Determining the impact of the RGS14-Gαi1-GDP complex on NHERF1 binding Public

Abstract

Regulator of G Protein Signaling 14 (RGS14) is a multifunctional signaling protein that integrates G protein, mitogen-activated kinase/extracellular regulated kinase (MAPK/ERK), and Ca++/CaM signaling pathways. Expressed in human hippocampal CA2 pyramidal cells, RGS14 plays a vital role in suppressing synaptic plasticity and long-term potentiation in the context of hippocampal-related memory. Human RGS14 contains an RGS domain that binds to Gαi/o-GTP, a tandem Ras/Rap binding domain (RBD) that binds active H-Ras/Rap2-GTP, a GRP motif that binds to Gαi1/3-GDP, and a poorly characterized PDZ-binding motif. While much is known about the RGS domain, RBD, and GPR motif using rodent models, little is known about the PDZ-binding motif due to its absence in rodent RGS14. 

Recent findings show that human RGS14 binds to NHERF1, a PDZ domain containing scaffolding protein located in postsynaptic spines, where it regulates GPCR-G signaling. RGS14 is well positioned to regulate both NHERF1 and Gαi1signaling at the plasma membrane. However, NHERF1 via its PDZ2 domain and Gαi1-GDP each bind RGS14 in very close proximity. How the binding of one protein impacts the binding of the other is unknown. In order to understand the interaction between the three proteins, we examined their direct binding as purified proteins. Using co-immunoprecipitation of human RGS14 with Gαi1-GDP, NHERF1, and the isolated PDZ2 domain of NHERF1, we tested the relative ratio and competition for successful binding between human RGS14, Gαi1-GDP, and the PDZ2 domain. The results suggested the importance of the order of protein binding for complex formation, as the binding of Gαi1-GDP first to human RGS14 reduces the apparent affinity of the PDZ2 domain towards the dimer. These findings imply a Gi-mediated mechanism for the regulation of the subcellular localization of RGS14 and NHERF1 and complex formation. Overall, our findings implicate RGS14 as an intermediate regulator of both NHERF1 and Gαi1 signaling in brain, providing more insights into its coupling mechanisms. Future studies will explore the impact of NHERF1 binding on RGS14-Gαi1-GDP complex formation (and vice versa) as purified proteins using size-exclusion chromatography and in live cells using BRET analysis. 

Table of Contents

INTRODUCTION...1

RGS14 and its binding partners in the brain...2

Human RGS14 binds NHERF1...3

NHERF1 regulates mGluR2/3 signaling in brain...4

Working hypothesis and goals for these studies...6

METHODS...7

Protein Transformation...7

His6-Gɑi Protein Expression and Purification...8

His6-NHERF1 Protein Expression and Purification...8

His6-MBP-TEV-human RGS14 Protein Expression and Purification...9

Co-Immunoprecipitation of Hu-RGS14 and its binding partners...10

Immunoblotting...10

RESULTS...11

Protein Purification...11

Complex Binding...12

DISCUSSION...17

Limitation and Future Direction...20

CONCLUSION...21

REFERENCES...23

About this Honors Thesis

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School	Emory College
Department	Neuroscience and Behavioral Biology
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Biology, Molecular Health Sciences, Pharmacology Biology, Neuroscience
Mot-clé	Group II metabotropic glutamate receptor GPCR NHERF1 mGluR2/3 RGS14 CA2 G protein Hippocampus
Committee Chair / Thesis Advisor	Hepler, John R., Emory University
Committee Members	Roesch, Leah, Emory University Manns, Joseph, Emory University Hall, Randy, Emory University

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