A Comprehensive Analysis of the RGS14 Interactome in the Context of Area CA2 of the Hippocampus translation missing: zh.hyrax.visibility.files_restricted.text

Gerber, Kyle (Spring 2019)

Permanent URL: https://etd.library.emory.edu/concern/etds/jw827c60c?locale=zh
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Abstract

Regulator of G protein signaling (RGS) proteins are key modulators of heterotrimeric G protein signaling downstream of G protein coupled receptors throughout the body. Many of these RGS proteins, including RGS2, RGS4, RGS7, RGS9, and RGS14, modulate synaptic signaling throughout the brain. RGS14 in particular, modulates synaptic plasticity in the CA2 region of the hippocampus. The hippocampus as a whole is essential for learning and memory but area CA2 has been largely ignored until recently due to its small size and lack of plasticity compared to the neighboring CA1 region. Many recent studies have taken advantage of proteins expressed specifically in CA2, including RGS14 and amigo-2, to identify and examine this enigmatic region of the brain. Using targeted dissections with the aid of enhanced green fluorescent protein (eGFP) expressed under the amigo-2 promoter, we were able to perform a proteomic analysis of area CA2 and CA1 to reveal a proteomic profile for CA2 (vs. CA1), identifying a wealth of potential targets for future studies that will allow us to understand the physiological and functional differences between area CA2 and CA1.

           One of the many proteins we found to be highly expressed in area CA2 was RGS14. RGS14 naturally suppresses long-term potentiation (LTP), a cellular correlate of learning and memory, in area CA2. The mechanism by which RGS14 suppresses this form of synaptic plasticity is unknown. By immunoprecipitating RGS14 out of mouse brain and analyzing all interacting proteins through mass spectrometry analysis, we revealed an RGS14 interactome containing functional interactors with RGS14 that will allow us to elucidate the mechanism by which RGS14 suppresses LTP. The top interactors with RGS14 were myosins and other actin-binding proteins that facilitate transport of cargo throughout the cell as well as control the structure and function of dendritic spines, which are essential for the proper expression of LTP. By combining our RGS14 interactome data with our CA2 proteome, we were able to identify myosin IIA and drebrin 1 as likely interactors with RGS14 that merit further study. Our interactome also showed that RGS14 interacts with 14-3-3 proteins, ubiquitously expressed proteins that typically bind phosphorylated serine or threonine residues at conserved motifs to affect substrate function, interactions, and localization. We find that RGS14 interacts with 14-3-3 at two unique sites. One site, downstream of RGS14’s RGS domain is phosphorylation-dependent. Binding of 14-3-3 inhibits active Gαi-GTP binding at the RGS domain, likely preventing RGS14 from catalyzing cleavage of the GTP on Gαi and ending the signal transduction cascade initiated by GPCR activation. The other 14-3-3 binding site on RGS14 is phosphorylation-independent. Binding of 14-3-3 to this site prevents RGS14 translocation into the nucleus, where it may modulate transcription. Here, we reveal both a proteome for area CA2 and an interactome for a CA2-expressed protein, RGS14. This interactome has revealed a functional interaction between RGS14 and 14-3-3 as well as the presence of many other potential functional interactors. Future studies examining the RGS14 interactome in the context of the CA2 proteome will elucidate the functional binding partners responsible for RGS14’s suppression of synaptic plasticity as well as other potential functions in other parts of the cell, including the nucleus. 

Table of Contents

Table of Contents

                                                                                                                                             Page

Chapter 1: Introduction                                                                                                          1

1.1 Overview of GPCR regulation of synaptic plasticity                                              2     

1.2 Overview of RGS protein regulation of synaptic plasticity                                    4     

1.2.1 RGS2 regulation of synaptic plasticity                                                     4

1.2.2 RGS4 regulation of synaptic plasticity                                                     8

1.2.3 RGS7 and RGS9-2 regulation of synaptic plasticity                              13

1.3 RGS14 protein architecture                                                                                   19

1.4 RGS14 interactions outside defined domains                                                       26

1.5 RGS14 is a native suppressor of LTP in area CA2                                               28

1.6 Overall hypothesis and objective of this research                                                 30

Chapter 2: Interactome Analysis Reveals Regulator of G Protein Signaling                  33

14 (RGS14) as a Complex Scaffold Protein at the Synapse

2.1 Introduction                                                                                                           34

2.2 Experimental Procedures                                                                                       36

2.3 Results                                                                                                                   39

2.4 Discussion                                                                                                             44

Chapter 3: Differential Proteomics and Systems Biology Approaches                            47

Reveal a Unique Hippocampal Area CA2 Proteome

3.1 Introduction                                                                                                           48

3.2 Experimental Procedures                                                                                       50

3.3 Results                                                                                                                   59

3.4 Discussion                                                                                                             71

Chapter 4: 14-3-3γ binds RGS14 at distinct sites to inhibit the                                        77

RGS14:Gαi-AlF4signaling complex and RGS14 nuclear localization

4.1 Introduction                                                                                                           78

4.2 Experimental Procedures                                                                                       81

4.3 Results                                                                                                                   89

4.4 Discussion                                                                                                           115

Chapter 5: Discussion                                                                                                         123

5.1 RGS14 is a complex scaffolding protein                                                             124

5.2 RGS14 interaction with 14-3-3                                                                           125

5.3 Finding the functional interactions in the RGS14 interactome                           126

5.4 RGS14 does what where                                                                                     130

5.5 RGS14 function throughout the brain                                                                 137

5.6 Concluding Remarks                                                                                           140

References                                                                                                                            142

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