Development and optimization of protocols to identify nuclear binding partners of RGS14 Público
Waters, Alaina (Spring 2023)
Abstract
Regulator of G protein signaling 14 (RGS14) is a multifunctional scaffolding protein that integrates GPCR-G, Ras/ERK, and Ca++/CaM signaling pathways in the brain linked to postsynaptic plasticity. At this point, there is significant research regarding the biochemical activity of RGS14 in the plasma membrane and cytosol of neurons, but nothing is known about the interactions of RGS14 within cell nuclei. Research has found that a subpopulation of native RGS14 regularly travels to the interior of the nuclei of neurons, which is unusual for a large scaffolding protein that is mainly involved in events occurring in the cell plasma membrane.
While there is significant shuttling of RGS14 in and out of the nucleus, the purpose of this is unknown as is any function for RGS14 within the nucleus.
To explore the function of RGS14 in the nucleus, we seek to identify nuclear-specific RGS14 interacting partners and determine what cellular processes these interacting partners are associated with. To achieve this goal, we will use TurboID, a proximity labeling system, to tag proteins that RGS14 interacts with, separating the cytosol of the cells from the nuclei, and comparing the tagged proteins. Additionally, wild-type and RGS14- L504R mutant mouse brains will be subjected to cellular fractionation and RGS14 immunoprecipitation to identify RGS-specific binding partners in each compartment.
At this time, hawse have successfully optimized protocols for cellular fractionation and biotin labeling of HEK293 cells, cellular fractionation of mouse hippocampi, and RGS14 immunoprecipitation from mouse brain hemispheres. To accomplish the goals of the study, successful collection of fractionated HEK293 cells that have been successfully transfected with plasmids expressing HA-Turbo, HA-Turbo-NLS, HA-Turbo-RGS14, and HA-Turbo-RGS14-L505R must be generated and/or whole mouse brain must be fractionated for the RGS14 immunoprecipitation. Once these goals are completed, samples taken will be sent for mass spectrometry and proteomic analysis with help from the Emory Proteomics core. This approach will provide insight into the binding partners of RGS14 in both the cytosol and nuclei, which may enhance our understanding of the compartment-specific function(s) of RGS14.
Table of Contents
Table of Contents
Chapter 1: Introduction........................................................................................................ 1Background of RGS14 and Relevant Prior Publications Goals of the Projects and Experimental Approach
Chapter 2: Methods............................................................................................................. 5Cell Line Management Generation of Constructs
Optimization of Cell Fractionation in HEK293 Cells Confirmation of Cell Fractionation through Western Blotting Confirmation of Construct Expression through Western Blotting Cell Transfection and Biotin Labeling
Confirmation of Construct Expression and Biotin Labeling through Western Blotting Optimization of Cell Fractionation in Mouse Brain Tissue
RGS14 Immunoprecipitation of Cytosolic and Nuclear Fractions of Mouse Brain Tissue Confirmation of RGS14 Immunoprecipitation through Western Blotting
Chapter 3: Results……………………………………………………………………..11Optimization of Cellular Fractionation in HEK293 Cells
Confirmation of Recombinant Turbo and Turbo RGS14 Construct Expression after Cellular Fractionation
Confirmation of Construct Expression and Biotin Labeling Optimization of Cell Fractionation in Mouse Brain Tissue Confirmation of RGS14 Immunoprecipitation
Chapter 4: Discussion………………………………………………………………26
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Development and optimization of protocols to identify nuclear binding partners of RGS14 () | 2023-04-19 16:58:29 -0400 |
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