A Comprehensive Analysis of the RGS14 Interactome in the Context of Area CA2 of the Hippocampus Open Access
Gerber, Kyle (Spring 2019)
Published
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-AlF4 – signaling 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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