SUBCELLULAR LOCALIZATION OF TSH PROCESSING

此申请旨在将NIRA（R23）奖转换为FIRST 奖励（R29）。 拟议研究的目的是 确定翻译后的亚细胞位置， 促甲状腺激素亚单位加工 前体 小鼠促甲状腺肿瘤将用作来源 甲状腺功能正常的垂体组织， 甲状腺切除的小鼠将是“休息”的来源， “刺激的”促甲状腺激素细胞。 肿瘤与垂体 将切碎的肉与（3 H）-或（35 S）甲硫氨酸在体外孵育， (3H)岩藻糖、（35 S）硫酸盐、（3 H）N-乙酰甘露糖胺或（14 C）-或 (3H)甘露糖，然后均质化和分级， 蔗糖逐步梯度法，我们已经使用了以前的 问题研究 粗面内质网富集的细胞组分 和高尔基体将通过电子显微镜来表征。 羧基 氰化物间氯苯腙（CCCP）和莫能菌素将被 用作亚细胞分离的鉴定，以进行推断 关于处理事件的亚细胞位置。 标记TSH 亚基将从细胞匀浆中免疫沉淀， 组分和培养基，并将通过SDS- 聚丙烯酰胺凝胶电泳 (3H)甘露糖标记的 低聚糖将从TSH亚单位释放， 糖苷内切酶H，并通过纸确定其结构 层析 双标记胰蛋白酶糖肽将被 通过HPLC表征。 我们将了解这三种组织 不同的类型在亚细胞位置或处理的性质 事件 我们将确定每种组织的每个隔室 类型的α-β亚基组合的程度和动力学， 亚基的摩尔比，以及岩藻糖的亚基含量， 唾液酸和硫酸盐;糖肽分析将确定 复合物的岩藻糖基化、唾液酸化或硫酸化程度 低聚糖，或高甘露糖结构，在每个 TSH的天冬酰胺基糖基化位点。 与研究不同， 糖蛋白加工的其他人，我们建议研究一个 免疫应答系统，其中 主要分泌的物质，TSH，被认为是重要的 生物活性 我们的发现将与罕见的 患有下丘脑甲状腺功能减退症和垂体TSH- 产生肿瘤。 首先，我们希望能为更好的 了解TSH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.