Date of Award

1-1-2011

Language

English

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

College/School/Department

Department of Biomedical Sciences

Content Description

1 online resource (iv, 119 pages) : illustrations (some color)

Dissertation/Thesis Chair

James A. Dias

Committee Members

Joseph Mazurkiewicz, Brian D. Cohen, Christina T. Egan, Douglas Conklin, William Lee

Keywords

Casein Kinase 2, CK2, Follicle Stimulating Hormone Receptor, FSHR, Steroid, Follicle-stimulating hormone, Luteinizing hormone releasing hormone, Protein kinase CK2

Subject Categories

Endocrinology | Molecular Biology

Abstract

Human reproduction in the female is a complex process requiring coordinated control of gonadotropic hormone signaling in a temporal, spatial and contextually defined manner. Paracrine and autocrine signaling between granulosa cells and oocytes of the developing ovarian follicle result in production of steroids, inhibin and growth factors that dictate maturation, ovulation or atresia. Follicular stimulating hormone (FSH) stimulation of the FSH receptor (FSHR), a G-protein coupled receptor (GPCR) is a key regulator of these processes. Recent developments in GPCR molecular and structural biology have helped define activation and signaling mechanisms that will aid in further defining the etiology of infertility and contribute to more effective treatment with fewer complications. We have analyzed the role of protein kinase CK2 in FSH mediated FSHR action using two different approaches: through genetic modification of the CK2 consensus sequence on hFSHR third loop and by inhibiting the kinase activity of CK2 in partially differentiated granulosa cells from DES treated rats and undifferentiated granulosa cells from untreated rats. The α-subunit of CK2 was shown to associate with the human FSHR in co-immunoprecipitation experiments indicating direct or indirect interaction. Mutation of the aspartic acid of the consensus sequence (hFSHR-D550A) resulted in increased intracellular accumulation of hFSH compared to WT-hFSHR and lower levels of cAMP production after stimulation in transfected HEK-293T cells. This mutation did not, however, affect CK2α/hFSHR association. When the kinase activity of CK2 was inhibited in undifferentiated and partially differentiated granulosa cells, differential effects on estradiol, progesterone and cAMP were discovered between distinct granulosa cell maturation stages. CK2 maintains low progesterone levels in undifferentiated, but not in partially differentiated granulosa cells. Secreted cAMP levels were reduced and intracellular cAMP increased in the presence of CK2 inhibitor in both granulosa cell stages. Conversely, CK2 inhibition resulted in increased total cAMP levels, but only in undifferentiated granulosa cells. The experiments provide evidence that the CK2 consensus sequence may function in FSH-mediated FSHR signaling and post endocytic regulation. These studies have uncovered a novel function for CK2 kinase activity in FSH-mediated regulation of steroidogenesis in a context specific manner whereby a role for CK2 in preventing premature ovulation is indicated.

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