overexpression in various cancers may be different with different tumors. Whereas many of the studies referred to in the following paragraph emphasize the growth stimulatory effects of BB₂ receptor on tumor cells, other studies, especially in colon cancer, support the conclusion that the ectopic expressing of the BB₂ receptor has a morphogenic effect rather than a mitogenic effect (Jensen et al., 2001). Whereas in normal colonic mucosal epithelial cells, the BB₂ receptor is not found (Preston et al., 1995; Ferris et al., 1997; Carroll et al., 1999b), in 40 to 100% of colon cancers (Carroll et al., 1999b) the BB₂ receptor is aberrantly expressed. BB₂ receptor activation on some colon cancer cells is reported to result in proliferation (Radulovic et al., 1991b; Frucht et al., 1992; Narayan et al., 1992). However, in detailed studies, although 62% of the tumors expressed both GRP and the BB₂ receptor, their coexpression was equally frequent in early- or late-stage cancers and was rarely detected in metastases (Carroll et al., 1999b). However, GRP/BB₂ receptor expression was seen in all well differentiated tumors, whereas poorly differentiated tumors never coexpressed GRP/BB₂ receptors (Carroll et al., 1999b). Furthermore, no difference in survival occurred in patients with cancers expressing or not expressing the GRP/BB₂ receptor (Carroll et al., 1999b). In a study (Carroll et al., 2000a) of BB₂ receptor knockout mice with colon tumors induced by azoxymethane, larger tumors were better differentiated in wild-type mice than in BB₂ receptor knockout mice. From these studies and others it was proposed that BB₂ receptor activation in these cells is functioning primarily as a morphogenic or differentiating factor (Carroll et al., 1999b; Jensen et al., 2001). More recent studies show that this morphogenic effect is mediated by activation of p125^FAK, which inhibits invasion/metastases by enhancing cell attachment (Glover et al., 2004), most likely by up-regulating the expression of intracellular adhesion protein-1 (Taglia et al., 2007). Subsequent studies showed that BB₂ receptor mutations occurred frequently in poorly differentiated colon tumor, resulting in the formation of inactive receptors, and the generation of these mutations correlated inversely with the differentiation of the tumor, suggesting that their production represents a new mechanism allowing for the differentiation of tumors (Carroll et al., 2000b; Glover et al., 2003).

A recent study (Ruginis et al., 2006) used a proteomic approach to identify proteins selectively up-regulated in human colorectal cancer cells subsequent to BB₂ receptor activation. This study took advantage of the fact that human colorectal cancer cells such as Caco-2 and HT-29 only secreted GRP and expressed BB₂ receptors when they are preconfluent and not when they are postconfluent (Glover et al., 2005). Total cellular proteins were isolated from preconfluent, GRP and BB₂ receptor-expressing cells in the presence and absence of the specific BB₂ receptor antagonist [d-Phe⁶]bombesin_6–13 methyl ester and from postconfluent cells not expressing GRP or BB₂ receptors. By using two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (Ruginis et al., 2006), at least six separate proteins were up-regulated subsequent to BB₂ signaling when this receptor is aberrantly expressed in human colorectal cancer: gephyrin, heat shock protein 70, heterochromatin associated protein 1, intracellular adhesion protein-1–1, and ThiL/acetyl-CoA acetyltransferase. These findings hold promise for further definition of the mechanism whereby aberrantly expressed BB₂ receptors in human colorectal cancer promote tumor cell differentiation and improve patient outcome.

I. BB₂ Receptor in Diseases

BB₂ receptor activation has been proposed to be important in the mediation of a number of human disorders including disorders of lung development, various pulmonary diseases, CNS disorders, and the growth/differentiation of human cancers. The tumor differentiation effects of BB₂ receptor activation were discussed in the previous section, and the growth effects and effects of BB₂ receptor overexpression will be considered here. BB₂ receptors are ectopically expressed or overexpressed in a large number of tumors including 85 to 100% of small cell lung cancers, 74 to 78% of non-small cell lung cancer cells, 38 to 72% of breast cancer, 75% of pancreatic cancer cell lines and 10% of pancreatic cancers, 62 to 100% of prostate cancers, 100% of head/neck squamous cell cancers, and 72 to 85% of neuroblastomas/glioblastomas (Jensen and Moody, 2006; Patel et al., 2006). Bn-like peptides are critical to the growth of some, but not all, small cell lung cancers (Cuttitta et al., 1985) and as discussed above have been shown to have an autocrine growth effect on a large number of tumors as well as stimulate growth in a large number of these tumors (Jensen and Moody, 2006; Patel et al., 2006). In some tumors such as prostate cancer, BB₂ receptor overexpression correlates with neoplastic transformation (Markwalder and Reubi, 1999). Bn peptides also function as proangiogenic factors in various tumors (Levine et al., 2003; Kanashiro et al., 2005; Martínez, 2006). The production of GRP-related peptides and/or overexpression of BB₂ receptors by tumors are playing a potential role in a number of aspects of the treatment and management of these tumors. These aspects include functioning as targets for antitumor treatment, as prognostic factors, as targets to image the tumors, and as targets to deliver cytotoxic treatment selectively to the tumor (Smith et al., 2003; Schally and Nagy, 2004; Jensen and Moody, 2006). Attempts to inhibit the autocrine growth effect of GRP-like peptides on tumor growth are reported in human and/or animal studies using monoclonal antibodies to GRP, antisense constructs, BB₂ receptor antagonists, or other inhibitors (Zhou et al., 2004). Infusion of the monoclonal antibody 2A11 directed against the biologically active COOH terminus of GRP is safe in humans (Chaudhry et al., 1999), and 2A11 was given to 13 patients with small cell lung cancer (Kelley et al., 1997). One patient had a complete remission and four patients had radiologically stable disease, and further evaluation was recommended (Kelley et al., 1997). In a recent study (Schwartsmann et al., 2006) the BB₂ receptor antagonist, RC-3095 was administered to 25 patients with different advanced malignancies. No side effects occurred, and there were no tumor responses; however, maximal doses could not be reached by the methods used despite dose escalation (Schwartsmann et al., 2006). EGF receptor transactivation upon BB₂ receptor stimulation may also be a target for therapeutic intervention. Experimental studies demonstrate that activation of the BB₂ receptor rescues tumor cells from the growth-inhibiting effect of the EGF receptor inhibitor, gefitinib, by stimulating the release of amphiregulin and activation of the Akt pathway (Liu et al., 2007). When a BB₂ receptor antagonist is combined with an EGF receptor inhibitor (erlotinib), there is marked enhanced antitumor activity (Zhang et al., 2007b), suggesting that such an approach may be useful in some cancers such as head/neck tumors or lung tumors. In a number of studies plasma levels of GRP precursors such as pro-GRP or assessment of GRP expression in tumors provides prognostic information (Hamid et al., 1990; Okusaka et al., 1997; Sunaga et al., 1999; Shibayama et al., 2001; Yonemori et al., 2005).

Recent clinical and laboratory studies with somatostatin receptors demonstrate that many endocrine tumors overexpress or ectopically express these receptors and that radiolabeled analogs of somatostatin can be used to localize these tumors as well as for somatostatin receptor-mediated cytotoxicity (Breeman et al., 2007; Van Essen et al., 2007). Somatostatin analogs coupled to ¹¹¹In are now widely used to image neuroendocrine tumors, and numerous studies have demonstrated that they have greater sensitivity than conventional imaging modalities (computed tomography, magnetic resonance imaging, angiography, or ultrasound) and that their routine use changes patient management in 20 to 47% of cases (Gibril et al., 1996; Gibril and Jensen, 2004; Breeman et al., 2007). Recent studies with somatostatin analogs coupled to ¹¹¹In, ⁹⁰Yt, and ¹⁷⁷Lu show promising results for somatostatin receptor-mediated cytotoxicity in patients with advanced neuroendocrine tumors, and they have entered phase 3 studies (Breeman et al., 2007; Forrer et al., 2007; Van Essen et al., 2007). Unfortunately, many common tumors (colon, pancreas, head/neck, prostate, and lung) may not overexpress somatostatin receptors; however they frequently overexpress Bn receptors, especially the BB₂ receptor (Jensen et al., 2001; Reubi et al., 2002; Moody et al., 2003a; Heuser et al., 2005; Jensen and Moody, 2006; Patel et al., 2006; Fleischmann et al., 2007). This observation has led to considerable interest in the possibility of developing radiolabeled analogs of Bn that could be use for localization of the tumors containing Bn receptors or the development of radiolabeled Bn analogs or Bn analogs coupled to cytotoxic agents that could be used to treat tumors overexpressing Bn receptors through bombesin receptor-mediated cytotoxicity (Breeman et al., 2002; de Jong et al., 2003; Cornelio et al., 2007; de Visser et al., 2007a). Numerous radiolabeled (¹¹¹In, ⁶⁸Ga, ¹⁷⁷Lu, ⁶⁴Cu, ⁸⁶Yt, ¹⁸F, and ^99mTc) GRP analogs with enhanced stability that bind with high affinity to BB₂ receptors have been reported, as well as their ability to image various human tumors in vivo using gamma detectors or positron emission tomography (Breeman et al., 2002; Nock et al., 2003; Smith et al., 2003, 2005; Johnson et al., 2006; Lantry et al., 2006; Zhang et al., 2006, 2007a; de Visser et al., 2007a; Dimitrakopoulou-Strauss et al., 2007; Garrison et al., 2007; Parry et al., 2007; Prasanphanich et al., 2007; Waser et al., 2007). In some preliminary studies in humans, tumors were imaged in the majority of patients, and in some cases, tumors that were not seen with other commonly used imaging modalities were detected using radiolabeled Bn analogs (De Vincentis et al., 2004; Scopinaro et al., 2004, 2005; Dimitrakopoulou-Strauss et al., 2007). At present no study has established the value of imaging using radiolabeled Bn analogs.

A number of Bn analogs coupled to radiolabeled compounds (e.g., ¹⁷⁷Lu) (Smith et al., 2005; Johnson et al., 2006; Lantry et al., 2006; Zhang et al., 2007a) and to cytotoxic agents (camptothecin, a topoisomerase inhibitor, doxorubicin analogs, and paclitaxel) (Schally and Nagy, 1999; Breeman et al., 2002; Moody et al., 2004, 2006b; Schally and Nagy, 2004; Engel et al., 2005; Buchholz et al., 2006; Nanni et al., 2006; Panigone and Nunn, 2006; Safavy et al., 2006; Engel et al., 2007) have been described. These analogs retain their high affinity for Bn receptors and are internalized by Bn receptor-bearing tissues, for the possibility of delivering Bn receptor-mediated tumoral cytotoxicity. Many of these compounds have been shown to cause tumor cytotoxicity in animal studies, and one study has provided evidence that it is due to specific interaction with the BB₂ receptors overexpressed on the tumor (Moody et al., 2006b). At present it is unclear whether this approach will be effective in vivo in human tumors whether alone or in combination with other antitumor treatments. A recent study using a chemically identical active and inactive cytotoxic GRP analog (i.e., camptothecin-L2-[d-Tyr⁶,β-Ala¹¹,Phe¹³,Nle¹⁴]bombesin_6–14 (where L2 = [N-(N-methyl-amino ethyl)glycine carbamate]) or its d-Phe¹³ inactive form, demonstrated that specific tumor receptor interaction is important in mediating the tumor cytotoxicity of these compounds (Moody et al., 2006b). Various studies have demonstrated that such an approach can inhibit the growth of pancreatic lung, prostate, and gastric cancers (Schally and Nagy, 1999, 2004; Breeman et al., 2002; Moody et al., 2004). At present the usefulness of GRP or the BB₂ receptor in management of human tumors in each of the areas discussed here has not been established (Jensen and Moody, 2006).

BB₂ receptor activation, GRP secretion, or abnormalities of either have been proposed to be important in a number of other diseases. In a recent study (Sun and Chen, 2007) evidence that activation of the BB₂-receptor in the dorsal spinal cord is important for mediating pruritus was presented. GRPR knockout mice showed significantly decreased scratching behavior in response to pruritogenic stimuli, whereas other responses were normal. Furthermore, administration of a BB₂ receptor antagonist into the spinal fluid inhibited scratching behavior in three different models of itching (Sun and Chen, 2007). The authors (Sun and Chen, 2007) point out that the BB₂ receptor may represent the first molecule identified that is dedicated to mediating the itch response in the spinal cord and may provide an important therapeutic target for the treatment of chronic pruritic conditions. Abnormalities of GRP, BB₂ receptors, and other bombesin-like peptides and/or their receptors are proposed to be important in normal lung development and mediation of the lung injury in premature infants with bronchopulmonary dysplasia (Li et al., 1994; Sunday et al., 1998; Emanuel et al., 1999; Cullen et al., 2000; Ashour et al., 2006; Ganter and Pittet, 2006; Subramaniam et al., 2007). In one recent study (Ashour et al., 2006) GRP given to newborn mice induced features of human BPD including interstitial pulmonary fibrosis and alveolarization. In a hyperoxic baboon model of BPD (Subramaniam et al., 2007) the early overproduction of Bn-like peptides correlated with the development of BPD-like histological features and the blockage of GRP partially reversed these effects, leading the authors to suggest that such an approach could have important implications for preventing BPD in premature infants. GRP has been shown to be protective to the small intestine in various injury models (Assimakopoulos et al., 2004, 2005a,b; Kinoshita et al., 2005; Kimura et al., 2006b), enhance gut barrier function, prevent the atrophy of enteric ganglia caused by FK506 in small bowel (Assimakopoulos et al., 2005a; Higuchi et al., 2006; Kimura et al., 2006a,b), and in a recent study (Fujimura et al., 2007) to prevent the atrophy of Peyer's patches and dysfunction of M cells in rabbits receiving long-term parenteral nutrition. These studies suggest that GRP agonists may have a potential therapeutic role in diseases causing this type of injury. Numerous studies in rodents provide evidence that GRP/BB₂ receptor activation is important for memory as well as for a number of social behaviors (learning, grooming, and stereotypy) (Roesler et al., 2006a,b). These results were supported by a recent study (Presti-Torres et al., 2007) in which the administration of BB₂-receptor antagonists in neonatal rats resulted in marked impairment of memory, and social interaction. These changes have led one group (Roesler et al., 2006a) to propose that the BB₂ receptor should be consider a therapeutic target in a subset of human CNS diseases, especially those involving memory, learning, and fear. Specifically, in the CNS it has been proposed that alterations in either the GRP and/or BB₂ receptor may be important in schizophrenia, Parkinson's disease, anxiety disorders, anorexia, bulimia, and mood disorders (Merali et al., 1999, 2006; Frank et al., 2001; Yeğen, 2003; Moody and Merali, 2004; Roesler et al., 2006a).

A. Early Studies of the BB₃ Receptor

Before the identification of the BB₃ receptor when it was cloned in 1992 from guinea pig uterus (Gorbulev et al., 1992), no pharmacological or functional studies suggested its existence.

B. Cloned BB₃ Receptor and Receptor Structure

The human BB₃ receptor is a 399-amino acid protein (Fathi et al., 1993b), and it shows 95% amino acid identities with the rhesus BB₃ receptor (Sano et al., 2004), 80% amino acid identity with the rat BB₃ receptor that shows 92% with the mouse BB₃ receptor, and 77% with the sheep BB₃ receptor (Liu et al., 2002) (Table 2). The human BB₃ receptor has 51% amino acid identities with the human BB₂ receptor and 47% with the human BB₁ receptor (Fathi et al., 1993b). The human BB₃ receptor has a predicted molecular mass of 44.4 kDa (Fathi et al., 1993b), and there are two potential N-linked glycosylation sites at Asn¹⁰ and Asn¹⁸ and a consensus site for potential PKC phosphorylation in the third cytoplasmic loop and carboxyl terminus (Fathi et al., 1993b; Whitley et al., 1999). A putative palmitoylation site existed at C³⁴⁷ and C³⁴⁸ (Fathi et al., 1993b; Whitley et al., 1999). Hydropathy plots yielded results consistent with a seven-transmembrane structure typical for a G protein-coupled receptor (Fathi et al., 1993b). The BB₃ receptor has been cloned from rat (Liu et al., 2002), mouse (Ohki-Hamazaki et al., 1997a), sheep (Whitley et al., 1999), and guinea pig (Gorbulev et al., 1992). In the chicken a receptor was cloned that has similarities to both the mammalian BB₃ receptor and the frog BB₄ receptor and has been termed the chBRS-3.5 receptor (Iwabuchi et al., 2003). No cross-linking studies have been performed on the mature BB₃ receptor so the extent of glycosylation or type is not known at present.

C. BB₃ Receptor Genomic Organization

The human BB₃ receptor gene is localized at human chromosome Xq25 and in the mouse on chromosome XA7.1–7.2 (Fathi et al., 1993b; Gorbulev et al., 1994; Weber et al., 1998). The human BB₃ receptor gene (Fathi et al., 1993b; Gorbulev et al., 1994; Weber et al., 1998) contained two introns and three exons similar to the sheep (Whitley et al., 1999), rhesus (Sano et al., 2004), mouse (Ohki-Hamazaki et al., 1997a), and rat BB₃ receptor genes (Liu et al., 2002). In the mouse the BB₃ receptor gene spanned more than 5 kb with exon 1 of the BB₃ gene separated from exon 2 by 1.6 kb and this in turn separated from exon 3 by 1.6 kb (Weber et al., 1998). In human, sheep, monkey, rat, mouse, and guinea pig the exon/intron splice sites occurred at Arg¹⁴⁵ in the second intracellular loop and at Ile²⁶³ in the third intracellular domain (Gorbulev et al., 1994; Weber et al., 1998; Sano et al., 2004).

D. BB₃ Receptor Expression

Expression levels of the BB₃ receptor mRNA have been reported in the rat (Fathi et al., 1993b; Liu et al., 2002; Jennings et al., 2003), sheep (Whitley et al., 1999), mouse (Ohki-Hamazaki et al., 1997a), monkey (Sano et al., 2004), and guinea pig (Gorbulev et al., 1992). In the monkey in which it was studied in detail, BB₃ mRNA is found in the greatest amount in the CNS and in the testis (Sano et al., 2004). This high expression in the testis is not seen in the sheep (Weber et al., 2003) or mouse (Ohki-Hamazaki et al., 1997a) but is similar to that in the rat (Fathi et al., 1993b) in which it was localized to the secondary spermatocytes and was not present in the Sertoli cells or different maturation stages of the spermatogonia (Fathi et al., 1993b). Detectable levels were also found in the monkey pancreas, thyroid, and ovary in peripheral tissues, and it was either undetectable or found in very low amounts in other tissues showing a very different distribution from that for the BB₁ receptor or BB₂ receptor (Fathi et al., 1993b; Sano et al., 2004).

In the CNS BB₃ receptor mRNA was expressed in a restricted distribution (Ohki-Hamazaki et al., 1997a; Liu et al., 2002; Jennings et al., 2003; Sano et al., 2004). In the rat and mouse (Ohki-Hamazaki et al., 1997a; Liu et al., 2002) the BB₃ receptor was present in the highest amounts in the hypothalamic area, notably the paraventricular, arcuate, striohypothalamic, dorsal hypothalamic, and dorsomedial hypothalamic nuclei, medial and lateral preoptic areas, and lateral/posterior hypothalamic areas. In the rat expression was also detected in the medial habenula nucleus in one study (Liu et al., 2002) and a second study (Jennings et al., 2003) in the nucleus accumbens and the thalamus. In the monkey brain (Sano et al., 2004) polymerase chain reaction quantitation showed that the BB₃ receptor mRNA was present in the highest amounts in the hypothalamus followed by the pituitary gland, amygdala, hippocampus, and caudate nucleus.

Specific BB₃ receptor antibodies have been used to localize the receptor in the tunica muscularis of the rat gastrointestinal tract (Porcher et al., 2005) and the rat CNS (Jennings et al., 2003). In the gastrointestinal tract tunica muscularis BB₃ receptor IR was observe in all regions studied (i.e., antrum, duodenum, ileum and colon) in nerves and non-neuronal cells but not in muscle cells (Porcher et al., 2005). It was detected in both myenteric and submucosal ganglia as well as in nerve fibers interconnecting myenteric ganglia (Porcher et al., 2005). BB₃ receptor IR was observed in the cell bodies and processes of the c-kit interstitial cells of Cajal, leading the authors to propose that the BB₃ receptor was probably involved in the regulation of gastrointestinal motility through the enteric nervous system and possibly in the pacemaker function of the gastrointestinal smooth muscle (Porcher et al., 2005). In the CNS, particularly strong BB₃ receptor IR was observed in the cerebral cortex, hippocampal formation, hypothalamus, and thalamus (Jennings et al., 2003).

With assessment of BB₃ mRNA (Fathi et al., 1993b) and/or binding studies (Reubi et al., 2002), BB₃ receptors have been shown to exist on a number of different human tumors (Fathi et al., 1993b; Reubi et al., 2002), including small cell and non-small cell lung cancers (Fathi et al., 1993b; Toi-Scott et al., 1996; Ryan et al., 1998b; Reubi et al., 2002), carcinoids (lung) (Fathi et al., 1993b; Reubi et al., 2002), renal cell cancers (Reubi et al., 2002), Ewing sarcomas (Reubi et al., 2002), pancreatic cancer (Schulz et al., 2006), pituitary tumors (Schulz et al., 2006), ovarian cancer (Sun et al., 2000b), and prostate cancer (Sun et al., 2000a; Schulz et al., 2006). BB₃ receptors have also been shown to exist on normal bronchial epithelial cells (DeMichele et al., 1994; Tan et al., 2006), human islets (Fleischmann et al., 2000), and rat kidney cells (Dumesny et al., 2004).

E. BB₃ Receptor Pharmacology

F. BB₃ Receptor Structural Basis of Receptor Binding/Activation

G. BB₃ Receptor Signaling, Activation, and Modulatory Processes (Internalization, Down-Regulation, and Desensitization)

The human BB₃ receptor (Fathi et al., 1993b; Ryan et al., 1996, 1998a; Wu et al., 1996), as well as the monkey (Sano et al., 2004) and rat BB₃ receptors (Liu et al., 2002) is coupled to phospholipase C, resulting in breakdown of phosphoinositides, mobilization of cellular calcium, and presumed activation of protein kinase C.

BB₃ receptor activation results in the stimulation of phospholipase D (Ryan et al., 1996) but does not activate adenylate cyclase (Ryan et al., 1996, 1998a). BB₃ receptor stimulation also results in activation of tyrosine kinases (Ryan et al., 1998a; Weber et al., 2001), stimulating tyrosine phosphorylation of p125^FAK by a mechanism that is not dependent on either limb of the phospholipase C cascade (i.e., activation of PKC or mobilization of cellular calcium) (Ryan et al., 1998a). Activation of BB₃ receptor also stimulates MAP kinase activation, resulting in rapid tyrosine phosphorylation of both 42- and 44-kDa forms, which is inhibited by the MEK-1 inhibitor PD98059 (Weber et al., 2001). In BB₃ receptor-transfected NCI-1299 lung cancer cells, activation of the BB₃ receptor by [d-Phe⁶,β-Ala¹¹,Phe¹³, Nle¹⁴]bombesin_6–14 resulted in stimulation of Elk-1 in a MEK-1-dependent manner as well as a 47-fold increase in c-fos mRNA (Weber et al., 2001). These results demonstrated that BB₃ receptor activation causes increased nuclear proto-oncogene expression and upstream events including activation of MAP kinase and Elk-1 activation (Weber et al., 2001). There have been no studies of BB₃ receptor modulatory processes (internalization, down-regulation, or desensitization).

H. BB₃ Receptor Function in Various Tissues and in Vivo

At present the function of the BB₃ receptor in normal physiology and pathological conditions is largely unknown because the natural ligand is still not known. An important insight into possible BB₃ receptor function was provided by studies of BB₃ receptor knockout mice. In the initial study (Ohki-Hamazaki et al., 1997b) mice lacking the BB₃ receptor developed mild obesity, associated with hypertension and impairment of glucose metabolism. These changes were associated with reduced metabolic rate, increased feeding behavior, a 5-fold increase in serum leptin levels, and hyperphagia (Ohki-Hamazaki et al., 1997b) and the results suggested that the BB₃ receptor might play an important role in the mechanisms responsible for energy balance and control of body weight. A number of studies have been performed subsequently on BB₃ receptor knockout mice to attempt to establish the mechanism of these effects. BB₃ receptor knockout mice were shown to have altered taste preference (Yamada et al., 1999), which was proposed to be due to the lack of BB₃ receptor expression in the medial and central nuclei of the amygdala and the hypothalamic nuclei, which are known to be involved in taste perception (Yamada et al., 1999) and to possibly be a contributory factor to the obesity. BB₃ receptors are present on pancreatic islets (Fleischmann et al., 2000), and BB₃ receptor knockout mice have a 2.3-fold increase in plasma insulin levels (Matsumoto et al., 2003) (Table 2). One study (Matsumoto et al., 2003) concluded that the BB₃ receptor contributes to regulation of plasma insulin concentration/secretion and that dysregulation in this contribution in these mice contributes to obesity (Matsumoto et al., 2003). In a second study (Nakamichi et al., 2004) it was concluded that the impaired glucose metabolism in BB₃ receptor knockout mice is mainly due to impaired glucose transporter 4 translocation in adipocytes.

I. BB₃ Receptor in Diseases

At present there are no diseases in which activation or alterations of the BB₃ receptor have been shown to be involved. BB₃ receptor activation has been proposed to be important in the mediation of a number of human disorders including disorders of lung development, various pulmonary diseases, CNS disorders, and the growth/differentiation of human cancers. The tumor differentiation effects of BB₃ receptor activation were discussed in the previous section; the growth effects and effects of BB₃ receptor overexpression will be considered here. In human cancer cells or cancers BB₃ receptors are not only ectopically expressed in a large number of tumors, as reviewed earlier (Fathi et al., 1993b, 1996; Toi-Scott et al., 1996; Sun et al., 2000b; Reubi et al., 2002; Schulz et al., 2006), but their activation alters lung cancer behavior by increasing MAP kinase activation and nuclear oncogene expression (Weber et al., 2001) and increasing adhesion of lung cancer tumor cells, which was proposed to contribute to increased tumor invasion and metastases by these tumors (Hou et al., 2006). In BB₃ knockout mice (Maekawa et al., 2004) the hyperphagic response to melanin-concentrating hormone (MCH) is impaired, but this impairment is not seen in BB₂ receptor knockout mice. Furthermore, the levels of the MCH receptor and prepro-MCH mRNAs in the hypothalamus of BB₃ receptor knockout mice were higher than those of controls, suggesting that up-regulation of the MCH receptor and MCH occurs in the knockout mice, which triggers hyperphagia and probably upsets the mechanism by which leptins decrease MCH receptors and feeding (Maekawa et al., 2004). Studies of BB₃ receptor knockout mice suggest that this receptor is important in various behavioral effects, including the neural mechanisms that regulate social isolation (Yamada et al., 2000a), and are important in modulating emotion including forms of anxiety (Yamada et al., 2002a).

BB₃ receptors as well as BB₁ receptor and BB₂ receptor are expressed in developing primate and murine fetal lungs (Emanuel et al., 1999; Shan et al., 2004). Studies (Tan et al., 2006, 2007) demonstrate that BB₃ receptors are expressed in the airway in response to ozone injury and that wound repair and proliferation of bronchial epithelial cells is accelerated by BB₃ receptor activation, suggesting that it may mediate wound repair. The mechanism of lung ozone injury mediation of the up-regulation of BB₃ receptors has been studied by examining proteins interacting with the BB₃ receptor gene promoter region (Tan et al., 2007). Activator protein-2α and peroxisome proliferator-activated receptor-α increased the ozone-inducible DNA binding on the BB₃ receptor gene promoter, suggesting that they are specifically involved in the BB₃ receptor up-regulation (Tan et al., 2007). BB₃ receptors are expressed on small cell and non-small cell lung cancers (Fathi et al., 1993b; Toi-Scott et al., 1996; Ryan et al., 1998b; Reubi et al., 2002) as well as lung carcinoids (Fathi et al., 1993b; Reubi et al., 2002). In the small cell lung cancer cell line, NCI-N417, which is known to possess functional BB₃ receptors (Ryan et al., 1998b), [d-Phe⁶,β-Ala¹¹,Phe¹³,Nle¹⁴]-bombesin_6–14 stimulated tumor cell adhesion, probably by stimulation of focal adhesion formation (Hou et al., 2006). It was proposed (Hou et al., 2006) that BB₃ receptor activation in these cells may be important for their invasion and development of metastases.

Although the function of BB₃ receptors in the gastrointestinal tract is largely unknown, specific BB₃ receptor antibodies localized the receptor in the tunica muscularis of the rat gastrointestinal tract (Porcher et al., 2005). BB₃ receptors were detected in both myenteric and submucosal ganglia as well as in nerve fibers interconnecting myenteric ganglia (Porcher et al., 2005). BB₃ receptor IR was observed in cell bodies and processes of the c-kit interstitial cells of Cajal, leading the authors to propose that the BB₃ receptor was probably involved in the regulation of gastrointestinal motility.

One study screened 104 Japanese obese men for defects in the BB₃ receptor gene, but no mutations or polymorphisms were found (Hotta et al., 2000), suggesting BB₃ receptor gene mutations are unlikely to be a major cause of obesity in humans.

The above studies and those reviewed in the previous section suggest that BB₃ receptor activation could be involved in human disorders of energy metabolism, including obesity, glucose homeostasis, blood pressure control, lung injury, tumor growth, and possibility motility disorders. However, all of these possibilities remain unproven at present.