The Subcellular Dynamics of the Gs-Linked Receptor GPR3 Contribute to the Local Activation of PKA in Cerebellar Granular Neurons.

Tatsuhiro Miyagi,Norio Sakai,Toshihiko Shirafuji,Shigeru Tanaka,Izumi Hide,Alexander G Obukhov

doi:10.1371/journal.pone.0147466

Abstract

G-protein-coupled receptor (GPR) 3 is a member of the GPR family that constitutively activates adenylate cyclase. We have reported that the expression of GPR3 in cerebellar granular neurons (CGNs) contributes to neurite outgrowth and modulates neuronal proliferation and survival. To further identify its role, we have analyzed the precise distribution and local functions of GPR3 in neurons. The fluorescently tagged GPR3 protein was distributed in the plasma membrane, the Golgi body, and the endosomes. In addition, we have revealed that the plasma membrane expression of GPR3 functionally up-regulated the levels of PKA, as measured by a PKA FRET indicator. Next, we asked if the PKA activity was modulated by the expression of GPR3 in CGNs. PKA activity was highly modulated at the neurite tips compared to the soma. In addition, the PKA activity at the neurite tips was up-regulated when GPR3 was transfected into the cells. However, local PKA activity was decreased when endogenous GPR3 was suppressed by a GPR3 siRNA. Finally, we determined the local dynamics of GPR3 in CGNs using time-lapse analysis. Surprisingly, the fluorescent GPR3 puncta were transported along the neurite in both directions over time. In addition, the anterograde movements of the GPR3 puncta in the neurite were significantly inhibited by actin or microtubule polymerization inhibitors and were also disturbed by the Myosin II inhibitor blebbistatin. Moreover, the PKA activity at the tips of the neurites was decreased when blebbistatin was administered. These results suggested that GPR3 was transported along the neurite and contributed to the local activation of PKA in CGN development. The local dynamics of GPR3 in CGNs may affect local neuronal functions, including neuronal differentiation and maturation.

Highlights

G-protein coupled receptors (GPRs) are the most abundant membrane proteins and have been reported to form a large family
We demonstrate the subcellular localization of GPR3 in cerebellar granular neurons (CGNs) for the first time
The movements of the GPR3 puncta were highly correlated with local PKA activation at the neurite tips

Summary

Introduction

G-protein coupled receptors (GPRs) are the most abundant membrane proteins and have been reported to form a large family. GPR3, GPR6, and GPR12 constitutively activate the Gs protein, which resulted in constitutively increased intracellular cAMP [1]. These receptors are largely expressed in the brain, with the exception of GPR3, which shows additional expression in oocytes and testis. GPR12 is expressed in the liver [2,3]. We have reported that GPR3 expression in cerebellar granular neurons (CGNs) is associated with neurite outgrowth, the modulation of premature neuronal proliferation, and neuronal survival [3,4,5]. The physiological function of GPR3 has not been fully elucidated

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