ER/K linked GPCR-G protein fusions systematically modulate second messenger response in cells

Rabia U Malik,Sivaraj Sivaramakrishnan,Matthew Dysthe,Roger K Sunahara,Michael Ritt

doi:10.1038/s41598-017-08029-3

Rabia U Malik, Sivaraj Sivaramakrishnan + Show 3 more

Open Access

https://doi.org/10.1038/s41598-017-08029-3

Copy DOI

Abstract

FRET and BRET approaches are well established for detecting ligand induced GPCR-G protein interactions in cells. Currently, FRET/BRET assays rely on co-expression of GPCR and G protein, and hence depend on the stoichiometry and expression levels of the donor and acceptor probes. On the other hand, GPCR-G protein fusions have been used extensively to understand the selectivity of GPCR signaling pathways. However, the signaling properties of fusion proteins are not consistent across GPCRs. In this study, we describe and characterize novel sensors based on the Systematic Protein Affinity Strength Modulation (SPASM) technique. Sensors consist of a GPCR and G protein tethered by an ER/K linker flanked by FRET probes. SPASM sensors are tested for the β2-, α1-, and α2- adrenergic receptors, and adenosine type 1 receptor (A1R), tethered to Gαs-XL, Gαi2, or Gαq subunits. Agonist stimulation of β2-AR and α2-AR increases FRET signal comparable to co-expressed FRET/BRET sensors. SPASM sensors also retain signaling through the endogenous G protein milieu. Importantly, ER/K linker length systematically tunes the GPCR-G protein interaction, with consequent modulation of second messenger signaling for cognate interactions. SPASM GPCR sensors serve the dual purpose of detecting agonist-induced changes in GPCR-G protein interactions, and linking these changes to downstream signaling.

Highlights

G protein coupled receptors relay detection of stimuli such as photons, neurotransmitters, hormones, or drugs from the extracellular milieu to the intracellular environment by binding and activating one or more functionally distinct heterotrimeric G proteins[1]
GPCR Systematic Protein Affinity Strength Modulation (SPASM) sensors involve expression of a single polypeptide encoding a GPCR tethered to a G protein via an ER/K α-helix/linker that is flanked by a pair of FRET probes (mCitrine (FRET acceptor) and mCerulean (FRET donor)) (Fig. 1a)
The SPASM module provides 1:1 stoichiometry of GPCR-Gα expression and consists of an ER/K α-helical linker flanked by a FRET pair[14]

Summary

Introduction

G protein coupled receptors relay detection of stimuli such as photons, neurotransmitters, hormones, or drugs from the extracellular milieu to the intracellular environment by binding and activating one or more functionally distinct heterotrimeric G proteins[1]. Direct GPCR-G protein fusions generated by tethering the N-terminus of the Gα to the GPCR’s C-tail either directly or with a short linker in between have been used to study the influence of tethering different G protein subunits on G protein activation and second messenger signaling[8, 9]. Such fusions have not been used to monitor the GPCR-G protein interaction using resonance energy transfer approaches[6].

Methods

Results

Conclusion