Imperial College London
Alternate

ProfessorGuyRutter

Faculty of Medicine, Department of Medicine

Visiting Professor
 
 
 
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Contact

 

+44 (0)20 7594 3340g.rutter Website

 
 
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Location

 

ICTEM buildingHammersmith Campus

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Summary

 

Publications

Citation

BibTex format

@article{Manchanda:2021:10.1101/2021.11.24.469908,
author = {Manchanda, Y and Ramchunder, Z and Shchepinova, MM and Rutter, GA and Inoue, A and Tate, EW and Jones, B and Tomas, A},
doi = {10.1101/2021.11.24.469908},
title = {Expression of mini-G proteins specifically halt cognate GPCR trafficking and intracellular signalling},
url = {http://dx.doi.org/10.1101/2021.11.24.469908},
year = {2021}
}
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RIS format (EndNote, RefMan)

TY  - JOUR
AB  - <jats:title>Abstract</jats:title><jats:p>Mini-G proteins are engineered thermostable variants of Gα subunits designed to specifically stabilise G protein-coupled receptors (GPCRs) in their active conformation for structural analyses. Due to their smaller size and ease of use, they have become popular tools in recent years to assess specific GPCR behaviours in cells, both as reporters of receptor coupling to each G protein subtype and for in-cell assays designed to quantify compartmentalised receptor signalling from a range of subcellular locations. Here, we describe a previously unappreciated consequence of the co-expression of mini-G proteins with their cognate GPCRs, namely a profound disruption in GPCR trafficking and intracellular signalling caused by the co-expression of the specific mini-G subtype coupled to the affected receptor. We studied the Gαs-coupled pancreatic beta cell class B GPCR glucagon-like peptide-1 receptor (GLP-1R) as a model to describe in detail the molecular consequences derived from this effect, including a complete halt in β-arrestin-2 recruitment and receptor internalisation, despite near-normal levels of receptor GRK2 recruitment and lipid nanodomain segregation, as well as the disruption of endosomal GLP-1R signalling by mini-G<jats:sub>s</jats:sub> co-expression. We also extend our analysis to a range of other prototypical GPCRs covering the spectrum of Gα subtype coupling preferences, to unveil a widely conserved phenomenon of GPCR internalisation blockage by specific mini-G proteins coupled to a particular receptor. Our results have important implications for the design of methods to assess intracellular GPCR signalling. We also present an alternative adapted bystander intracellular signalling assay for the GLP-1R in which we substitute the mini-G<jats:sub>s</jats:sub> by a nanobody, Nb37, with specificity for active Gαs:GPCR complexes and no deleterious effect o
AU  - Manchanda,Y
AU  - Ramchunder,Z
AU  - Shchepinova,MM
AU  - Rutter,GA
AU  - Inoue,A
AU  - Tate,EW
AU  - Jones,B
AU  - Tomas,A
DO  - 10.1101/2021.11.24.469908
PY  - 2021///
TI  - Expression of mini-G proteins specifically halt cognate GPCR trafficking and intracellular signalling
UR  - http://dx.doi.org/10.1101/2021.11.24.469908
ER  -
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