Visualization of endogenous G proteins on endosomes and other organelles
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eLife assessment
This important study investigates the intracellular localization patterns of G proteins involved in GPCR signaling, presenting convincing evidence for their preference for plasma and lysosomal membranes over endosomal, endoplasmic reticulum, and Golgi membranes. This discovery has significant implications for understanding GPCR action and signaling from intracellular locations. This research will interest cell biologists studying protein trafficking and pharmacologists exploring localized signaling phenomena.
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Abstract
Classical G protein-coupled receptor (GPCR) signaling takes place in response to extracellular stimuli and involves receptors and heterotrimeric G proteins located at the plasma membrane. It has recently been established that GPCR signaling can also take place from intracellular membrane compartments, including endosomes that contain internalized receptors and ligands. While the mechanisms of GPCR endocytosis are well understood, it is not clear how internalized receptors are supplied with G proteins. To address this gap we use gene editing, confocal microscopy, and bioluminescence resonance energy transfer to study the distribution and trafficking of endogenous G proteins. We show here that constitutive endocytosis is sufficient to supply newly internalized endocytic vesicles with 20-30% of the G protein density found at the plasma membrane. We find that G proteins are present on early, late, and recycling endosomes, are abundant on lysosomes, but are virtually undetectable on the endoplasmic reticulum, mitochondria, and the medial Golgi apparatus. Receptor activation does not change heterotrimer abundance on endosomes. Our results provide a detailed subcellular map of endogenous G protein distribution, suggest that G proteins may be partially excluded from nascent endocytic vesicles, and are likely to have implications for GPCR signaling from endosomes and other intracellular compartments.
Article activity feed
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Reviewer #1 (Public Review):
Summary:
The manuscript by Jang et al. describes the application of new methods to measure the localization of GTP-binding signaling proteins (G proteins) on different membrane structures in a model mammalian cell line (HEK293). G proteins mediate signaling by receptors found at the cell surface (GPCRs), with evidence from the last 15 years suggesting that GPCRs can induce G-protein mediated signaling from different membrane structures within the cell, with variation in signal localization leading to different cellular outcomes. While it has been clearly shown that different GPCRs efficiently traffic to various intracellular compartments, it is less clear whether G proteins traffic in the same manner, and whether GPCR trafficking facilitates "passenger" G protein trafficking. This question was a blind spot …
Reviewer #1 (Public Review):
Summary:
The manuscript by Jang et al. describes the application of new methods to measure the localization of GTP-binding signaling proteins (G proteins) on different membrane structures in a model mammalian cell line (HEK293). G proteins mediate signaling by receptors found at the cell surface (GPCRs), with evidence from the last 15 years suggesting that GPCRs can induce G-protein mediated signaling from different membrane structures within the cell, with variation in signal localization leading to different cellular outcomes. While it has been clearly shown that different GPCRs efficiently traffic to various intracellular compartments, it is less clear whether G proteins traffic in the same manner, and whether GPCR trafficking facilitates "passenger" G protein trafficking. This question was a blind spot in the burgeoning field of GPCR localized signaling in need of careful study, and the results obtained will serve as an important guidepost for further work in this field. The extent to which G proteins localize to different membranes within the cell is the main experimental question tested in this manuscript. This question is pursued through two distinct methods, both relying on genetic modification of the G-beta subunit with a tag. In one method, G-beta is modified with a small fragment of the fluorescent protein mNG, which combines with the larger mNG fragment to form a fully functional fluorescent protein to facilitate protein trafficking by fluorescent microscopy. This approach was combined with the expression of fluorescent proteins directed to various intracellular compartments (different types of endosomes, lysosome, endoplasmic reticulum, Golgi, mitochondria) to look for colocalization of G-beta with these markers. These experiments showed compelling evidence that G-beta co-localizes with markers at the plasma membrane and the lysosome, with weak or absent co-localization for other markers. A second method for measuring localization relied on fusing G-beta with a small fragment from a miniature luciferase (HiBit) that combines with a larger luciferase fragment (LgBit) to form an active luciferase enzyme. Localization of G-beta (and luciferase signal) was measured using a method known as bystander BRET, which relies on the expression of a fluorescent protein BRET acceptor in different cellular compartments. Results using bystander BRET supported findings from fluorescence microscopy experiments. These methods for tracking G protein localization were also used to probe other questions. The activation of GPCRs from different classes had virtually no impact on the localization of G-beta, suggesting that GPCR activation does not result in the shuttling of G proteins through the endosomal pathway with activated receptors.
Strengths:
The question probed in this study is quite important and, in my opinion, understudied by the pharmacology community. The results presented here are an important call to be cognizant of the localization of GPCR coupling partners in different cellular compartments. Abundant reports of endosomal GPCR signaling need to consider how the impact of lower G protein abundance on endosomal membranes will affect the signaling responses under study.
The work presented is carefully executed, with seemingly high levels of technical rigor. These studies benefit from probing the experimental questions at hand using two different methods of measurement (fluorescent microscopy and bystander BRET). The observation that both methods arrive at the same (or a very similar) answer inspires confidence about the validity of these findings.
Weaknesses:
The rationale for fusing G-beta with either mNG2(11) or SmBit could benefit from some expansion. I understand the speculation that using the smallest tag possible may have the smallest impact on protein performance and localization, but plenty of researchers have fused proteins with whole fluorescent proteins to provide conclusions that have been confirmed by other methods. Many studies even use G proteins fused with fluorescent proteins or luciferases. Is there an important advantage to tagging G-beta with small tags? Is there evidence that G proteins with full-size protein tags behave aberrantly? If the studies presented here would not have been possible without these CRISPR-based tagging approaches, it would be helpful to provide more context to make this clearer. Perhaps one factor would be interference from newly synthesized G proteins-fluorescent protein fusions en route to the plasma membrane (in the ER and Golgi).
As noted by the authors, they do not demonstrate that the tagged G-beta is predominantly found within heterotrimeric G protein complexes. If there is substantial free G-beta, then many of the conclusions need to be reconsidered. Perhaps a comparison of immunoprecipitated tagged G beta vs immunoprecipitated supernatant, with blotting for other G protein subunits would be informative.
Additional context and questions:
(1) There exists some evidence that certain GPCRs can form enduring complexes with G-beta-gamma (Pubmed: 23297229, 27499021). That would seem to offer a mechanism that would enable receptor-mediated transport of G protein subunits. It would be helpful for the authors to place the findings of this manuscript in the context of these previous findings since they seem somewhat contradictory.
(2) There is some evidence that GaS undergoes measurable dissociation from the plasma membrane upon activation (see the mechanism of the assay in Pubmed: 35302493). It seems possible that G-alpha (and in particular GaS) might behave differently than the G-beta subunit studied here. This is not entirely clear from the discussion as it now stands.
(3) The authors say "The presence of mNG-b1 on late endosomes suggested that some G proteins may be degraded by lysosomes". The mechanism of lysosomal degradation by proteins on the outside of the lysosome is not clear. It would be helpful for the authors to clarify.
(4) Although the authors do a good job of assessing G protein dilution in endosomal membranes, it is unclear how this behavior compares to the measurement of other lipid-anchored proteins using the same approach. Is the dilution of G proteins what we would expect for any lipid-anchored protein at the inner leaflet of the plasma membrane?
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Reviewer #2 (Public Review):
This is an interesting method that addresses the important problem of assessing G protein localization at endogenous levels. The data are generally convincing.
Specific comments
Methods:
The description of the gene editing method is unclear. There are two different CRISPR cell lines made in two different cell backgrounds. The methods should clearly state which CRISPR guides were used on which cell line. It is also not clear why HiBit is included in the mNG-β1 construct. Presumably, this is not critical but it would be helpful to explicitly note. In general, the Methods could be more complete.Results:
The explanation of validation experiments in Figures 1 C and D is incomplete and difficult to follow. The rationale and explanation of the experiments could be expanded. In addition, because this is an …Reviewer #2 (Public Review):
This is an interesting method that addresses the important problem of assessing G protein localization at endogenous levels. The data are generally convincing.
Specific comments
Methods:
The description of the gene editing method is unclear. There are two different CRISPR cell lines made in two different cell backgrounds. The methods should clearly state which CRISPR guides were used on which cell line. It is also not clear why HiBit is included in the mNG-β1 construct. Presumably, this is not critical but it would be helpful to explicitly note. In general, the Methods could be more complete.Results:
The explanation of validation experiments in Figures 1 C and D is incomplete and difficult to follow. The rationale and explanation of the experiments could be expanded. In addition, because this is an interesting method, it would be helpful to know if the endogenous editing affects normal GPCR signaling. For example, the authors could include data showing an Iso-induced cAMP response. This is not critical to the present interpretation but is relevant as a general point regarding the method. Also, it may be relevant to the interpretation of receptor effects on G protein localization.Discussion:
The conclusion that beta-gamma subunits do not redistribute after GPCR activation seems new and different from previous reports. Is this correct? Can the authors elaborate on how the results compare to previous literature?Can the authors note that OpenCell has endogenously tagged Gβ1 and reports more obvious internal localization? Can the authors comment on this point?
Is this the first use of CRISPR / HiBit for BRET assay? It would be helpful to know this or cite previous work if not. Also, as this is submitted as a tools piece, the authors might say a little more about the potential application to other questions.
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Reviewer #3 (Public Review):
Summary:
This article addresses an important and interesting question concerning intracellular localization and dynamics of endogenous G proteins. The fate and trafficking of G protein-coupled receptors (GPCRs) have been extensively studied but so far little is known about the trafficking routes of their partner G proteins that are known to dissociate from their respective receptors upon activation of the signaling pathway. The authors utilize modern cell biology tools including genome editing and bystander bioluminescence resonance energy transfer (BRET) to probe intracellular localization of G proteins in various membrane compartments in steady state and also upon receptor activation. Data presented in this manuscript shows that while G proteins are mostly present on the plasma membrane, they can be also …
Reviewer #3 (Public Review):
Summary:
This article addresses an important and interesting question concerning intracellular localization and dynamics of endogenous G proteins. The fate and trafficking of G protein-coupled receptors (GPCRs) have been extensively studied but so far little is known about the trafficking routes of their partner G proteins that are known to dissociate from their respective receptors upon activation of the signaling pathway. The authors utilize modern cell biology tools including genome editing and bystander bioluminescence resonance energy transfer (BRET) to probe intracellular localization of G proteins in various membrane compartments in steady state and also upon receptor activation. Data presented in this manuscript shows that while G proteins are mostly present on the plasma membrane, they can be also detected in endosomal compartments, especially in late endosomes and lysosomes. This distribution, according to data presented in this study, seems not to be affected by receptor activation. These findings will have implications in further studies addressing GPCR signaling mechanisms from intracellular compartments.
Strengths:
The methods used in this study are adequate for the question asked. Especially, the use of genome-edited cells (for the addition of the tag on one of the G proteins) is a great choice to prevent the effects of overexpression. Moreover, the use of bystander BRET allowed authors to probe the intracellular localization of G proteins in a very high-throughput fashion. By combining imaging and BRET authors convincingly show that G proteins are very low abundant on early endosomes (also ER, mitochondria, and medial Golgi), however seem to accumulate on membranes of late endosomal compartments.
Weaknesses:
While the authors provide a novel dataset, many questions regarding G protein trafficking remain open. For example, it is not entirely clear which pathway is utilized to traffic G proteins from the plasma membrane to intracellular compartments. Additionally, future studies should also address the dynamics of G protein trafficking, for example by tracking them over multiple time points.
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eLife assessment
This important study investigates the intracellular localization patterns of G proteins involved in GPCR signaling, presenting convincing evidence for their preference for plasma and lysosomal membranes over endosomal, endoplasmic reticulum, and Golgi membranes. This discovery has significant implications for understanding GPCR action and signaling from intracellular locations. This research will interest cell biologists studying protein trafficking and pharmacologists exploring localized signaling phenomena.
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