SIGNAL TRANSDUCTION IN SALIVARY GLAND

乙酰胆碱、去甲肾上腺素和P物质控制唾液 唾液腺泡细胞表面受体的分泌 以产生特定的细胞内信号。两种信号转导途径 已经确定了两条途径：一条涉及cAMP合成，另一条涉及cAMP合成。 包括肌醇磷脂的水解和钙离子的动员。营地 途径，包括G蛋白作为转导蛋白的作用，是很好的 明白了。对肌醇磷脂/钙离子途径知之甚少 尽管有证据表明G蛋白也参与了这一过程 路径。G蛋白的身份尚不清楚；它缺乏 对霍乱和百日咳毒素的敏感性似乎排除了 目前已纯化的G蛋白中的任何一种都参与其中。 这项提议的目的是鉴定这种新的G蛋白， 暂定名为G(P)，并对其生化和生物化学特性进行了研究 功能属性。具体目标是：1)隔离和 百日咳毒素不敏感菌株G的生化特性 颌下腺中可与之发生功能相互作用的蛋白质(S) SP受体；2)与P物质相关的G蛋白的鉴定 SP受体，化学交联法；3)功能分析 纯化的G与SP受体和磷脂酶C的相互作用 同源GP的部分氨基酸序列分析。 将使用两种新的实验方法。唾液腺 膜，通过处理耗尽其内源G蛋白 碱性缓冲液，将与获得的活性G蛋白重组 在层析分离马G蛋白的过程中 颌下腺。该方法简便、灵敏，可用于检测G 能与SP受体功能相互作用的蛋白质 结合GTP敏感、高亲和力激动剂的第二种方法将 涉及使用一种新型的SP的光反应类似物。(125)I-标签 Phe8(PBZ)-SP可以有效地被光结合到G-偶联SP中 受体。然后将GP化学交联到光标记的SP上 受体和所产生的放射性标记的共价加合物 用十二烷基硫酸钠-PAGE和免疫检测技术进行成分分析。 有一些疾病状态是由G蛋白改变引起的 功能。理解G在肌醇磷脂/钙离子中的作用 通路将允许对某些特定的改变的转导进行评估 唾液腺功能障碍的状况。

Acetylcholine, norepinephrine and substance P control salivary secretion by binding to receptors on the surface of salivary acinar cells to produce specific intracellular signals. Two signal transduction pathways have been identified: one involves cAMP synthesis and the other involves phosphoinositide hydrolysis and Ca2+ mobilization. The cAMP pathway, including the role of G proteins as transducers, is well understood. Less is known about the phosphoinositide /Ca2+ pathway although there is evidence that a G protein is also involved in this pathway. The identity of the G protein is unknown; its lack of sensitivity to cholera and pertussis toxins would seem to rule out the involvement of any of the G proteins that have been purified to date. The objectives of this proposal are to identify this novel G protein, provisionally termed G(p), and to characterize its biochemical and functional properties. The specific aims are: 1) isolation and biochemical characterization of a pertussis toxin-insensitive G protein(s) from submaxillary glands that can functionally interact with the SP receptor; 2) identification of the G protein associated with the SP receptor, by chemical crosslinking; 3) analysis of the functional interactions of purified G with the SP receptor and phospholipase C; 4) partial amino acid sequence analysis of homogeneous GP. Two new experimental approaches will be used. Salivary gland membranes, depleted of their endogenous G proteins by treatment with alkaline buffer, will be reconstituted with active G proteins obtained during chromatographic fractionation of G proteins from horse submaxillary gland. This convenient and sensitive assay detects G proteins that can functionally interact with SP receptors to restore GTP-sensitive, high affinity agonist binding The second approach will involve the use of a novel photoreactive analog of SP. (125)I-labelled Phe8(pBz)-SP can be photoincorporated efficiently into the G -coupled SP receptor. Gp will then be chemically crosslinked to the photolabelled SP receptor and the resulting radiolabelled covalent adduct subjected to compositional analysis by SDS-PAGE and immunodetection techniques. There are disease states that result from altered G protein function. Understanding the role of G in the phosphoinositide/Ca2+ pathway will permit an assessment of altered transduction in certain states of salivary gland dysfunction.