Effector membrane translocation biosensors reveal G protein and βarrestin coupling profiles of 100 therapeutically relevant GPCRs
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Evaluation Summary:
A challenge to understanding the physiology and therapeutic potential of G protein-coupled receptors (GPCRs) is to understand the range of their couplings to different G proteins. Avet et al developed of a novel set of biosensors to assess the coupling specificity of 100 therapeutically relevant G protein-coupled receptors (GPCRs) to various G protein isoforms and arrestins. The novel screen and results obtained with reference ligands will have broad use for researchers studying GPCRs, potentially impacting discovery of new physiological pathways, understanding adverse effects of currently marketed therapeutics, and discovery of novel, safer therapeutics.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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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 βarrestins 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 G s ). 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 1500 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.
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Evaluation Summary:
A challenge to understanding the physiology and therapeutic potential of G protein-coupled receptors (GPCRs) is to understand the range of their couplings to different G proteins. Avet et al developed of a novel set of biosensors to assess the coupling specificity of 100 therapeutically relevant G protein-coupled receptors (GPCRs) to various G protein isoforms and arrestins. The novel screen and results obtained with reference ligands will have broad use for researchers studying GPCRs, potentially impacting discovery of new physiological pathways, understanding adverse effects of currently marketed therapeutics, and discovery of novel, safer therapeutics.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes …
Evaluation Summary:
A challenge to understanding the physiology and therapeutic potential of G protein-coupled receptors (GPCRs) is to understand the range of their couplings to different G proteins. Avet et al developed of a novel set of biosensors to assess the coupling specificity of 100 therapeutically relevant G protein-coupled receptors (GPCRs) to various G protein isoforms and arrestins. The novel screen and results obtained with reference ligands will have broad use for researchers studying GPCRs, potentially impacting discovery of new physiological pathways, understanding adverse effects of currently marketed therapeutics, and discovery of novel, safer therapeutics.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
The goal of health scientists is to find better ways to treat patients for the various ailments. One of the most significant challenges we face is developing safer and more efficacious therapeutics. In creating safer therapeutics our goal as scientists is to understand and minimize adverse effects, usually caused by off-target effects. In some cases, the adverse effects are caused by on-target but related to a complex signal transduction pathway. To dissect this complexity, Avet et al developed a novel set of biosensors to assess the coupling specificity of 100 therapeutically relevant G protein-coupled receptors (GPCRs) to various G protein isoforms and arrestins. In their manuscript, the authors tested reference ligands defining each receptor and assessed their capacity to interact with a 12 different G …
Reviewer #1 (Public Review):
The goal of health scientists is to find better ways to treat patients for the various ailments. One of the most significant challenges we face is developing safer and more efficacious therapeutics. In creating safer therapeutics our goal as scientists is to understand and minimize adverse effects, usually caused by off-target effects. In some cases, the adverse effects are caused by on-target but related to a complex signal transduction pathway. To dissect this complexity, Avet et al developed a novel set of biosensors to assess the coupling specificity of 100 therapeutically relevant G protein-coupled receptors (GPCRs) to various G protein isoforms and arrestins. In their manuscript, the authors tested reference ligands defining each receptor and assessed their capacity to interact with a 12 different G proteins and 2 arrestins, each contributing to distinct signal transduction pathways. The novel screen and results obtained with reference ligands will have a direct impact on understand adverse effects of currently marketed therapeutics but also on the discovery of novel, safer therapeutics.
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Reviewer #2 (Public Review):
The authors describe unique resources for studying GPCR coupling to different G proteins. The manuscript describes a full set of "effector membrane translocation assay" (EMTA) tools that can be used to assess activation of Gi/o, Gq and G12/13 families, and pair this with a Gs membrane translocation assay to assess activation of 12 G proteins and 2 arrestins by 100 GPCRs. The data presented represents a valuable resource for investigators interested in the precise signaling pathways that mediate physiological events, and the tools described are likely to be useful for many specific studies of individual receptors, including efforts to discover ligands that display functional selectivity (bias) between G protein pathways or between G proteins and arrestins. The authors make the case that their system has a …
Reviewer #2 (Public Review):
The authors describe unique resources for studying GPCR coupling to different G proteins. The manuscript describes a full set of "effector membrane translocation assay" (EMTA) tools that can be used to assess activation of Gi/o, Gq and G12/13 families, and pair this with a Gs membrane translocation assay to assess activation of 12 G proteins and 2 arrestins by 100 GPCRs. The data presented represents a valuable resource for investigators interested in the precise signaling pathways that mediate physiological events, and the tools described are likely to be useful for many specific studies of individual receptors, including efforts to discover ligands that display functional selectivity (bias) between G protein pathways or between G proteins and arrestins. The authors make the case that their system has a unique advantage in that most of the G proteins studied need not be modified. While this advantage is not empirically demonstrated in the manuscript (assay systems are not directly compared), the availability of multiple assay systems based on different principles is obviously advantageous. The manuscript also includes some very nice observations, such as the ability to detect signals produced by endogenous receptors and G proteins, and a near-universal assay system that combines Gz and G15 assays. The overall utility of the assay system, which should have significant impact on the field, is well-supported by the data.
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Reviewer #3 (Public Review):
Building on previous work (Namkung, Nat communications 2016 & Science Signaling 2018) the authors now develop a set of new enhance bystander BRET (ebBRET) biosensors to measure the GPCR coupling with all G proteins classes and barrestin. Using this set, they then map the effector coupling possibilities of 100 GPCRs in HEK293 cells. The biosensors optimally function upon heterologous expression of GPCRs and the relevant G proteins, although the authors show that they can be used when endogenous expression levels and/or effector coupling are sufficient. The authors claim that since GPCR and g proteins are untagged that these biosensors represent potential advantages over alternative developed technologies, although it is not clear if a systematic comparison has been done and differences revealed. From their …
Reviewer #3 (Public Review):
Building on previous work (Namkung, Nat communications 2016 & Science Signaling 2018) the authors now develop a set of new enhance bystander BRET (ebBRET) biosensors to measure the GPCR coupling with all G proteins classes and barrestin. Using this set, they then map the effector coupling possibilities of 100 GPCRs in HEK293 cells. The biosensors optimally function upon heterologous expression of GPCRs and the relevant G proteins, although the authors show that they can be used when endogenous expression levels and/or effector coupling are sufficient. The authors claim that since GPCR and g proteins are untagged that these biosensors represent potential advantages over alternative developed technologies, although it is not clear if a systematic comparison has been done and differences revealed. From their work, the authors show that combining two biosensors to measure Galpha15 and GalphaZ coupling provides a quasi-universal biosensor to measure GPCR activation that could be useful to identify off-target activation or polypharmacology.
Strengths:
This is an impressive quantity of work describing the possible GPCR coupling for 100 GPCRs (in one cell type), which are an important class of drug targets. The authors have now assembled a collection of ebBRET sensors (some previously reported ones and some new ones) that monitor activation of all heterotrimeric G proteins and b-arrestin that could be used to profile ligands and uncover functional selectivity at G proteins or b-arrestin pathways. These new sensors called EMTA, are based on the plasma membrane translocation of effectors. EMTA was found to be suitable for the detection of constitutive activity, inverse agonism, biased signaling and polypharmacology. The uncovering that combining Galpha15 and GalphaZ biosensors could enable monitoring activation of virtually all GPCRs could also lead to a new screening platform and/or allow to identify off-target activation or polypharmacology properties of various ligands.
Weaknesses:
Although one can imagine potential use for the information reported in the manuscript and from the new tools to study GPCR signaling, the present study is mostly descriptive and stops short of exploring some new biology made possible by these new technological advances. Given that in most contexts biosensors are used with overexpression of GPCRs and G proteins, some concerns exist about the relevance of the information presented, especially in the absence of validation.
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