MEMBRANE TRAFFIC IN PEPTIDE HORMONE SECRETING CELLS

肽激素分泌细胞中的膜运输

• 批准号: 3232346
• 负责人: REGIS B KELLY
• 金额: $ 11.33万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-04-01 至 1991-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3232346
• 关键词: adrenocorticotropic hormone; antibody formation; antibody specificity; cell cell interaction; chemical structure function; chromosome deletion; cyclic AMP; endonuclease; gene expression; genetic manipulation; high performance liquid chromatography; immunofluorescence technique; immunoprecipitation; insulin; laboratory mouse; laboratory rabbit; laboratory rat; membrane permeability; membrane proteins; monoclonal antibody; mutagens; nucleic acid hybridization; pancreatic islet function; peptide hormone; peptide hormone biosynthesis; proinsulin; protein transport; proteolysis; radioimmunoassay; radiotracer; secretion; surface antigens

A pituitary tumor cell line (AcT-20) from the mouse packages mature ACTH into secretory vesicles but also leaks the longer precursor form, proopiomelanocortin (POMC) into the medium by a non-regulated or constitutive pathway. Human proinsulin, human growth hormone and rat trypsinogen, encoded by transfected DNA also take two pathways to the surface but other proteins such as the endogenous laminin or the truncated viral G protein, encoded by transfected DNA, only exit constitutively. Since the truncated viral G protein can be targeted to the secretory vesicle by fusing it with human growth hormone, we propose that peptide hormones, and trypsinogen, share a common sorting domain responsible for correct targeting to the secretory vesicle. To identify such domains we plan to delete by oligonucleotide mutagenesis candidate domains from rat trypsinogen, taking advantage of the known three-dimensional structure of rat trypsinogen and other proteases. To examine the properties of the constitutive pathway, DNA encoding immunoglobulin light and heavy chains will be transfected into AtT-20 cells. Since immunoglobulins must assemble before secretion, we hope to gain insight into rules of protein assembly that will facilitate the engineering of proteins capable of secretion by mammalian cells. The immediate goal is to link fragments of peptide hormone to immunoglobulin chains, and re-direct them to secretory vesicles. To allow us to begin a molecular explanation of protein secretion we have developed an AtT-20 variant into the cytoplasm of which macromolecules can be delivered with high efficiency using red blood cell fusion. Antibodies to vesicle and cytoskeletal components will be introduced to try to inhibit secretory vesicle formation, and vesicle movement to the cell surface. Evidence that secretory vesicles show a selective association with cytoskeletal elements will be sought. The ability to study secretory vesicle movement, secretory vesicle formation and exocytosis in a cell line that can be readily transfected with appropriate expression vectors, and whose cytoplasm can be readily modified by red blood cell microinjection gives an unusually rich opportunity to study the nature of protein secretion and its disorders.

来自小鼠的垂体肿瘤细胞系 (AcT-20) 包装成熟的 ACTH 进入分泌囊泡，但也泄漏出较长的前体形式， 阿黑皮质素原 (POMC) 通过非调节或 构成途径。 人胰岛素原、人生长激素和大鼠 由转染 DNA 编码的胰蛋白酶原也通过两条途径到达 表面但其他蛋白质，如内源性层粘连蛋白或截短的 由转染DNA编码的病毒G蛋白仅以组成型形式存在。 由于截短的病毒 G 蛋白可以靶向分泌 通过将其与人类生长激素融合来形成囊泡，我们提出肽 激素和胰蛋白酶原共享一个共同的排序域，负责 正确靶向分泌囊泡。 为了识别此类域，我们 计划通过寡核苷酸诱变从大鼠中删除候选结构域 胰蛋白酶原，利用已知的三维结构 大鼠胰蛋白酶原和其他蛋白酶。 要检查的属性 组成型途径，编码免疫球蛋白轻链和重链的 DNA 将转染至 AtT-20 细胞中。 由于免疫球蛋白必须组装 在分泌之前，我们希望深入了解蛋白质组装的规则 这将有助于工程化能够分泌的蛋白质 哺乳动物细胞。 当前的目标是连接肽片段 激素到免疫球蛋白链，并将它们重新定向到分泌囊泡。 为了让我们开始对蛋白质分泌进行分子解释，我们有 将AtT-20变体开发到细胞质中，其大分子可以 使用红细胞融合进行高效递送。 抗体 将引入囊泡和细胞骨架成分以试图抑制 分泌囊泡的形成和囊泡运动到细胞表面。 有证据表明分泌囊泡与 将寻找细胞骨架元素。 研究分泌的能力 细胞系中的囊泡运动、分泌囊泡形成和胞吐作用 可以很容易地用适当的表达载体转染，并且 其细胞质可以通过红细胞显微注射轻松修饰 为研究蛋白质的性质提供了异常丰富的机会 分泌及其紊乱。