SUBCELLULAR LOCALIZATION OF TSH PROCESSING

此应用程序寻求将NIRA(R23)奖转换为第一个 奖项(R29)。拟议研究的目标是 确定翻译后的亚细胞位置 促甲状腺激素(TSH)亚单位的加工 先驱物。将利用小鼠促甲状腺肿瘤作为来源 分泌活跃的促甲状腺激素；正常甲状腺和 切除甲状腺的小鼠将成为“休息”和 分别是“刺激”的促甲状腺激素细胞。肿瘤与脑下垂体 肉末将在体外与(~3H)-或(35S)蛋氨酸孵化， (3H)岩藻糖、(35S)硫酸盐、(3H)N-乙酰甘露糖胺或(14C)-或 (3H)甘露糖，然后均质并通过 我们以前使用的蔗糖阶梯度法 学习。富含粗面内质网的细胞组分 电子显微镜将对高尔基体进行表征。羧基 氰化物间氯苯肼(CCCP)和莫能菌素 用作亚细胞分馏的辅助物以作出推断 关于处理事件的亚细胞位置。标记TSH 亚基将从细胞匀浆、细胞 馏分和介质，并将用十二烷基硫酸钠- 聚丙烯酰胺凝胶电泳法。(3H)甘露糖标记 低聚糖将从TSH亚基中释放出来 内切糖苷酶H及其纸法结构测定 层析法。双标记胰酶糖肽将成为 用高效液相色谱法对其进行了表征。我们将了解到这三种组织是否 类型在亚细胞位置或处理的性质上不同 事件。我们将确定每个组织的每个隔间 键入α-β亚基结合的程度和动力学， 亚基的摩尔比和岩藻糖的亚基含量， 唾液酸和硫酸盐；糖肽分析将确定 络合物的岩藻糖化、唾液酸化或硫酸盐化程度 低聚糖，或高甘露糖结构，在每个 TSH的天冬酰胺糖基化位点。不同于对 对于糖蛋白的加工，我们建议研究一种 激素反应系统，在这个系统中，低聚糖 主要的分泌物种TSH被认为对 生物活性。我们的发现将在临床上与罕见的 下丘脑甲状腺功能减退症和垂体TSH- 产生肿瘤。首先，我们希望为更好的 了解促甲状腺激素生物合成的基本细胞生物学。

This application seeks to convert a NIRA (R23) Award to a FIRST Award (R29). The objective of the proposed research is to determine the subcellular locations of the post-translational processing of thyroid-stimulating hormone (TSH) subunit precursors. Mouse thyrotropic tumor will be utilized as a source of actively secreting thyrotrophs; pituitary tissue of euthyroid and thyroidectomized mice will be sources of "resting" and "stimulated" thyrotrophs, respectively. Tumor and pituitary minces will be incubated in vitro with (3H)-or (35S) methionine, (3H) fucose, (35S) sulfate, (3H) N-acetylmannosamine, or (14C)-or (3H) mannose, and then homogenized and fractionated by the sucrose step-gradient method that we have used for previous studies. Cell fractions enriched in rough endoplasmic reticulum and Golgi will be characterized by electron microscopy. Carboxyl cyanide m-chlorophenylhydrazone (CCCP) and monensin will be used as adjuncts to subcellular fractionation to make inferences about the subcellular location of processing events. Labeled TSH subunits will be immunoprecipitated from cell homogenates, cell fractions, and media, and will be characterized by SDS- polyacrylamide gel electrophoresis. (3H) mannose-labeled oligosaccharides will be released from TSH subunits by endoglycosidase H, and their structures determined by paper chromatography. Dual-labeled tryptic glycopeptides will be characterized by HPLC. We will learn whether the three tissue types differ in the subcellular locations or nature of processing events. We will determine for each compartment of each tissue type the extent and kinetics of alpha-beta subunit combination, the molar ratios of subunits, and the subunit content of fucose, sialic acid, and sulfate; glycopeptide analyses will determine the degree of fucosylation, sialylation, or sulfation of the complex oligosaccharides, or the high mannose structures, at each asparaginyl glycosylation site of TSH. Unlike studies of glycoprotein processing by others, we propose to study a hormonally-responsive system in which the oligosaccharides of a major secreted species, TSH, are believed to be important for biological activity. Our findings will be clinically relevant to rare patients with hypothalamic hypothyroidism and pituitary TSH- producing tumors. Primarily, we hope to contribute to a better understanding of the basic cell biology of TSH biosynthesis.