The impact of rare human genetic variants and disease-linked mutations of RGS14 on the RGS14-Rap2A interactions and the regulation of JNK signaling Open Access

Terzioglu, Gizem (Spring 2021)

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The Regulator of G Protein Signaling 14 (RGS14) is a multifunctional signaling protein that integrates G protein, MAP kinase (MAPK), and Ca++/CaM signaling pathways in host cells. Expressed primarily in the hippocampus, RGS14 is a natural suppressor of synaptic plasticity and long-term potentiation (LTP) in area CA2 neurons and is linked to hippocampal-based learning and memory. The RGS14 domain structure consists of an RGS domain that binds active Gαi/o-GTP, a tandem (R1/R2) Ras/Rap binding domain (RBD), and a GoLoco/GPR motif that binds inactive Gαi1/3-GDP. While much is known about RGS14 regulation of H-Ras, much less is known about RGS14 regulation of Rap2A, which stimulates the TNIK-JNK activity implicated in synaptic plasticity. In order to understand how RGS14 regulates Rap2A, we identified rare human variants of RGS14 that we predicted to disrupt RGS14 interactions with Rap2A as experimental tools. Using co-immunoprecipitation and bioluminescence resonance energy transfer (BRET) assays to measure RGS14 functions, we identified two rare variants of RGS14, D310N and K334N, that block RGS14 interactions with active Rap2A. We tested the impact of wild-type and the variants of RGS14 on the regulation of Rap2A-mediated JNK MAPK signaling by analyzing phosphorylated JNK (pJNK) levels in whole-cell lysates of HEK293 cells transfected with different combinations of constitutively active Rap2A, TNIK, wild-type and the variant RGS14 via Western immunoblotting. Our results suggest that RGS14 downregulates JNK signaling by negatively regulating active Rap2A, whereas the variants fail to do so. Overall, our findings implicate RGS14 as a signaling protein that may provide neuroprotection against ischemia- and excitotoxicity-induced cell death and Alzheimer’s disease pathogenesis in CA2 pyramidal neurons. This may occur by blocking AMPA receptor trafficking as a potential mechanism by which RGS14 suppresses synaptic plasticity in the area CA2. Future studies will explore the potential impacts of RGS14 genetic variants on synaptic plasticity, neuron physiology, behavior and disease states for human carriers.

Table of Contents

Background and Significance...1


Hypotheses and Aims...6

Materials and Methods...7

Cell Culture and Transfection...7

RGS14 Genetic Variants and Constructs...7

Co-Immunoprecipitation and Western Blotting...8

BRET in Live Cells...9

Analysis of Phosphorylated JNK (pJNK)...10


Naturally occurring RGS14 variants D310N and K334N fail to bind active Rap2A in vitro...14

RGS14 suppresses active Rap2A and downregulates the phosphorylation of JNK, the variants D310N and K334N disrupt this regulation...18


Potential implications of RGS14 regulation of JNK signaling...23

RGS14’s regulation of synaptic plasticity...25

Rare human genetic variants of RGS14: Human health and disease...27


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