Mono-ADP-ribosylation of the G Protein βγ Dimer Is Modulated by Hormones and Inhibited by Arf6

Nadia Dani,Emilia Mayo,Annalisa Stilla,Adriano Marchegiani,Simone Di Paola,Daniela Corda,Maria Di Girolamo

doi:10.1074/jbc.m110.112466

Abstract

Mono-ADP-ribosylation is a reversible post-translational modification that can modulate the functions of target proteins. We have previously demonstrated that the β subunit of heterotrimeric G proteins is endogenously mono-ADP-ribosylated, and once modified, the βγ dimer is inactive toward its effector enzymes. To better understand the physiological relevance of this post-translational modification, we have studied its hormonal regulation. Here, we report that Gβ subunit mono-ADP-ribosylation is differentially modulated by G protein-coupled receptors. In intact cells, hormone stimulation of the thrombin receptor induces Gβ subunit mono-ADP-ribosylation, which can affect G protein signaling. Conversely, hormone stimulation of the gonadotropin-releasing hormone receptor (GnRHR) inhibits Gβ subunit mono-ADP-ribosylation. We also provide the first demonstration that activation of the GnRHR can activate the ADP-ribosylation factor Arf6, which in turn inhibits Gβ subunit mono-ADP-ribosylation. Indeed, removal of Arf6 from purified plasma membranes results in loss of GnRHR-mediated inhibition of Gβ subunit mono-ADP-ribosylation, which is fully restored by re-addition of purified, myristoylated Arf6. We show that Arf6 acts as a competitive inhibitor of the endogenous ADP-ribosyltransferase and is itself modified by this enzyme. These data provide further understanding of the mechanisms that regulate endogenous ADP-ribosylation of the Gβ subunit, and they demonstrate a novel role for Arf6 in hormone regulation of Gβ subunit mono-ADP-ribosylation.

Highlights

The heterotrimeric G proteins are the most common transducers for G protein-coupled receptors (GPCRs),4 the largest family of cell-surface receptors
Mono-ADP-ribosylation of the G Protein ␤ Subunit Is Modulated by GPCRs—We have previously demonstrated that hormones that act on different GPCRs, including thrombin, serotonin, and cholecystokinin, can increase the levels of the ADP-ribosylated ␤ subunit in in vitro ADP-ribosylation assays [5]
Arf6-GTP Is Mono-ADP-ribosylated by the Chinese hamster ovary (CHO) Plasma Membrane-associated Mono-ADP-ribosyltransferase—We have provided evidence that Arf6 can act as a competitive inhibitor for the endogenous CHO plasma membrane-associated ART activity

Summary

Introduction

The heterotrimeric G proteins are the most common transducers for G protein-coupled receptors (GPCRs),4 the largest family of cell-surface receptors. Stimulation of plasma membranes from wild-type CHO cells (which do not express ␣2-adrenoreceptor, ␤2-adrenoreceptor, and GnRHR) with clonidine, isoproterenol, and GnRH did not modulate this labeling of the G␤ subunit (Fig. 1A), confirming the specificity of this agonist-mediated modulation of G␤ subunit ADP-ribosylation.

Results

Conclusion