Cellular and Subcellular Localization of the RGS7/Gβ5/R7BP Complex in the Cerebellar Cortex.

Carolina Aguado,Ana Fajardo-Serrano,Rafael Luján,Cesare Orlandi,Mercedes Gil-Minguez,Kirill A Martemyanov

doi:10.3389/fnana.2016.00114

Carolina Aguado, Ana Fajardo-Serrano + Show 4 more

Open Access

https://doi.org/10.3389/fnana.2016.00114

Copy DOI

Abstract

A member of regulator of G-protein signaling family, RGS7, is an essential modulator of signaling through GABAB receptors. RGS7 functions as a macromolecular complex with type 5 G protein β (Gβ5) and R7 binding protein (R7BP) to control the localization and function of the resultant heterotrimeric complexes. Here, we used co-immunoprecipitation, in situ hybridization, histoblot and immunohistochemical techniques at the light and electron microscopic level to advance understanding of RGS7-Gβ5-R7BP complexes in the central nervous system, focusing on distinct neuronal populations in the cerebellar cortex. Histoblot analysis showed that RGS7, Gβ5 and R7BP proteins were widely expressed in the brain, with mostly an overlapping pattern and showing a high expression level in the molecular layer of the cerebellar cortex. Co-immunoprecipitation experiments established that the RGS7/Gβ5 forms complexes with R7BP in the cerebellum. At the cellular level, RGS7 and R7BP mRNAs were expressed at the highest level in Purkinje cells (PCs) and Golgi cells, and at low levels in granule cells. Immunohistochemistry confirmed that labeling for RGS7, Gβ5 and R7BP were present in the three neuronal populations and concentrated in dendrites and spines. At the electron microscopic level, immunolabeling for RGS7, Gβ5 and R7BP proteins was found both at postsynaptic and presynaptic sites and showed similar distribution patterns. Immunoreactivity for the three proteins was mostly localized along the extrasynaptic plasma membrane of dendritic shafts and spines of PCs and to a lesser extent, in axon terminals (AT) establishing excitatory synapses. Quantitative analysis of immunogold particles for RGS7, Gβ5 and R7BP revealed that they are non-uniformly distributed along the surface of PCs, and show enrichment around excitatory synapses on dendritic spines. We further report that deletion of R7BP in mice reduced the targeting of both RGS7 and Gβ5 to the plasma membrane. Altogether, these data support the existence of macromolecular complexes composed of RGS7-Gβ5-R7BP in PCs. The location at post- and pre-synaptic sites in PCs spines-parallel fiber synapses suggests their involvement in the modulation of glutamatergic neurotransmission in the cerebellar cortex.

Highlights

G protein-coupled receptors (GPCRs) form a large and diverse superfamily of integral membrane proteins whose main function is to transduce extracellular stimuli into intracellular signals
Distribution of Proteins Relative to Glutamate Release Sites To determine the relative abundance of RGS7, Gβ5 and R7 binding protein (R7BP) in dendritic spines of Purkinje cells (PCs) and their association with excitatory synapses, immunoparticles identified in each reference area and present in dendritic spines were counted
RGS7, a member of the R7 regulators of G-protein signaling (RGS) family, and Gβ5 form obligate heterodimeric complexes that are required for normal regulation of GPCR function in the brain (Xie et al, 2010; Fajardo-Serrano et al, 2013; Masuho et al, 2013; Ostrovskaya et al, 2014)

Summary

Introduction

G protein-coupled receptors (GPCRs) form a large and diverse superfamily of integral membrane proteins whose main function is to transduce extracellular stimuli into intracellular signals. GPCRs play a vital role in sensory reception, neurotransmission, cell differentiation and regulation of neuronal excitability (Wettschureck and Offermanns, 2005). Upon activation by a specific ligand, the GPCR undergoes a conformational change and activates heterotrimeric G proteins by promoting the exchange of GDP to GTP associated with the Gα subunit. This leads to the dissociation of Gα-GTP and Gβγ subunits which subsequently modulate downstream effectors responsible for initiating unique intracellular signaling responses (Smrcka, 2008). Termination of G protein signaling relies on the action of the regulators of G-protein signaling (RGS) proteins, which accelerate the rate of G-protein deactivation by acting as GTPase-activating proteins (GAPs) for Gα subunits and resulting in faster response deactivation (Ross and Wilkie, 2000; Hollinger and Hepler, 2002; Anderson et al, 2009b)

Methods

Results

Conclusion