Functional Reconstitution of an Atypical G Protein Heterotrimer and Regulator of G Protein Signaling Protein (RGS1) from Arabidopsis thaliana

Janice C Jones,Brenda R.S Temple,Alan M Jones,Henrik G Dohlman

doi:10.1074/jbc.m110.190355

Janice C Jones, Brenda R.S Temple + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m110.190355

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Abstract

It has long been known that animal heterotrimeric Gαβγ proteins are activated by cell-surface receptors that promote GTP binding to the Gα subunit and dissociation of the heterotrimer. In contrast, the Gα protein from Arabidopsis thaliana (AtGPA1) can activate itself without a receptor or other exchange factor. It is unknown how AtGPA1 is regulated by Gβγ and the RGS (regulator of G protein signaling) protein AtRGS1, which is comprised of an RGS domain fused to a receptor-like domain. To better understand the cycle of G protein activation and inactivation in plants, we purified and reconstituted AtGPA1, full-length AtRGS1, and two putative Gβγ dimers. We show that the Arabidopsis Gα protein binds to its cognate Gβγ dimer directly and in a nucleotide-dependent manner. Although animal Gβγ dimers inhibit GTP binding to the Gα subunit, AtGPA1 retains fast activation in the presence of its cognate Gβγ dimer. We show further that the full-length AtRGS1 protein accelerates GTP hydrolysis and thereby counteracts the fast nucleotide exchange rate of AtGPA1. Finally, we show that AtGPA1 is less stable in complex with GDP than in complex with GTP or the Gβγ dimer. Molecular dynamics simulations and biophysical studies reveal that altered stability is likely due to increased dynamic motion in the N-terminal α-helix and Switch II of AtGPA1. Thus, despite profound differences in the mechanisms of activation, the Arabidopsis G protein is readily inactivated by its cognate RGS protein and forms a stable, GDP-bound, heterotrimeric complex similar to that found in animals.

Highlights

Exceptions to the G protein signaling paradigm were discovered in the plant model organism Arabidopsis thaliana
Whereas animal G␣ proteins have a slow rate of nucleotide exchange in the absence of an activated G protein-coupled receptors (GPCRs) or other guanine nucleotide exchange factor, the Arabidopsis G␣ (AtGPA1, referred to as GPA1) rapidly releases GDP without any stimulus [5]
Whereas most animal Regulators of G protein signaling (RGS) proteins are present in the cytoplasm, the RGS protein from Arabidopsis is fused to a predicted seven-transmembrane domain reminiscent of GPCRs and localizes to the plasma membrane [7]

Summary

Introduction

Exceptions to the G protein signaling paradigm were discovered in the plant model organism Arabidopsis thaliana. Animal G␤␥ dimers diminish G protein activation by acting as a guanine nucleotide dissociation inhibitor to prevent spontaneous GDP release from G␣ proteins and thereby help maintain the inactive state [8]. Direct interaction between G protein subunits has not been shown previously with purified components, and possible guanine nucleotide dissociation inhibitor activity of plant G␤␥ proteins toward GPA1 had not been investigated; it is not known whether GPA1 retains its self-activation property within the heterotrimeric complex.

Results

Conclusion