Effector membrane translocation biosensors reveal G protein and βarrestin coupling profiles of 100 therapeutically relevant GPCRs
- University of Montreal, Canada
- Domain Therapeutics North America, Canada
- University of Copenhagen, Denmark
- Pfizer Worldwide Research, United States
- Pfizer Global R&D, United States
- Domain Therapeutics, France
Abstract
The recognition that individual GPCRs can activate multiple signaling pathways has raised the possibility of developing drugs selectively targeting therapeutically relevant ones. This requires tools to determine which G proteins and barrestins are activated by a given receptor. Here, we present a set of BRET sensors monitoring the activation of the 12 G protein subtypes based on the translocation of their effectors to the plasma membrane (EMTA). Unlike most of the existing detection systems, EMTA does not require modification of receptors or G proteins (except for Gs). EMTA was found to be suitable for the detection of constitutive activity, inverse agonism, biased signaling and polypharmacology. Profiling of 100 therapeutically relevant human GPCRs resulted in 1,500 pathway-specific concentration-response curves and revealed a great diversity of coupling profiles ranging from exquisite selectivity to broad promiscuity. Overall, this work describes unique resources for studying the complexities underlying GPCR signaling and pharmacology.
Data availability
All data generated or analysed during this study are included in the manuscript and supporting file; Source Data files have been provided for Figures 2, 4, 5, 6 and 7 and associated figure supplements. Supplementary Table 1 contains the numerical data used to generate Figure 2-figure supplement 1 and Figure 2-figure supplement 3. Supplementary Table 2 contains the numerical data used to generate figure 3.
-
G protein selectivity - a unified meta-analysisAccompanying dataset paper in BioRXiv (https://doi.org/10.1101/2021.09.07.459250) and www.gpcrdb.org (https://doi.org/10.1093/nar/gkab852).
Article and author information
Author details
Alexander Sebastian Hauser
Hiroyuki Kobayashi
Funding
Canada Research Chairs
- Michel Bouvier
Canadian Institutes of Health Research (FDN-148431)
- Michel Bouvier
Lundbeckfonden (R218-2016-1266)
- David E Gloriam
Lundbeckfonden (R313-2019-526)
- David E Gloriam
Novo Nordisk Fonden (NNF18OC0031226)
- David E Gloriam
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- William I Weis, Stanford University School of Medicine, United States
Version history
- Preprint posted: April 24, 2020 (view preprint)
- Received: September 21, 2021
- Accepted: March 17, 2022
- Accepted Manuscript published: March 18, 2022 (version 1)
- Version of Record published: April 12, 2022 (version 2)
Copyright
© 2022, Avet et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 5,231
- Page views
-
- 1,039
- Downloads
-
- 51
- Citations
Article citation count generated by polling the highest count across the following sources: Scopus, Crossref, PubMed Central.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Effector membrane translocation biosensors reveal G protein and βarrestin coupling profiles of 100 therapeutically relevant GPCRs
eLife 11:e74101.
https://doi.org/10.7554/eLife.74101
Further reading
-
- Biochemistry and Chemical Biology
- Cell Biology
Nitric oxide (NO), as a gaseous therapeutic agent, shows great potential for the treatment of many kinds of diseases. Although various NO delivery systems have emerged, the immunogenicity and long-term toxicity of artificial carriers hinder the potential clinical translation of these gas therapeutics. Mesenchymal stem cells (MSCs), with the capacities of self-renewal, differentiation, and low immunogenicity, have been used as living carriers. However, MSCs as gaseous signaling molecule (GSM) carriers have not been reported. In this study, human MSCs were genetically modified to produce mutant β-galactosidase (β-GALH363A). Furthermore, a new NO prodrug, 6-methyl-galactose-benzyl-oxy NONOate (MGP), was designed. MGP can enter cells and selectively trigger NO release from genetically engineered MSCs (eMSCs) in the presence of β-GALH363A. Moreover, our results revealed that eMSCs can release NO when MGP is systemically administered in a mouse model of acute kidney injury (AKI), which can achieve NO release in a precise spatiotemporal manner and augment the therapeutic efficiency of MSCs. This eMSC and NO prodrug system provides a unique and tunable platform for GSM delivery and holds promise for regenerative therapy by enhancing the therapeutic efficiency of stem cells.
-
- Biochemistry and Chemical Biology
- Cell Biology
The ATPase p97 (also known as VCP, Cdc48) has crucial functions in a variety of important cellular processes such as protein quality control, organellar homeostasis, and DNA damage repair, and its de-regulation is linked to neuromuscular diseases and cancer. p97 is tightly controlled by numerous regulatory cofactors, but the full range and function of the p97–cofactor network is unknown. Here, we identify the hitherto uncharacterized FAM104 proteins as a conserved family of p97 interactors. The two human family members VCP nuclear cofactor family member 1 and 2 (VCF1/2) bind p97 directly via a novel, alpha-helical motif and associate with p97-UFD1-NPL4 and p97-UBXN2B complexes in cells. VCF1/2 localize to the nucleus and promote the nuclear import of p97. Loss of VCF1/2 results in reduced nuclear p97 levels, slow growth, and hypersensitivity to chemical inhibition of p97 in the absence and presence of DNA damage, suggesting that FAM104 proteins are critical regulators of nuclear p97 functions.
