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) 和神经内毒素B(NMB)，调节一系列不同的生物学效应 回应 在正常组织中，并刺激一些肺的生长， 胰腺， 和前列腺癌。我们的实验室克隆并鉴定了 三 结构和药理上不同的人弹丸 受体： 胃泌素释放肽受体(GRP-R，或BB2)； 神经内科 B受体(NMB-R或BB1)；蛙皮素受体亚型3(BRS-3， 或 BB3)。这三种受体都是G蛋白偶联的成员 受体 超家族，偶联到激活异三聚体的G蛋白 磷脂酶C.我们对磷脂酶C的理解的几个进展 功能 对这些受体的调节发生在上一次 年份：(1) 我们在其跨膜结构域中鉴定出四个残基。 这个 对蛙皮素的高亲和力结合至关重要的受体 受体 使用定点突变的激动剂；(2)我们使用了基因 靶向策略，创造缺乏GRP-R的小鼠，以及 进展情况 在我们努力培育缺乏NMB-R或BRS-3的类似小鼠的过程中。 这个 这些突变体的表型将在来年进行研究； (3)我们 已经开发出一种协议，允许我们生成非耦合的 受体 嵌入在正常的生物膜中。这一准备工作使 我们要 重组与纯化的G蛋白的偶联，检查 选择性 和受体催化的核苷酸交换的同工酶 异三聚体GTP结合蛋白。这些研究表明， GRP-R 选择性地偶联到G蛋白的Q亚家族，而不是 大兵，去吧， 或者换能器。同样的制剂也用于研究受体。 G蛋白受体在体外的磷酸化，显示 那 GRK1以配体依赖的方式磷酸化GRP-R，与 所有的 先前在体内确定的GRP-R磷酸化的性质； (4) 我们正在检测自闭症患者的GRP-R基因 确定 是否有任何等位基因变异与这种疾病有关。这个 这些变体的功能性质(配体结合、偶联、 内化、敏化、糖基化、磷酸化) 将会是 仔细检查了一下。

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. Our laboratory 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 identified four residues in the transmembrane domains of the receptor that are crucial for high-affinity binding of bombesin receptor agonists using site-directed mutagenesis; (2) we have used gene targeting strategies to create mice which lack the GRP-R, and progressed in our efforts to generate similar mice that lack NMB-R, or BRS-3. The phenotype of these mutants will be studied in the upcoming year; (3) 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 purified G-proteins, examining the selectivity and emzymology of receptor-catalyzed nucleotide exchange on heterotrimeric GTP-binding proteins. These studies show that the GRP-R couples selectively to the q subfamily of G-proteins, and not to Gi, Go, or transducin. This same preparation is also used to study receptor phosphorylation by G-protein receptor kinases in vitro, showing that GRK1 phosphorylates the GRP-R in a ligand dependent manner, with all the properties determined previously for GRP-R phosphorylation in vivo; (4) we are examining the GRP-R gene from patients with autism to determine if any allelic variants are associated with this disorder. The functional properties of these variants (ligand binding, coupling, internalization, sensitization, glycosylation, phosphorylation) will be examined in detail.