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

项目摘要/摘要--项目3(JUNAS) 该项目的目标是了解卵泡刺激素(FSH)随年龄的变化。 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(Bousfield)报告了FSH糖基化模式随年龄的变化；部分减少 糖基化卵泡刺激素(FSH18/21)和完全糖基化卵泡刺激素(FSH24)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，FSH18/21显示更快的结合 并且比FSH24更有效地激活信号。然而，衰老和卵泡发生是如何发生的仍不清楚。 细胞微环境的依赖性变化影响FSH糖形式依赖性FSHR的调节， 这个项目将解决这个问题。调节G蛋白偶联受体的两种重要机制 功能是GPCR二聚体和寡聚体的形成，以及受体的转运。我们最近出版的 研究表明，FSH糖型对FSHR寡聚有不同的调节作用，对cAMP有影响 发信号。此外，我们的初步数据表明，FSH糖形式可能对FSHR有不同的控制作用 内吞作用。因此，这个项目的首要目标是了解FSH糖类是如何调节的 在卵泡发生和衰老过程中，FSHR在初级颗粒细胞中的寡聚和运输。这个 工作假说是FSH糖型特异性调节FSHR寡聚导致不同的 贩运和信令签名，具有明确的功能后果。具体目标是：1. FSH糖型对幼年和幼鼠颗粒细胞FSHR复合体的特异性调节作用 繁殖能力较强的小鼠。我们的工作假设是FSH对FSHR的糖形调节 同源异构体和信号输出将受到年龄和卵泡发育阶段的不同调控。我们 将利用N端标记的FLAG-FSHR敲入小鼠来检验这一假设。2.确定角色 内体FSHR在指导FSH糖形式依赖的功能选择性中的作用。我们的工作 假设FSH糖形式依赖的FSHR内化引导信号中的功能选择性 输出和接头蛋白结合。我们将利用原代颗粒细胞和多色荧光共聚焦 和TIRF显微镜确定FSH糖型和衰老调节的FSHR的时空性质 贩卖人口。3.了解FSH糖型如何通过调节和调节FSHR相互作用 适配蛋白。我们的工作假设是卵泡刺激素糖体通过 信号机制和调节蛋白的差异性招募。FSHR下拉策略和LC- MS/MS将确定FSH糖形式依赖的FSHR相互作用组。FSHR的互动功能 蛋白质将通过siRNA/过度表达来评估，并确定年龄相关的调节。该项目 结果将阐明FSH糖型的年龄依赖性调节如何调节FSHR功能，以 突出治疗进展的新的调节战略，以改善妇女的健康和福祉。

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.