SUBCELLULAR LOCALIZATION OF TSH PROCESSING

TSH 加工的亚细胞定位

• 批准号: 3462842
• 负责人: JAMES MAGNER
• 金额: $ 8.83万
• 依托单位: MICHAEL REESE HOSP & MED CTR (CHICAGO)
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-07-20 至 1990-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3462842
• 关键词: Golgi apparatus; cell components; electron microscopy; endoplasmic reticulum; fucose; gel electrophoresis; glycopeptides; high performance liquid chromatography; hypothyroidism; laboratory mouse; mannose; oligosaccharides; paper chromatography; peptide hormone biosynthesis; pituitary gland; pituitary neoplasms; sialate; sulfation; thyroid neoplasm; thyroidectomy; thyrotropin; thyrotropin releasing hormone

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.

此申请旨在将 NIRA (R23) 奖转换为 FIRST 奖（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 (TSH) 亚基的加工 前体。 小鼠促甲状腺肿瘤将被用作来源 积极分泌促甲状腺素；甲状腺功能正常的垂体组织和 甲状腺切除小鼠将成为“休息”和 分别“刺激”促甲状腺素。 肿瘤和垂体 肉末将在体外与 (3H)-或 (35S) 蛋氨酸一起孵育， (3H)岩藻糖、(35S)硫酸盐、(3H)N-乙酰甘露糖胺、或(14C)-或 (3H) 甘露糖，然后均质化并通过 我们之前使用过的蔗糖阶梯梯度法 研究。 富含粗面内质网的细胞组分 和高尔基体将通过电子显微镜进行表征。 羧基 氰化物间氯苯腙 (CCCP) 和莫能菌素 用作亚细胞分离的辅助物以进行推论 关于处理事件的亚细胞位置。 标记促甲状腺激素 亚单位将从细胞匀浆、细胞 级分和培养基，并将通过 SDS- 进行表征 聚丙烯酰胺凝胶电泳。 (3H) 甘露糖标记 寡糖将从 TSH 亚基中释放出来 糖苷内切酶 H 及其纸质结构测定 色谱法。 双标记胰蛋白酶糖肽将 通过HPLC表征。 我们将了解这三种组织是否 类型在亚细胞位置或处理性质上有所不同 事件。 我们将确定每个组织的每个隔室 输入 α-β 亚基组合的程度和动力学， 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 复合物的岩藻糖基化、唾液酸化或硫酸化程度 寡糖，或高甘露糖结构，在每个 TSH 的天冬酰胺酰化位点。 与研究不同的是 其他人的糖蛋白加工，我们建议研究 激素响应系统，其中寡糖 主要分泌物质 TSH 被认为对于 生物活性。 我们的研究结果将与罕见的临床相关 下丘脑甲状腺功能减退症和垂体 TSH- 患者 产生肿瘤。 首先，我们希望为更好的 了解 TSH 生物合成的基本细胞生物学。