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 glyc...