Characterization And Pharmacology Of Receptors For Bombe

蛙皮素相关肽（[gastrin-releasing peptide [GRP]，neuromedin B）与两种不同的受体（GRP-R，NMB-R）相互作用，在胃肠道（GI）、中枢神经系统（CNS）以及正常和肿瘤组织的生长中介导多种效应。此外，最近已经描述了两种相关受体，哺乳动物孤儿受体（BRS-3），其与GRP-R具有60%的同源性，以及两栖动物中的新受体BB-4-R。该项目的目的是了解这些受体的药理学，分子药理学和细胞生物学，以及开发可用于确定其生理作用的特定激动剂和拮抗剂。正在进行的研究包括这些受体在稳定细胞系中的表达，这些细胞系在细胞生物学和药理学上类似于天然受体;研究使用定点诱变和受体嵌合体来确定受体结构决定因素的配体选择性和特异性的激动剂和拮抗剂，BN相关肽的药理学研究以鉴定选择性激动剂/拮抗剂和天然细胞和转染细胞的研究，以确定这些受体的转导级联。使用定点突变和嵌合受体方法，我们已经检查了激动剂GRP（2）、高GRPR肽拮抗剂（4）和NMB受体拮抗剂PD 168368（3）选择性的分子基础。这些结果表明，第三胞外结构域（EC-3）中的关键氨基酸负责GRP选择性，GRPR的EC 4中的三个氨基酸负责拮抗剂的高GRPR选择性，并且NMBR的上第五跨膜区中的氨基酸负责PD 168368的选择性。分子模拟和定点突变研究表明，这种选择性主要是由这些配体和受体之间的氢键和π-阳离子相互作用介导的。使用受体建模和诱变研究，我们最近设计了BRS-3受体的选择性激动剂（1）。在BRS-3研究之前，BRS-3受体不存在限制研究其生理作用的能力的选择性配体。使用构象限制类似物[B-Ala 11]蛙皮素和建模，我们确定了一个氨基-3-苯基丙酸类似物作为第一个选择性BRS-3激动剂。

Bombesin-related peptides ([gastrin-releasing peptide [GRP], neuromedin B) interact with two distinct receptors (GRP-R, NMB-R) to mediate a number of effects in the gastrointestinal tract (GI), central nervous sytem (CNS) and on growth of normal and neoplastic tissues. Furthermore, two related receptors, a mammalian orphan receptor (BRS-3), having 60% homology to GRP-R and a novel receptor in amphibians, BB-4-R has been described recently. The aims of this project are to understand the pharmacology, molecular pharmacology, and cell biology of these receptors as well as to develop specific agonists and antagonists that can be used to determine their physiological roles. Investigations being performed include expression of these receptors in stable cell lines that resemble native receptors in their cell biology and pharmacology; investigations using site-directed mutagenesis and receptor chimeras to define receptor structural determinants of ligand selectivity and specificity for agonists and antagonists, pharmacological studies of BN-related peptides to identify selective agonists/antagonists and studies of native cells and transfected cells to define the transduction cascades of these receptors. Using site directed mutagenesis and a chimeric receptor approach, we have examined the molecular basis of the selectivity of agonist GRP (2), high GRPR peptide antagonists (4) and the NMB receptor antagonist, PD168368 (3). These results demonstrate the critical amino acids in the 3rd extracellular domain (EC-3) are responsible for GRP selectivity, three amino acids in EC4 of the GRPR responsible for the antagonist's high GRPR selectivity and the amino acids in upper 5th transmembrane region of the NMBR responsible for PD168368's selectivity. Molecular modeling and site-directed mutagenesis studies showed this selectivity was primarily mediated by hydrogen bonding and pi-cation interactions between these ligands and the receptor. Using receptor modeling and mutagenesis studies we have recently designed a selective agonist for the BRS-3 receptor (1). Prior to the BRS-3 study, no selective ligands existed for the BRS-3 receptor restricting the ability to investigate its physiological roles. Using conformationally restricted analogues of [B-Ala11] bombesin and modeling we identified an amino-3 phenyl propionic acid analogue as the first selective BRS-3 agonist.