Structure, Function and Regulation of Bombesin Receptors

哺乳动物蛙皮素样神经肽，胃泌素释放肽（GRP）和神经介肽B（NMB），介导正常组织中的多种生物学反应，并刺激一些肺癌、胰腺癌和前列腺癌的生长。 我们实验室克隆并鉴定了三种结构上不同的人铃蟾肽受体：胃泌素释放肽受体（GRP-R，或bb 2）;神经介肽B受体（NMB-R，或bb 1）;铃蟾肽受体亚型3（BRS-3，或bb 3）。所有三种受体都是G蛋白偶联受体超家族的成员，与激活磷脂酶C的异源三聚体G蛋白偶联。在过去的一年里，我们对这些受体的功能和调节的理解取得了一些进展：（1）我们使用基因靶向策略来创造缺乏GRP-R或BRS-3的小鼠。 缺乏GRP-R的小鼠不表现出蛙皮素诱导的饱腹感，暗示GRP-R是食欲的调节剂。 随着缺乏GRP-R的小鼠年龄的增长，GRP-R靶向小鼠更有可能变得肥胖。相反，缺乏BRS-3的小鼠在出生后的最初几个月内变得肥胖。我们目前正在研究人类BRS-3基因的等位基因变异可能导致人类肥胖的假设，特别是男性个体。(2)我们已经开发了一种协议，使我们能够产生嵌入正常生物膜的非偶联受体。 这种制备方法使我们能够重建与纯化G蛋白的偶联，研究异源三聚体GTP结合蛋白上受体催化的核苷酸交换的选择性和酶学。 使用这种方法，我们表明，磷酸化的丝氨酸和苏氨酸的GRP-R的C-末端结构域抑制耦合到异源三聚体G-蛋白，从而建立配体刺激的受体磷酸化是关闭受体信号的开关。(3)我们已经使用酵母双杂交系统来寻找与C-末端结构域相互作用的分子，并确定了几个候选人进行进一步研究，包括最近已被证明与相应的视紫红质C-末端结构域相互作用的一个。(4)使用定点诱变，我们已经表明，有残基在第六跨膜结构域是关键的选择性激动剂和拮抗剂的结合，和其他残基在这个相同的域，调节GRP-R的构象变化。总之，这些研究暗示第六跨膜结构域作为GRP-R功能的整合者和协调者。

Mammalian bombesin-like neuropeptides, gastrin releasing peptide (GRP) and neuromedin B (NMB), mediate a diverse range of biological responses in normal tissues, and stimulate the growth of some lung, pancreatic, and prostate carcinomas. Ourlaboratory cloned and characterized three structurally and pharmacologically distinct human bombesin receptors: the gastrin-releasing peptide receptor (GRP-R, or bb2); the neuromedin B receptor (NMB-R, or bb1); and bombesin receptor subtype 3 (BRS-3, or bb3). All three receptors are members of the G-protein coupled receptor superfamily, coupling to heterotrimeric G-proteins that activate phospholipase C. Several advances in our understanding of the function and regulation of these receptors has occurred within the last year: (1) we have used gene targeting strategies to create mice which lack either GRP-R or BRS-3. Mice lacking GRP-R do not exhibit bombesin-induced satiety, implicating the GRP-R as a regulator of appetite. As mice lacking GRP-R age, GRP-R targeted mice are more likely to become obese. In contrast, mice lacking BRS-3 become obese within the first few months of life.We are currently examining the hypothesis that allelic variation in the human BRS-3 gene may contribute to obesity in humans, particulary male individuals. (2) We have developed a protocol that allows us to generate uncoupled receptors embedded in a normal biological membrane. This preparation allows us to reconstitute coupling to purifiedG-proteins, examining the selectivity and enzymology of receptor-catalyzed nucleotide exchange on heterotrimeric GTP-binding proteins. Using this assay, we show that phosphorylation of serines and threonines in the C-terminal domain of the GRP-R inhibits coupling to heterotrimeric G-proteins thereby establishing that ligand-stimulated receptor phosphorylation is the switch that turns off receptor signalling. (3) We have used the yeast two-hybrid system to search for molecules that interact with the C-terminal domain, and have identified several candidates for further study, including one that has been recently shown to interact with the corresponding C-terminal domain of rhodopsin. (4) Using site-directed mutagenesis, we have shown that there are residues in the sixth transmembrane domain that are critical for selective agonist and antagonist binding, and other residues in this same domain that regulate conformational change of the GRP-R. Taken together, these studies implicate the sixth transmembrane domain as an integrator and coordinator of GRP-R function.