SUBCELLULAR LOCALIZATION OF TSH PROCESSING

拟议的研究的目标是确定亚细胞 促甲状腺激素翻译后加工的定位 激素(TSH)亚单位前体。将利用小鼠促甲状腺肿瘤 作为活跃分泌促甲状腺激素的来源；脑下垂体组织 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。肿瘤和垂体腺组织切片 将与[S-35]蛋氨酸、[H-3]岩藻糖、[S-35]体外孵育 硫酸盐、[H-3]N-乙酰甘露糖胺或[H-3]甘露糖，然后均质 用我们用过的蔗糖梯度法进行分馏 以前的研究。富含粗面内质网的细胞组分， 近端高尔基体、远端高尔基体和分泌颗粒的特征 通过电子显微镜，并分析“细胞器特有的”酶，如 半乳糖基转移酶。标记前驱体的顺序运动 将分析在脉冲追逐研究期间这些隔室之间的距离。 将使用羧基氰化物、间氯苯肼(CCCP)和莫能菌素 作为亚细胞分馏的辅助成分，以推断 处理事件的亚蜂窝位置。标记的TSH亚基将是 免疫沉淀来自细胞匀浆、细胞部分和培养液，以及 用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法进行鉴定。[H-3] 甘露糖标记的低聚糖将从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。我们将确定这三种组织类型是否在 新合成的TSH亚基在亚细胞间的分布 间隔、处理事件的位置或动力学，或 亚基在隔室之间的移位。我们将为每个人确定 每种组织类型的间隔α-β的范围和动力学 亚基组合、亚基的摩尔比、亚基含量 某些残基(岩藻糖、唾液酸和硫酸盐)，以及 亚基的高甘露糖低聚糖。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，其中主要的低聚糖 分泌性物种TSH被认为对生物学意义重大。 活动。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促甲状腺激素生成性肿瘤。首先， 我们希望为更好地了解基本的细胞生物学做出贡献 促甲状腺激素的生物合成。

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 tissue minces will be incubated in vitro with [S-35] methionine, [H-3] fucose, [S-35] sulfate, [H-3]N- acetylmannosamine, or [H-3] 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, proximal Golgi, distal Golgi, and secretory granules will be characterized by electron microscopy, and assayed for "organelle-specific" enzymes, such as galactosyltransferase. The sequential movement of labeled precursors between these compartments during pulse-chase studies will be analyzed. 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. [H-3] mannose-labeled oligosaccharides will be released from TSH subunits by endoglycosidase H, and their structures determined by paper chromatography. We will determine whether the three tissue types differ in their distribution of newly synthesized TSH subunits among subcellular compartments, the locations or kinetics of processing events, or the translocation of subunits between compartments. We will determine for each compartment of each tissue type the extent and kinetics of alpha-beta subunit combination, the molar ratios of subunits, the subunit content of certain residues (fucose, sialic acid, and sulfate), and the structures of the high mannose oligosaccharides of subunits. 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.