Date of Award

1-1-2016

Document Type

Masters Thesis

Degree Name

M.S.

First Advisor

Joseph K. Angleson, Ph.D.

Second Advisor

Scott Barbee

Third Advisor

Nancy Lorenzon

Keywords

AKAP150, Anterior pituitary cells, Fluorescence recovery after photobleaching, FRAP, Gonadotropes, LβT2 cells

Abstract

Cellular communication occurs as a result of changes in signaling pathways. A well-studied signaling pathway is through G protein coupled receptors (GPCRs). In gonadotropes, GPCR stimulation by GnRH leads to the activation of protein kinase A (PKA). Activated PKA can phosphorylate ion channels, potentially causing an influx of calcium, depolarization and secretion of hormones. A scaffolding protein known as AKAP150 anchors PKA near L-type calcium channels. In addition, AKAP150 anchors phosphatases, which provides temporal control during signaling events. It was recently shown that AKAP150 is mobile in neuronal dendrites, providing regulation to where the signaling cascade occurs in the cell. It is not yet known what AKAP150 binds to in gonadotropes or how it translocates. In order to investigate the mobility of this protein in pituitary cells, a variety of methods were used in this study. Time-lapse imaging and permeabilization assays suggested that AKAP150-GFP was mobile in LbetaT2 cells, but more sensitive methods were needed to study temporal and spatial resolution. Quantitative FRAP and FLIP analysis provided strong qualitative evidence that AKAP150-GFP was mobile in LbetaT2 cells and the protein moved laterally along the cell rim. Lastly, comparing rim intensity in LbetaT2 cells immunostained for endogenous AKAP150 and actin indicated the scaffolding protein could cluster with to actin.

Publication Statement

Copyright is held by the author. User is responsible for all copyright compliance.

Rights Holder

Kristen E. Dew

Provenance

Received from ProQuest

File Format

application/pdf

Language

en

File Size

83 p.

Discipline

Cellular Biology, Molecular Biology

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