SIGNAL TRANSDUCTION IN SALIVARY GLAND

乙酰胆碱、去甲肾上腺素和P物质控制唾液 与唾液腺泡细胞表面受体结合分泌 产生特定的细胞内信号。 两种信号转导 已确定的途径：一个涉及cAMP合成和其他 涉及磷酸肌醇水解和Ca 2+动员。 营地 通路，包括G蛋白作为转换器的作用，是很好的 明白 关于磷酸肌醇/Ca 2+途径知之甚少 尽管有证据表明G蛋白也参与其中， 通路 G蛋白的身份是未知的;它缺乏 对霍乱和百日咳毒素的敏感性似乎排除了 涉及迄今为止已经纯化的任何G蛋白。 本提案的目的是鉴定这种新的G蛋白， 暂时称为G（p），并表征其生化和 功能特性 具体目标是：1）隔离， 百日咳毒素不敏感G 来自颌下腺的蛋白质，其可以在功能上与 SP受体; 2）鉴定与SP受体相关的G蛋白， SP受体，通过化学交联; 3）功能分析 纯化的G与SP受体和磷脂酶C的相互作用; 4） 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，耗尽其内源性G蛋白的处理， 碱性缓冲液，将用获得的活性G蛋白复溶 在马G蛋白的色谱分离过程中 颌下腺 这种方便和灵敏的检测方法检测G 这些蛋白质可以与SP受体功能性相互作用， GTP敏感的高亲和力激动剂结合 涉及使用一种新的SP的光反应类似物。（125）I标记的 Phe 8（pBz）-SP可以有效地光掺入到G -偶联的SP中， 受体的 然后GP将与光标记的SP化学交联 受体和得到的放射性标记的共价加合物， 通过SDS-PAGE和免疫检测技术进行组成分析。 有些疾病是由G蛋白改变引起的 功能了解G在磷酸肌醇/Ca 2+中的作用 通路将允许评估某些细胞中改变的转导， 唾液腺功能障碍的状态。

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.