Regulator of G Protein Signaling 8 (RGS8) Requires Its NH2 Terminus for Subcellular Localization and Acute Desensitization of G Protein-gated K+ Channels

Osamu Saitoh,Ikuo Masuho,Ion Terakawa,Satoshi Nomoto,Tomiko Asano,Yoshihiro Kubo

doi:10.1074/jbc.m006917200

Abstract

Functional roles of the NH(2)-terminal region of RGS (regulators of G protein signaling) 8 in G protein signaling were studied. The deletion of the NH(2)-terminal region of RGS8 (DeltaNRGS8) resulted in a partial loss of the inhibitory function in pheromone response of yeasts, although Galpha binding was not affected. To examine roles in subcellular distribution, we coexpressed two fusion proteins of RGS8-RFP and DeltaNRGS8-GFP in DDT1MF2 cells. RGS8-RFP was highly concentrated in nuclei of unstimulated cells. Coexpression of constitutively active Galpha(o) resulted in translocation of RGS8 protein to the plasma membrane. In contrast, DeltaNRGS8-GFP was distributed diffusely through the cytoplasm in the presence or absence of active Galpha(o). When coexpressed with G protein-gated inwardly rectifying K(+) channels, DeltaNRGS8 accelerated both turning on and off similar to RGS8. Acute desensitization of G protein-gated inwardly rectifying K(+) current observed in the presence of RGS8, however, was not induced by DeltaNRGS8. Thus, we, for the first time, showed that the NH(2) terminus of RGS8 contributes to the subcellular localization and to the desensitization of the G protein-coupled response.

Highlights

RGS1 proteins comprise a large family of more than 20 members that modulate heterotrimeric G protein signaling [1, 2]
It was shown that several RGS proteins (RGS1, RGS3, RGS4, and GAIP) attenuate G protein signaling in cultures [4, 6, 7]
The NH2-terminal Domain of RGS8 Is Required for Its Full Ability to Function in Yeasts—In the NH2-terminal region of RGS4 and RGS16 outside of the RGS domains, the sequence conservation was found, and it was shown that the deletion of this region reduced the effect to attenuate pheromone signaling in yeasts [17, 18]

Summary

Introduction

RGS1 (regulators of G protein signaling) proteins comprise a large family of more than 20 members that modulate heterotrimeric G protein signaling [1, 2]. To examine roles in subcellular distribution, we coexpressed two fusion proteins of RGS8-RFP and ⌬NRGS8-GFP in DDT1MF2 cells. Expression levels of wild and mutant RGS8 proteins in yeast cells grown in galactose were determined by Western blotting using anti-Myc antibody (Fig. 2C).

Results

Conclusion