Emily Armlin

Human Follitropin Receptor Lipid Raft Residency is Mediated by its Caveolin Binding Motif

The follicle stimulating hormone (FSH) is a necessary hormone involved in reproductive pathways in both males and females. Previous work in our lab has shown that its receptor (FSHR) contains a caveolin binding motif (CBM) which we believe is responsible for localization of the receptor to lipid raft domains of the membrane and for proper receptor signaling. Mutations were introduced to this motif to provide a deeper understanding of this interaction and identify the importance of specific sites within the CBM in FSHR. Discontinuous sucrose gradients were used to separate lipid raft from non-lipid raft portions of the membrane and western blotting was used to visualize the localization of the FSHR within the gradient. We found that disruption of the CBM of FSHR resulted in altered localization of the receptor relative to the wild type FSHR. Receptors with mutations F497L, F481L, and F486L, as well as F497L, F481L, F486L, and F489L were found to localize only to non-raft portions of the membrane, while receptors with mutations F481L, F486L, and F489L were found in both lipid raft and non-raft portions of the membrane. From this we concluded that F479 may play a more significant role in lipid raft localization and signaling. These findings are consistent with previous research within the lab, which show higher levels of basal signaling in CBM mutants compared to the wild type. The results of this study suggest that the CBM is important in proper signaling of FSH, and that certain sites within the CBM may be more important in this interaction than others. These findings are important in understanding FSHR function which could lead to the development of novel treatments for infertility.

Ella Harper-Schiehl

Investigating Genetic Underpinnings of Depression
Current estimates of worldwide depression incidence are over 280 million people, with women and those over 50 at even greater risk. Research has found numerous potential genetic links, including those within the hypothalamic-pituitary-adrenal (HPA) axis, specifically with relation to glucocorticoids, and within the immune system, particularly as it relates to cytokine production and function. Based on this, we have hypothesized that single nucleotide polymorphisms (SNPs) in glucocorticoid receptors (GR) and corticotropin releasing hormone receptor 1 (CRHR1), as well as interleukin 1 (IL-1B) and cyclooxygenase 2 (COX-2) may predict incidence of depression. Our study investigated the genotypic frequency of several SNPs related to both HPA axis and immune system function. Buccal swabs provided DNA and were acquired from both patients being treated from psychiatric illness, and from a control population. Extracted DNA was analyzed using a combination of allele-specific polymerase chain reaction (asPCR), as well as quantitative polymerase chain reaction (qPCR) to determine allelic frequency associated with each SNP. Participants also completed the Center for Epidemiologic Studies Depression Scale (CES-D) and the Mini Mood and Anxiety Symptom Questionnaire (Mini-MASQ). Participant scores on these measurements spanned the range of potential scores, including non-symptomatic, sub-clinical, and clinical. Understanding the connection between depression and genotypic variation can help shape diagnosis and treatment in the future, with the goal of improving patient outcomes.

Isabel Lubin

Effect of lipid raft disrupting agents on FSHR-dependent cAMP production

Infertility affects millions of individuals worldwide and the causes of infertility are often unexplained. There are a number of signaling pathways that are involved in reproduction and defects in these pathways could contribute to infertility. Specifically, we are interested in studying how FSHR (follicle stimulating hormone receptor) responds to human follicle stimulating hormone (hFSH) because having the ability to enhance or downregulate its signaling has implications for fertility treatments. When FSH binds to its cognate G-protein-coupled receptor (GPCR) it activates the production of cAMP as a second messenger. Previous work in our lab has demonstrated that FSHR needs to be embedded in a specialized microdomain of the cellular membrane called a lipid raft to mediate this response. After treating FSHR-expressing cells with lipid raft-disrupting drugs, decreased cAMP was observed as a decrease in activated (phosphorylated) cAMP response-element binding protein (pCREB) within the Gα/cAMP/PKA pathway. The amount of pCREB is an indirect measurement of cAMP production. To establish a direct relationship between FSHR signaling and cAMP production, a quantitative luciferase assay was developed. Luciferase is an enzyme found in fireflies that allows them and other bioluminescent organisms to glow. A cell line expressing hFSHR (HEK293-hFSHR) was transiently transfected with a plasmid to express luciferase under the control of multiple, tandem cAMP response elements. This acts as a quantitative reporter for cAMP because it produces more luminescence as the amount of cAMP in the cells increases. Cells were treated with Methyl-β-cyclodextrin (MβCD) which acts as a lipid raft-disrupting agent by depleting cholesterol from cells and destroying lipid rafts. All cells were stimulated with various amounts of FSH to create a dose-response curve. MβCD-treated cells produced less cAMP in a dose-dependent manner compared to the untreated cells. These results differ from previous observations of the FSH-induced activation of p44/42 MAP kinase (ERK1/2). It was found that treatment with the lipid raft disrupting agents resulted in increased basal cAMP production (as measured by pCREB activation). Future studies will test different lipid raft-disrupting agents to further investigate the role of lipid rafts in FSH induced cAMP production from FSHR stimulation. Studying and characterizing FSHR behavior is essential to furthering our understanding of infertility and improving the possible treatments for both men and women who are struggling to conceive.