Complementary biosensors reveal different G-protein signaling modes triggered by GPCRs and non-receptor activators

Mikel Garcia-Marcos

doi:10.7554/elife.65620

Abstract

It has become evident that activation of heterotrimeric G-proteins by cytoplasmic proteins that are not G-protein-coupled receptors (GPCRs) plays a role in physiology and disease. Despite sharing the same biochemical guanine nucleotide exchange factor (GEF) activity as GPCRs in vitro, the mechanisms by which these cytoplasmic proteins trigger G-protein-dependent signaling in cells have not been elucidated. Heterotrimeric G-proteins can give rise to two active signaling species, Gα-GTP and dissociated Gβγ, with different downstream effectors, but how non-receptor GEFs affect the levels of these two species in cells is not known. Here, a systematic comparison of GPCRs and three unrelated non-receptor proteins with GEF activity in vitro (GIV/Girdin, AGS1/Dexras1, and Ric-8A) revealed high divergence in their contribution to generating Gα-GTP and free Gβγ in cells directly measured with live-cell biosensors. These findings demonstrate fundamental differences in how receptor and non-receptor G-protein activators promote signaling in cells despite sharing similar biochemical activities in vitro.

Highlights

Heterotrimeric G-proteins are ubiquitous molecular switches that transduce extracellular signals into intracellular cascades of biochemical reactions to steer cellular responses (Gilman, 1987)
The premise to establish control over the input is that triggering the relocalization of G-protein regulators from the cytosol to the plasma membrane would allow their action on their constitutively membrane-anchored Gi protein substrates by virtue of increasing the local concentration of the reactants. This was achieved by implementing chemically induced dimerization with rapamycin, which has been successfully applied in the past to rapidly modulate Gi signaling with some GAPs and non-receptor guanine nucleotide exchange factor (GEF) (Muntean and Martemyanov, 2016; Parag-Sharma et al, 2016)
Activity is reported as an increase in bioluminescence resonance energy transfer (BRET) due to binding of fluorescent protein (FP)-tagged G-protein to a luciferase-tagged protein module that binds to either dissociated Gbg or GTP-bound Gai

Summary

Introduction

Heterotrimeric G-proteins are ubiquitous molecular switches that transduce extracellular signals into intracellular cascades of biochemical reactions to steer cellular responses (Gilman, 1987). Similar biochemical reductionist approaches have been useful to define the enzymatic activities of cytoplasmic regulators of G-proteins like GAPs, GDIs, and non-receptor GEFs, but have left several open questions about how these regulatory mechanisms operate in cells. The second experimental issue is that approaches to directly detect G-protein activity in cells have typically relied on the detection of Ga-Gbg dissociation instead of detecting Ga-GTP While these two signaling events correlate well in the process of GPCR-mediated G-protein activation, this is not necessarily the case for many cytoplasmic regulators of G-proteins, making evident the need for detecting both free Gbg and Ga-GTP formation to understand how they operate in cells. This newly developed approach allowed to pinpoint key differences between the modes of G-protein signaling regulation in cells exerted by various proteins with GEF activity, including both receptor (i.e., GPCRs) and non-receptor proteins

Results

Discussion

Materials and methods

Funding Funder