Mechanisms of Hormonal Release by Endocrine Cells

内分泌细胞释放激素的机制

• 批准号: 7057284
• 负责人: GIULIA BALDINI
• 金额: $ 21.18万
• 依托单位: UNIV OF ARKANSAS FOR MED SCIS
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-04-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7057284
• 关键词: PC12 cells; binding proteins; calcium binding protein; cell membrane; density gradient ultracentrifugation; endocrine gland /system; exocytosis; fluorescence microscopy; granule; hormone biosynthesis; hormone regulation /control mechanism; immunoelectron microscopy; immunoprecipitation; membrane proteins; peptide hormone biosynthesis; protein protein interaction; synaptotagmin; tissue /cell culture; transfection; yeast two hybrid system

DESCRIPTION (provided by the applicant): The long-term objective of the proposal is to further elucidate the molecular mechanism of regulated hormone release. Hormone release requires sequential steps of granule positioning at the cell cortex, docking to the plasma membrane, priming and fusion. Although many key components of this process have been identified, progress has been hindered because there have been no available in vitro assays that can measure docking of granules to the plasma membrane as a separate event. We have designed a novel in vitro assay that measures docking of granules to the plasma membrane. As one of the first results of the in vitro assay, it was found that docking is stimulated by Ca2+ and requires an interaction between SNAP-25 and Synaptotagmin 1. It is also known that other Synaptotagmins function in hormone release. We propose to determine, by using the in vitro assay in combination with immunoprecipitation experiments, whether the interaction of multiple Synaptotagmins with SNAP-25 is necessary for granule docking. SNAP-25 is expressed in neurons and endocrine cells, while Syndet/SNAP-23 is ubiquitous. Both SNAP-25 and Syndet/SNAP-23 are predominantly localized at the plasma membrane. The molecular mechanisms underlying differences in docking of granules and other vesicles of the regulated and constitutive pathway to the plasma membrane are unknown. By using the in vitro assay, we will determine the specifics of SNAP-25 and Sydet/SNAP-23 dependent docking. We will also study whether low molecular weight GTPases, or Rab3 proteins, and the putative Rab3 protein effectors, Rabphilin and Noc2, control the docking step. Furthermore, we will test the hypothesis that the Rab3-Rabphilin functions to position granules within the cortical actin by using immunofluorescence and electron microscopy analysis of transfected PC 12 cells. The molecular events involved in the recruitment of dense core granules near the cell surface are largely unknown. This study of hormone release will help elucidate possible defects underlying endocrine diseases such as Diabetes Mellitus and Cushing syndrome.

描述（由申请人提供）：该提案的长期目标是进一步阐明受调节激素释放的分子机制。激素释放需要在细胞皮质上定位的顺序步骤，该颗粒位于质膜，启动和融合。尽管已经确定了此过程的许多关键组成部分，但由于没有可用的体外测定法可以测量颗粒对质膜的离子对接，但进展受到阻碍。我们设计了一种新型的体外测定法，该测定法将颗粒对接到质膜。作为体外测定的第一个结果之一，发现对接是由Ca2+刺激的，并且需要SNAP-25和SynaptotoDagmin 1之间的相互作用。也众所周知，激素释放中的其他突触型函数。我们建议，通过将体外测定与免疫沉淀实验结合使用，是否需要多种突触量25与SNAP-25的相互作用。 SNAP-25在神经元和内分泌细胞中表达，而syndet/snap-23无处不在。 SNAP-25和SYNDET/SNAP-23均主要位于质膜。颗粒和其他囊泡的分子机制和质膜构成途径的其他囊泡的分子机制是未知的。通过使用体外测定，我们将确定SNAP-25和SYDET/SNAP-23依赖对接的细节。我们还将研究低分子量GTPase或RAB3蛋白，以及推定的RAB3蛋白效应子Rabphilin和Noc2是否控制对接步骤。此外，我们将通过使用免疫荧光和对转染的PC 12细胞的电子显微镜分析来检验Rab3- rabphilin的作用在皮质肌动蛋白中定位的假设。在细胞表面附近募集密集核颗粒的分子事件在很大程度上未知。这项对激素释放的研究将有助于阐明内分泌疾病（例如糖尿病和库欣综合征）的潜在缺陷。

项目成果

Role of the cysteine-rich domain of the t-SNARE component, SYNDET, in membrane binding and subcellular localization.

t-SNARE 成分 SYNDET 的富含半胱氨酸结构域在膜结合和亚细胞定位中的作用。

• DOI: 10.1074/jbc.274.13.9053
• 发表时间: 1999
• 期刊: The Journal of biological chemistry
• 作者: Koticha,DK;Huddleston,SJ;Witkin,JW;Baldini,G
• 通讯作者: Baldini,G

Rabphilin localizes with the cell actin cytoskeleton and stimulates association of granules with F-actin cross-linked by {alpha}-actinin.

Rabphilin 定位于细胞肌动蛋白细胞骨架，并刺激颗粒与通过 {α}-肌动蛋白交联的 F-肌动蛋白结合。

• DOI: 10.1074/jbc.m502695200
• 发表时间: 2005
• 期刊: The Journal of biological chemistry
• 作者: Baldini,Giovanna;Martelli,AlbertoM;Tabellini,Giovanna;Horn,Chad;Machaca,Khaled;Narducci,Paola;Baldini,Giulia
• 通讯作者: Baldini,Giulia

Localization of the small monomeric GTPases Rab3D and Rab3A in the AtT-20 rat pituitary cell line.

小单体 GTP 酶 Rab3D 和 Rab3A 在 AtT-20 大鼠垂体细胞系中的定位。

• DOI: 10.4081/1643
• 发表时间: 2001
• 期刊: European journal of histochemistry : EJH
• 作者: Tabellini,G;Baldini,G;Baldini,G;Bortul,R;Bareggi,R;Narducci,P;Martelli,AM
• 通讯作者: Martelli,AM