Project 3: Understanding the Role of FSHR Oligomerization and Trafficking in Transducing Age-dependent Changes in FSH Glycoforms

项目概要/摘要-项目3（乔纳斯） 该项目的目标是了解促卵泡激素（FSH）的年龄依赖性变化 糖基化是由其G蛋白偶联受体FSH受体（FSHR）转导的。按项目分类的研究 4例（Bousfield）报告了FSH糖基化模式的年龄依赖性变化; 糖基化FSH（FSH 18/21）和完全糖基化FSH（FSH 24）随年龄增长而增加。这些 FSH糖基化修饰具有显著的功能影响，FSH 18/21显示更快的结合 并且比FSH24更有效的信号激活。然而，仍然不清楚衰老和卵泡生成- 细胞微环境的依赖性变化影响FSH糖型依赖性FSHR调节， 这将通过这个项目来解决。调节G蛋白偶联受体的两种重要机制 功能是形成GPCR二聚体和寡聚体以及受体运输。我们最近出版的 研究表明，FSH糖型对FSHR寡聚化有不同的调节作用， 发信号。此外，我们的初步数据表明，FSH糖型可能差异控制FSHR 内吞作用因此，本项目的首要目标是了解FSH糖型如何调节 卵泡发生和衰老过程中初级颗粒细胞中FSHR的寡聚化和运输的 工作假设是FSH糖型特异性调节FSHR寡聚化导致不同的 交易和信号签名，具有定义的功能后果。具体目标是：1。 评估FSH糖型特异性调节年轻人和成年人颗粒细胞中FSHR复合物的作用 繁殖能力很强的老鼠我们的工作假设是FSH糖型调节FSHR 同聚体和信号输出将受到年龄和卵泡发育阶段的不同调节。我们 将在小鼠中利用N末端标记的FLAG-FSHR敲除来测试该假设。2.确定角色 内体FSHR在指导FSH糖型依赖性功能选择性中的作用。我们的工作 一种假说是FSH糖型依赖性FSHR内化指导信号传导的功能选择性 输出和衔接蛋白结合。我们将利用原代颗粒细胞和荧光共聚焦显微镜， 和TIRF显微镜，以确定FSH糖型和衰老调节的FSHR的时空性质 贩卖人口3.了解FSH糖型如何调节FSHR与调节性和 衔接蛋白我们的工作假设是FSH糖型通过以下途径驱动功能选择性： 信号机制和调节蛋白的差异募集。FSHR下拉策略和LC- MS/MS将确定FSH糖型依赖性FSHR相互作用组。FSHR的功能-相互作用 通过siRNA/过表达评估蛋白质，并确定年龄依赖性调节。项目 结果将阐明FSH糖型的年龄依赖性调节如何调节FSHR功能， 突出了改善妇女健康和福祉的治疗进展的新型调节策略。

Project Summary/Abstract – Project 3(Jonas) The goal of this project is to understand how age-dependent changes in follicle stimulating hormone (FSH) glycosylation are transduced by its G protein-coupled receptor, FSH receptor (FSHR). Studies by Project 4(Bousfield) reported age-dependent changes in the glycosylation pattern of FSH; with a reduction in partially glycosylated FSH (FSH18/21) and an increase in fully glycosylated FSH (FSH24) observed with aging. These modifications in FSH glycosylation have significant functional impact, with FSH18/21 displaying faster binding and more potent signal activation than FSH24. However, it remains unknown how aging and folliculogenesis- dependent changes in the cellular microenvironment impacts FSH glycoform-dependent FSHR modulation, which will be addressed by this project. Two important mechanisms that modulate G protein-coupled receptor function is the formation of GPCR dimers and oligomers, and receptor trafficking. Our recently published studies suggest that FSH glycoforms differentially modulate FSHR oligomerization, with impact on cAMP signaling. Moreover, our pilot data suggests that FSH glycoforms may differentially control FSHR endocytosis. The overarching aim of this project is therefore to understand how FSH glycoforms regulate FSHR oligomerization and trafficking in primary granulosa cells, during folliculogenesis and aging. The working hypothesis is that FSH glycoform-specific modulation of FSHR oligomerization results in distinct trafficking and signaling signatures, with defined functional consequences. The Specific Aims are: 1. Assessing FSH glycoform-specific regulation of FSHR complexes in granulosa cells of young and reproductively advanced mice. Our working hypothesis is that FSH glycoform modulation of FSHR homomers and signal output will be differentially regulated by aging and stage of follicle development. We will utilize an N terminal tagged FLAG-FSHR knock in mouse to test this hypothesis. 2. Determining the role of endosomal FSHR in directing FSH glycoform-dependent functional selectivity. Our working hypothesis is that FSH glycoform-dependent FSHR internalization directs functional selectivity in signal output and adapter protein binding. We will utilize primary granulosa cells and multicolor fluorescent confocal and TIRF microscopy to determine the spatial-temporal nature of FSH glycoform and aging modulated FSHR trafficking. 3. Understanding how FSH glycoforms modulate FSHR interactions with modulatory and adapter proteins. Our working hypothesis is that FSH glycoforms drive functional selectivity through differential recruitment of signaling machinery and modulatory proteins. A FSHR pulldown strategy and LC- MS/MS will determine the FSH glycoform-dependent FSHR interactome. The function of FSHR- interacting proteins will be assessed by siRNA/over-expression, and age-dependent modulation determined. The project outcomes will elucidate how age-dependent regulation of FSH glycoforms modulates FSHR functions, to highlight novel modulatory strategies for therapeutic advances to improve women’s health and wellbeing.