BB3 receptor

Overview

Prior to the identification of the BB₃ receptor when it was cloned in 1992 from the guinea pig uterus [1], no pharmacological or functional data suggested its presence. The BB₃... [read more] receptor has now been cloned from rat [2], mouse [3], sheep [4], monkey [5] and human [6]. Each of these receptors, when expressed, have low affinity for the mammalian peptides GRP and NMB, and in the case of the human BB₃ receptor, it was shown to have low affinity for all known naturally occurring bombesin related peptides [2,7,8,9,10,11,12]. Therefore at present the natural ligand of this receptor is unknown.

The human BB₃ receptor is a 399 amino acid protein [6] and it shows 95% amino acid identities with the monkey [5] and 80% with the rat BB₃ receptor [13]. The human BB₃ receptor has 47% amino acid identities with the human BB₁ receptor and 51% with the human BB₂ receptor [6]. The human BB₃ receptor has a predicted molecular weight of 44.4 kDa and has two potential N-linked glycosylation sites, but there are no cross-linking studies of the mature BB₃ receptor.

The human BB₃ receptor gene is located at chromosome Xq25 and in the mouse at XA7.1-7.2 [6,14,15]. The human BB₃ receptor gene contains two introns and three exons [6,15].

Expression levels of the BB₃ receptor mRNA are reported in rat, sheep, mouse, monkey and guinea pig [1,2,4,5,6,16,17,18]. Detectable amounts are found in the monkey thyroid, pancreas and ovary [5]. Detailed studies show that BB₃ receptor mRNA is present in the islets in multiple species including human [19]. In the CNS BB₃ receptor mRNA is expressed in a more restricted distribution than BB₁ receptor or BB₂ receptor [2,5,16,17], although in a recent study it is more widely distributed [20]. In the rat and mouse [2,17,20] and in the monkey brain BB₃ receptor mRNA is present in the highest amounts in the hypothalamus [5,20] and is particularly high in the preoptic nucleus, the PVN, the dorsomedial hypothalmic nucleus and the arcuate nucleus, supporting the hypothesis that the BB₃ receptor may be important for the hypothalamic regulation of energy homeostasis [20]. In the monkey brain BB₃ receptor mRNA is present in the highest amounts, after the hypothalamus [5], in the pituitary gland, amygdala, hippocampus and caudate nucleus [5]. In the mouse and rat brains [20], no BB₃ receptor mRNA can be detected in the cerebral cortex, olfactory bulb, hippocampal formation, cerebellum, substantia nigra or ventral tegmental area. In rat and mouse brains [20], double in situ hydridization studies show that BB₃ receptor mRNA colocalizes frequently with glutamatergic neurons, and to a lesser extent with cholinergic or GABAergic neurons, and CRF or GHRH, but not with NPY, POMC, orexin/hypocretin, MCH,GnRH or kisspentin. Specific BB₃ receptor antibodies have been used to localise the receptor in the tunica muscularis of the gastrointestinal tract [21] and in the rat CNS [16]. In the gastrointestinal tract the BB₃ receptor was detected in myenteric and submucosal ganglia as well as the interstitial cells of Cajal [21]. In the CNS particularly strong staining was detected in the cerebral cortex, hippocampus, hypothalamus and thalamus [21].

The natural ligand for the BB₃ receptor remains unknown. Recently two Drosphilia G-protein coupled receptors (CG30106,CG14593) have been identified and belong to the BB₃ receptor family [22,23,24]. These receptors interact with two ligands CCHamide-1[SCLEYGHSCWGAH-NH₂] and CCHamide-2[GCQAYGHVCYGGH-NH₂], which when administered to blowflies increased feeding [22]. Unfortunately, neither of the CCHamide peptides activated the BB₃ receptor[22] and each was found to have a very low affinity for the human BB₃ receptor (Jensen, unpublished data). Using a bacterial two-hybrid system, the BB₃ receptor was found to interact with a novel 354 amino acid protein, named bombesin receptor activated protein (BRAP) [25]. BRAP is found in both the cellular cytoplasm and nucleus, its expression promotes G1 to S phase transition, accelerates DNA snythesis, and promotes the proliferation of bronchial epithelial cells [25]. This has led to the proposal that BRAP may be important in BB₃ receptor mediated changes in cell cycle regulation and in wound healing in bronchial epithelial cells [25]. Overexpression of BRAP [26] in bronchial epithelial cells inhibits the ability of these cells to take up antigen suggesting it may play an important role in the antigen presenting process of bronchial epithelium.

Even although no high affinity natural ligands exist for this receptor it has been found that the synthetic bombesin ligand, [D-Phe⁶,β-Ala¹¹,Phe¹³,Nle¹⁴]bombesin(6-14) binds and activates both cells containing the natural occurring [9] and expressed BB₃ receptors with high affinity/potency [7,8,11,27,28]. When the rat BB₃ receptor was cloned [2] it was surprisingly found that [D-Phe⁶,β-Ala¹¹,Phe¹³,Nle¹⁴]bombesin(6-14) had low affinity for this receptor, whereas it had high affinity for the monkey BB₃ receptor, similar to the human receptor [5]. Using a chimeric receptor approach [2] in which the individual extracellular loops of the rat BB₃ receptor were replaced with the corresponding human sequences the important residues were localised to the 4^th extracellular domain (1^st=N-terminus). Within this region, using site-directed mutagenesis [2], the mutation of Y²⁹⁸E²⁹⁹S³³⁰ (rat) to S²⁹⁸Q²⁹⁹T³⁰⁰ (human) or of D³⁰⁶V³⁰⁷H³⁰⁸ (rat) to A³⁰⁶M³⁰⁷H³⁰⁸ (human) partially mimic the effect of switching the entire 4^th extracellular domain. These results indicate that variations in the 4^th extracellular domains of the rat and human BB₃ receptor are responsible for the differences in affinity for [D-Phe⁶,β-Ala¹¹,Phe¹³,Nle¹⁴]bombesin_6-14 [2].Subsequent studies demonstrated the synthetic bombesin analogue, [D-Phe⁶,β-Ala¹¹,Phe¹³,Nle¹⁴]bombesin_6-14, in addition to having high affinity for monkey and human BB₃ receptors, also had high affinity for all BB₁ and all BB₂ receptors, as well as a frog BB₄ subtype which is not found in mammals [7,8,9,11,27,28,29]. Due to the lack of selectivity of the high affinity agonist, [D-Phe⁶,β-Ala¹¹,Phe¹³, Nle¹⁴]bombesin_6-14 for the human BB₃ receptor, a number of groups have attempted to develop more selective BB₃ receptor ligands. In one study [30] rational peptide design was used by substituting conformationally restricted amino acids and a number of BB₃ receptor selective agonists were identified with two peptides with either an (R) or (S)-amino-3-phenylpropionic acid substitution for β-Ala¹¹ in the prototype ligand having the highest selectivity (i.e.17-19-fold) [30]. Molecular modeling demonstrated these two selective BB₃ receptor ligands had a unique conformation of the position of the 11 β-amino acids, which likely accounted for their selectivity [30]. In a second study [31] two strategies were used to attempt to develop a more selective BB₃ receptor ligand: substitutions on the phenyl ring of Ala¹¹ and the substitution of additional conformationally restricted amino acids into the position 11 of [D-Phe⁶,β-Apa¹¹,Phe¹³,Nle¹⁴]bombesin_6-14 or its D-Tyr⁶ analogue. One analogue, [D-Tyr⁶,Apa-4Cl¹¹,Phe¹³,Nle¹⁴]bombesin_6-14 retained high affinity for BB₃ receptor and was 227-fold selective for the BB₃ receptor over the human BB₂ receptor and 800-fold selective over the human BB₁ receptor [31]. Using [D-Phe⁶,β-Ala,sup>11,Phe¹³,Nle¹⁴]bombesin_6-14 or its D-Tyr⁶ analogue as the prototype, three studies [32,33,34] reported shortened analogues with selectivity for BB₃ receptor assessed by calcium or FIPR calcium assays. A recent study has assessed the selectivity of four of the most selective of these shortened [D-Phe⁶,β-Ala¹¹,Phe¹³,Nle¹⁴]bombesin(6-14) analogues by binding assays as well as assessment of phospholipase C potencies [35]. Only the novel compound Ac-Phe,Trp,Ala,His(tBzl),Nip,Gly,Arg-NH₂ (compound 34 in reference [34]) had a 14-fold higher affinity for BB₃ receptor than BB₁ receptor and >20 fold for BB₂ receptor [35], however it was less BB₃ receptor selective than [D-Tyr⁶,Apa-4Cl¹¹,Phe¹³,Nle¹⁴] bombesin_6-14 (i.e. >100 fold selectivity) [31,35].

Recently a number of BB₃ receptor nonpeptide agonists have been described including derivatives of omeprazole [36], benzodiazepine sulfonamide analogues [37], derivatives of an analogue found to have activity during high throughput screening [7-benzyl-5-(piperidin-1-yl)-6,7,8,9-tetrahydro-3H-pyrazolo[3,4-c]- [2,7]naphthyridin-1-ylamine] [38,39], substituted biphenyl imidazole analogues [40], 2-biarylethylimidazole analogues [41], pyridinesulfonylureas and pyridinesulfonamide analogues [42]. Of these the selective nonpeptide high affinity agonist, MK-5046 [(2S)-1,1,1-trifluoro-2-[4-(1H-pyrazol-1-yl)phenyl]-3-(4-[[1-(trifluoromethyl)cyclopropyl] methyl]-1H-imidazol-2-yl)propan-2-ol ] [37-39], or Bag-1, (2-(4-[2-[5-(2,2-dimethylbutyl)-1H-imidazol-2-yl] ethyl]phenyl) pyridine (compound 9 in [41]) have been the most widely used for agonist studies of the BB₃ receptor. Also a high affinity selective BB₃ receptor peptide antagonist, Bantag-1 [Boc-Phe-His-4-amino-5-cyclohexyl-2,4,5-trideoxypentonyl-Leu-(3-dimethylamino) benzylamide N-methylammonium trifluoroacetate] has been described [19,43] MK-5046 is orally active and shown to be active in dog, mice, rat, monkey and human [44,45]. In a recent comparison of their selectivity for human BB₃ receptor cells expressing native or transfected receptors, MK-50946 and Bantag-1 were found to have high affinity for the human BB₃ receptor (17.7-18.8, 1-1.6 nM, resectively) and to be 555-fold and 6250-fold selective, respectively, based on affinities for the human BB₃ receptor over either the human BB₁ or BB₂ receptor. In terms of selectivity for activation of the human BB₃ receptor (phospholipase C activation), MK-5046 had a selective potency of >10,000-fold over the human BB₁ and human BB₂ receptors [46]. A number of studies using site-directed mutageneis have examined the molecular basis for agonist activation and selectivity for the human BB₃ receptor [11,47,48]. In one study [47] epitopes determining the agonist property of [D-Tyr⁶,(R)-Apa-Cl¹¹,Phe¹³,Nle¹⁴]bombesin(6-14) or Ac-Phe,Trp,Ala,His(tBzl),Nip,Gly,Arg-NH₂ (compound 34 in reference [34]) were examined. The mutational map for Ac-Phe,Trp,Ala,His(ψBzl),Nip,Gly,Arg-NH₂ spanned the entire binding pocket, whereas that for [D-Tyr⁶,(R)-Apa-Cl¹¹,Phe¹³,Nle¹⁴]bombesin(6-14) was confined to the center of the pocket encompassing the opposing faces of the extracelluar segment of TM-111,TM-V1 and TM-VIII [47]. Using a chimeric receptor approach combined with site-directed mutagenesis [48], the selectivity of [D-Tyr⁶,(R)-Apa-Cl¹¹,Phe¹³,Nle¹⁴]bombesin(6-14), Ac-Phe,Trp,Ala,His(ψBzl),Nip,Gly,Arg-NH₂, and [D-Tyr⁶,(R)-Apa¹¹,Phe¹³,Nle¹⁴]bombesin(6-14) for the human BB₃ receptor was examined. Even though these three human BB₃ receptor agonists were developed from the same template peptide, [D-Tyr⁶,β-Ala¹¹,Phe¹³,Nle¹⁴]bombesin(6-14), their molecular determinants of selectivity/high affinity varied considerably [48].

References

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