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Browsing by Author "Ashley, Ryan L., author"

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    Identification and characterization of two ovine membrane receptors for progesterone
    (Colorado State University. Libraries, 2007) Ashley, Ryan L., author; Nett, Terry M., advisor
    Classically, progesterone (P4) has been thought to act only through the well-known genomic pathway involving hormone binding to nuclear receptors (PR) and subsequent modulation of gene expression. Alternatively, there is increasing evidence for rapid, nongenomic effects of P4 in a variety of mammalian tissues; however the receptor responsible for these actions is yet to be characterized. The likelihood of a membrane P4 receptor (mPR) causing these events is quite plausible. Sheep are the experimental model often used in our laboratory, because regulation of reproductive function is very similar in ewes and women. Nongenomic actions of P4 have been described in sheep, but the receptor(s) responsible has not been identified. The objective of this dissertation was to isolate and characterize an ovine mPR distinct from the nuclear PR. A membrane protein found to bind P4 was first isolated from porcine liver and is now known as P4 receptor membrane component 1 (PGRMC1). Since PGRMC1 is expressed in a variety of species and tissues, I hypothesized that PGRMC1 was the protein responsible for the nongenomic effects of P4 in sheep. As such, the purpose of my initial studies was to determine if PGRMC1 was expressed in the sheep and further verify if PGRMC1 was present in the plasma membrane of the ovine corpus luteum (CL). A protein lacking homology to the nPRs but homologous to other reported PGRMC1 proteins was isolated from the sheep and contains a single transmembrane domain at the N-terminus. Despite the transmembrane domain, the ovine PGRMC1 displays predominant localization in the endoplasmic reticulum. During my initial studies, another putative mPR was cloned from the seatrout that displayed seven transmembrane domains, indicative of a G-protein-coupled receptor (GPCR) and upon ligand activation, also caused rapid induction of second messenger pathways. As such, I wanted to determine if this mPR was also present in the sheep. An ovine mPR distinct from the nuclear PR was isolated and characterized. The ovine mPR is a 350 amino acid protein that, based on predicted structural analysis, possesses seven transmembrane domains and is expressed in the hypothalamus, pituitary, uterus, ovary and CL. The first characterization of mPR expression throughout the ovine estrous cycle in the hypothalamus, pituitary, uterus, ovary, and CL is also reported. In CHO cells that overexpress a mPR-GFP fusion protein the ovine mPR was uniquely localized to the endoplasmic reticulum and not the plasma membrane. The ovine mPR specifically binds only progestins. Progesterone, 20α-hydroxyprogesterone and 17α-hydroxyprogesterone significantly (P < 0.001) displaced binding of 3H-P4 to membrane fractions from CHO cells expressing ovine mPR. Additionally, the ovine mPR directly induces Ca2+ release from the endoplasmic reticulum upon ligand activation. Further, the ovine mPR appears to activate the MAPK pathway, specifically by phosphorylation of JNK1/2 upon ligand activation. This novel method of signaling at the endoplasmic reticulum adds to the intricacy of signaling in cells and provides a mechanistically unique method for initiating actions of P4 that may alter classical dogma regarding the mechanisms by which steroid hormones act.