Endogenous RGS14 is a cytoplasmic-nuclear shuttling protein that localizes to juxtanuclear membranes and chromatin-rich regions of the nucleus.

Mary Rose Branch,John R Hepler

doi:10.1371/journal.pone.0184497

Abstract

Regulator of G protein signaling 14 (RGS14) is a multifunctional scaffolding protein that integrates G protein and H-Ras/MAPkinase signaling pathways to regulate synaptic plasticity important for hippocampal learning and memory. However, to date, little is known about the subcellular distribution and roles of endogenous RGS14 in a neuronal cell line. Most of what is known about RGS14 cellular behavior is based on studies of tagged, recombinant RGS14 ectopically overexpressed in unnatural host cells. Here, we report for the first time a comprehensive assessment of the subcellular distribution and dynamic localization of endogenous RGS14 in rat B35 neuroblastoma cells. Using confocal imaging and 3D-structured illumination microscopy, we find that endogenous RGS14 localizes to subcellular compartments not previously recognized in studies of recombinant RGS14. RGS14 localization was observed most notably at juxtanuclear membranes encircling the nucleus, at nuclear pore complexes (NPC) on both sides of the nuclear envelope and within intranuclear membrane channels, and within both chromatin-poor and chromatin-rich regions of the nucleus in a cell cycle-dependent manner. In addition, a subset of nuclear RGS14 localized adjacent to active RNA polymerase II. Endogenous RGS14 was absent from the plasma membrane in resting cells; however, the protein could be trafficked to the plasma membrane from juxtanuclear membranes in endosomes derived from ER/Golgi, following constitutive activation of endogenous RGS14 G protein binding partners using AlF4¯. Finally, our findings show that endogenous RGS14 behaves as a cytoplasmic-nuclear shuttling protein confirming what has been shown previously for recombinant RGS14. Taken together, the findings highlight possible cellular roles for RGS14 not previously recognized that are distinct from the regulation of conventional GPCR-G protein signaling, in particular undefined roles for RGS14 in the nucleus.

Highlights

Heterotrimeric G proteins couple to cell surface receptors to transduce signals by hormones and neurotransmitter across the plasma membrane and into the interior of cells to mediate all aspects of cell and organ physiology [1,2]
We further found that the Regulator of G protein signaling 14 (RGS14) antibody recognizes overexpressed GFP-RGS14 in transiently transfected B35 cells, as antibody staining completely colocalized with intrinsic GFP fluorescence (S1 Fig)
These findings demonstrate that the polyclonal RGS14 antibody recognizes endogenous RGS14 expressed in rat B35 neuroblastoma cells and validate its use in both western blot and immunofluorescence experiments

Summary

Introduction

Heterotrimeric G proteins couple to cell surface receptors to transduce signals by hormones and neurotransmitter across the plasma membrane and into the interior of cells to mediate all aspects of cell and organ physiology [1,2]. Whereas most RGS proteins are relatively simple proteins that act as dedicated GAPs activated Gα subunits, others are more complex with various domains for binding partners that mediate unconventional G protein signaling events. One such RGS protein is RGS14, a multifunctional scaffolding protein that interacts with specific G protein alpha subunits (Gαi/o) and activated H-Ras to integrate G protein and MAP kinase signaling pathways [4,5,6,7,8,9]. Due to the key role that RGS14 serves in regulating synaptic plasticity, we felt it to be critically important that we determine where endogenous RGS14 operates within a neuronal cell line for a better understanding of its cellular roles and functions and the possible mechanism(s) by which it limits LTP

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