RNA Editing Induces Variation in Desensitization and Trafficking of 5-Hydroxytryptamine 2c Receptor Isoforms

Sébastien Marion,David M Weiner,Marc G Caron

doi:10.1074/jbc.m308742200

Abstract

The 5-hydroxytryptamine2c receptor (5-HT2cR) is subjected to RNA editing, in the second intracellular loop, generating 14 different isoforms in human brain. This post-transcriptional event markedly alters the signaling properties of the receptor by reducing its ability to couple to G-proteins. Although the non-edited form of the receptor is essentially fully constitutively active, edited forms show lesser degrees of constitutive activity. We have used two extensively edited receptor isoforms, VGV and VSV, and the non-edited INI isoform to investigate how variations in constitutive receptor activity affect the trafficking and the interaction of these isoforms with components of the desensitization machinery in HEK 293 cells. We found that cell surface expression of the 5-HT2cR decreased in parallel with increased constitutive activity of the isoforms. The subcellular distribution of the various isoforms was dependent of their ability to interact with betaarrestin2, which correlated with the constitutive activity level of each isoform. We observed that the agonist-independent interaction of betaarrestin2 with constitutively active 5-HT2cR isoforms was reversed by inverse agonist treatments promoting receptor redistribution to the cell surface. Overexpression of a G-protein-coupled receptor kinase (GRK2) was able to stabilize the interaction of betaarrestin2 with constitutively active 5-HT2cR isoforms even in the presence of inverse agonists. Taken together, our observations indicate that the constitutively active 5-HT2cR isoforms are spontaneously internalized in an agonist-independent manner. This endocytosis process is mediated by a GRK/betaarrestin-dependent mechanism and is directly correlated with the constitutive activity status of the RNA edited receptor variants. Thus the ultimate physiological output of constitutively active receptors may be determined not only by their agonist-independent activity but also by their interactions with GRKs and betaarrestin.

Highlights

The 5-hydroxytryptamine2c receptor (5-HT2cR) is subjected to RNA editing, in the second intracellular loop, generating 14 different isoforms in human brain
We observed that the agonist-independent interaction of ␤arrestin2 with constitutively active 5-HT2cR isoforms was reversed by inverse agonist treatments promoting receptor redistribution to the cell surface
Stimulation of the 5-HT2cRINI-green fluorescent protein (GFP) with serotonin did not induce any noticeable variation of the vesicular distribution of this GFP-tagged isoform, suggesting that the agonist can induce no further internalization of this isoform (Fig. 1C, middle panel)

Summary

Introduction

The 5-hydroxytryptamine2c receptor (5-HT2cR) is subjected to RNA editing, in the second intracellular loop, generating 14 different isoforms in human brain. We observed that the agonist and inverse agonist treatments for 30 min at 37 °C of cells expressing the FLAG5-HT2cR-VSV resulted in a 29% decrease but a 1.8-fold increase of cell surface expression of this isoform respectively, suggesting that serotonin can induce internalization of cell surface receptors but that not all the receptors are expressed at the plasma membrane in the absence of stimulation (Fig. 2B).

Results

Conclusion