Jingyi Zhao’s research while affiliated with Boston University and other places

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Publications (12)


A sensitive biosensor of endogenous Gα i activity enables the accurate characterization of endogenous GPCR agonist responses
  • Article

March 2025

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48 Reads

Science Signaling

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Remi Janicot

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Jingyi Zhao

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[...]

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Mikel Garcia-Marcos

The activation of heterotrimeric G proteins (Gαβγ) by G protein-coupled receptors (GPCRs) is a mechanism broadly used by eukaryotes to transduce signals across the plasma membrane and a target for many clinical drugs. Many optical biosensors commonly used for measuring GPCR-stimulated G protein activity rely on exogenously expressed GPCRs and/or G proteins, which compromise readout fidelity. Biosensors that measure endogenous signaling may interfere with the signaling process under investigation or have a limited dynamic range of detection , hindering applicability. Here, we developed an optical BRET-based biosensor, Gα i bONE-GO, that detects endogenous GTP-bound (active) Gα i upon stimulation of endogenous GPCRs more robustly than existing sensors of endogenous activity. Its design leverages the Gα i-binding protein GINIP as a high-affinity and specific detector of Gα i-GTP. We optimized this design to prevent interference with downstream G i-dependent signaling and to enable implementation in different experimental systems having endogenous GPCRs, including adenosine receptors in primary astroglial cells and opioid receptors in cell lines. In a neuronal cell line, Gα i bONE-GO revealed activation profiles indicating that several natural opioid neuropeptides acted as partial agonists, in contrast with their characterization as full agonists using biosensors that depend on exogenously expressed receptors and G proteins. The Gα i bONE-GO biosensor is a direct and sensitive detector of endogenous activation of Gα i proteins by GPCRs in different experimental settings but does not interfere with the subsequent propagation of signaling.


Direct detection of endogenous Gαi activity in cells with a sensitive conformational biosensor
  • Preprint
  • File available

August 2024

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43 Reads

Activation of heterotrimeric G-proteins (Gαβγ) by G-protein-coupled receptors (GPCRs) is not only a mechanism broadly used by eukaryotes to transduce signals across the plasma membrane, but also the target for a large fraction of clinical drugs. However, approaches typically used to assess this signaling mechanism by directly measuring G-protein activity, like optical biosensors, suffer from limitations. On one hand, many of these biosensors require expression of exogenous GPCRs and/or G-proteins, compromising readout fidelity. On the other hand, biosensors that measure endogenous signaling may still interfere with the signaling process under investigation or suffer from having a small dynamic range of detection, hindering broad applicability. Here, we developed an optical biosensor that detects the endogenous G-protein active species Gαi-GTP upon stimulation of endogenous GPCRs more robustly than current state-of-the-art sensors for the same purpose. Its design is based on the principle of bystander Bioluminescence Resonance Energy Transfer (BRET) and leverages the Gαi-binding protein named GINIP as a high affinity and specific detector module of the GTP-bound conformation of Gαi. We optimized this design to prevent interference with Gi-dependent signaling (cAMP inhibition) and to enable implementation in different experimental systems with endogenous GPCRs, including neurotransmitter receptors in primary astroglial cells or opioid receptors in cell lines, which revealed opioid neuropeptide-mediated activation profiles different from those observed with other biosensors involving exogenous GPCRs and G-proteins. Overall, we introduce a biosensor that directly and sensitively detects endogenous activation of G-proteins by GPCRs across different experimental settings without interfering with the subsequent propagation of signaling.

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Direct interrogation of context-dependent GPCR activity with a universal biosensor platform

January 2024

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57 Reads

G protein-coupled receptors (GPCRs) are the largest family of druggable proteins in the human genome, but progress in understanding and targeting them is hindered by the lack of tools to reliably measure their nuanced behavior in physiologically-relevant contexts. Here, we developed a collection of compact ONE vector G -protein O ptical (ONE-GO) biosensor constructs as a scalable platform that can be conveniently deployed to measure G-protein activation by virtually any GPCR with high fidelity even when expressed endogenously in primary cells. By characterizing dozens of GPCRs across many cell types like primary cardiovascular cells or neurons, we revealed new insights into the molecular basis for G-protein coupling selectivity of GPCRs, pharmacogenomic profiles of anti-psychotics on naturally-occurring GPCR variants, and G-protein subtype signaling bias by endogenous GPCRs depending on cell type or upon inducing disease-like states. In summary, this open-source platform makes the direct interrogation of context-dependent GPCR activity broadly accessible.


Fig. 1. Small-molecule screening to identify inhibitors of the GIV-Gαi interaction. (A) Diagram depicting the rationale for targeting the GIV-Gαi interaction with small molecules. (B) Scheme of the full screening campaign. (C) Confirmation of hit compounds that inhibit the GIV-Gαi interaction in two orthogonal biochemical assays, FP and AS. (D) Triage of compounds based on unfavorable chemical properties and availability of quality controlled molecules. (E) Test of 69 IGGi compounds (100 μM) on MDA-MB-231 cell migration and viability. Red, <30% reduction; blue, >30% reduction. Mean ± SEM (N = 4). (F) Comparison of the effect of IGGi compounds (100 μM) on the viability of three breast cell lines, MDA-MB-231, MCF-7, and MCF-10A (mean of N = 3).
Fig. 3. Lack of effect of IGGi-11 on G-protein coupling to GPCRs and effectors. (A) Diagram of key steps and protein interactions involved in Gαi-subunit functions. (B) IGGi-11 does not dissociate Gβγ from Gαi3 in membranes isolated from HEK293T cells expressing a BRET-based biosensor for free Gβγ, whereas two positive controls do (a GoLoco peptide derived from RGS12, R12 GL, 25 μM; and GTPγS 300 μM). (C-F) IGGi-11 does not affect GPCR-mediated activation of G i3 as determined by the dissociation of Gαi3-Gβγ heterotrimers (C and D) or the formation of Gαi3-GTP (E and F) using BRET-based biosensors. In C and E, membranes isolated from HEK293T cells expressing the α2 A adrenergic receptor were treated with the indicated concentrations of IGGi-11 with (green) or without (blue) stimulation with a receptor agonist (brimonidine, 1 μM) for 2 min before BRET measurements. In D and F, BRET was continuously measured in real time in the presence of 100 μM IGGi-11 or vehicle (1% dimethyl sulfoxide (DMSO), v:v). (G) IGGi-11 does not interfere with G-protein-mediated regulation of adenylyl cyclase. Membranes isolated from HEK293T cells expressing adenylyl cyclase 5 were treated with IGGi-11 (100 μM), purified Gαs (0.5 μM), purified myristoylated Gαi1 (Gαi, 1 μM), and forskolin (Fsk, 10 μM) in the combinations indicated in the graphs. Mean ± SEM (N ≥ 3). **P < 0.01, ANOVA.
Small-molecule targeting of GPCR-independent noncanonical G-protein signaling in cancer

April 2023

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87 Reads

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11 Citations

Proceedings of the National Academy of Sciences

Activation of heterotrimeric G-proteins (Gαβγ) by G-protein-coupled receptors (GPCRs) is a quintessential mechanism of cell signaling widely targeted by clinically approved drugs. However, it has become evident that heterotrimeric G-proteins can also be activated via GPCR-independent mechanisms that remain untapped as pharmacological targets. GIV/Girdin has emerged as a prototypical non-GPCR activator of G proteins that promotes cancer metastasis. Here, we introduce IGGi-11, a first-in-class small-molecule inhibitor of noncanonical activation of heterotrimeric G-protein signaling. IGGi-11 binding to G-protein α-subunits (Gαi) specifically disrupted their engagement with GIV/Girdin, thereby blocking noncanonical G-protein signaling in tumor cells and inhibiting proinvasive traits of metastatic cancer cells. In contrast, IGGi-11 did not interfere with canonical G-protein signaling mechanisms triggered by GPCRs. By revealing that small molecules can selectively disable noncanonical mechanisms of G-protein activation dysregulated in disease, these findings warrant the exploration of therapeutic modalities in G-protein signaling that go beyond targeting GPCRs.


Small-molecule targeting of GPCR-independent non-canonical G protein signaling inhibits cancer progression

February 2023

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25 Reads

Activation of heterotrimeric G-proteins (Gαβγ) by G-protein-coupled receptors (GPCRs) is a quintessential mechanism of cell signaling widely targeted by clinically-approved drugs. However, it has become evident that heterotrimeric G-proteins can also be activated via GPCR-independent mechanisms that remain untapped as pharmacological targets. GIV/Girdin has emerged as a prototypical non-GPCR activator of G proteins that promotes cancer metastasis. Here, we introduce IGGi-11, a first-in-class small-molecule inhibitor of non-canonical activation of heterotrimeric G-protein signaling. IGGi-11 binding to G-protein α-subunits (Gαi) specifically disrupted their engagement with GIV/Girdin, thereby blocking non-canonical G-protein signaling in tumor cells, and inhibiting pro-invasive traits of metastatic cancer cells in vitro and in mice. In contrast, IGGi-11 did not interfere with canonical G-protein signaling mechanisms triggered by GPCRs. By revealing that small molecules can selectively disable non-canonical mechanisms of G-protein activation dysregulated in disease, these findings warrant the exploration of therapeutic modalities in G-protein signaling that go beyond targeting GPCRs.



Naturally-occurring hotspot cancer mutations in Gα13 promote oncogenic signaling

October 2020

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60 Reads

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25 Citations

Journal of Biological Chemistry

s> Heterotrimeric G-proteins are signaling switches broadly divided into four families based on the sequence and functional similarity of their Gα subunits: Gs, Gi/o, Gq/11 and G12/13. Artificial mutations that activate Gα subunits of each of these families have long been known to induce oncogenic transformation in experimental systems. With the advent of next-generation sequencing, activating hotspot mutations in Gs, Gi/o or Gq/11 proteins have also been identified in patient tumor samples. In contrast, patient tumor-associated G12/13 mutations characterized to date lead to inactivation rather than activation. By using bioinformatic pathway analysis and signaling assays, here we identified cancer-associated hotspot mutations in Arg-200 of Gα13 (encoded by GNA13) as potent activators of oncogenic signaling. First, we found that components of a G12/13-dependent signaling cascade that culminates in activation of the Hippo pathway effectors YAP and TAZ is frequently altered in bladder cancer. Upregulation of this signaling cascade correlates with increased YAP/TAZ activation transcriptional signatures in this cancer type. Among the G12/13 pathway alterations were mutations in Arg-200 of Gα13, which we validated to promote YAP/TAZ-dependent (TEAD) and MRTF-A/B-depedent (SRE.L) transcriptional activity. We further showed that this mechanism relies on the same RhoGEF-RhoGTPase cascade components that are upregulated in bladder cancers. Moreover, Gα13 Arg-200 mutants induced oncogenic transformation in vitro as determined by foci formation assays. In summary, our findings on Gα13 mutants establish that naturally-occurring hotspot mutations in Gα subunits of any of the four families of heterotrimeric G-proteins are putative cancer drivers.


Citations (4)


... ; https://doi.org/10.1101/2025.04.30.651471 doi: bioRxiv preprint whereas "pulse-chase" stimulation with the highest ligand concentration importantly reduced the maximal cAMP level detected downstream β2AR and V2R compared to continuous stimulation, it did not significantly change the maximal cAMP level detected downstream LHR (figures 1A-F, S1). To assess whether sustained cAMP dynamics by LHR relies on Gs activation or decreased PDEs activity, we used the ONE-GO Gs biosensor to monitor the Gs-GTP dynamics 21 . Following the stimulation of LHR with 10 nM hCG in a continuous or transient manner, we observed a rapid Gs activation followed by a slow decrease (figure 1G, 1H). ...

Reference:

LHR and Gαs trafficking drive sustained cAMP signalling from endosomes to control steroidogenesis
Direct interrogation of context-dependent GPCR activity with a universal biosensor platform
  • Citing Article
  • February 2024

Cell

... Subsequent studies have also discovered de novo missense variants, point mutations, or deletions of Brd4 in ASD patients and developmental delay cases, correlating with neuropsychiatric phenotypes [84,85]. Furthermore, the remaining five candidate genes demonstrate multidimensional associations with neurodevelopmental disorders through distinct mechanisms as follows: epigenetic regulation (Dnmt3a [86]), cytoskeletal dynamics (Ccdc88a [87]), autophagy (Becn1 [88]), proteostasis (Psmb6 [89]), and transcriptional control (Gng2 [90]). Our PheWAS analysis corroborated these pathwayspecific associations. ...

Small-molecule targeting of GPCR-independent noncanonical G-protein signaling in cancer

Proceedings of the National Academy of Sciences

... Several lines of evidence indicate that dominant-active Gα13 promotes cell transformation and can be oncogenic in solid cancers [1,3,54,55]. However, few studies have assessed the effect of the naturally existing wild-type Gα13 on tumorigenic phenotypes in vitro or in vivo [7,12,13]. ...

Naturally-occurring hotspot cancer mutations in Gα13 promote oncogenic signaling

Journal of Biological Chemistry

... To generate GINIP protein used in HDX-MS experiments, a second bacterial expression plasmid was generated using a human GINIP sequence codon optimized for expression in E. coli, which was inserted into pET21a(+) without a TEV cleavage site preceding the His-tag (pET21a(+)-coGINIP-His). Plasmids for the bacterial expression of rat His-Gαi3 (pET28b-Gαi3) and GST-Gαi3 (pGEX-4T-1-Gαi3) have been described previously (Garcia-Marcos et al., 2009;Garcia-Marcos et al., 2020;Marivin et al., 2020). The pbb131 plasmid encoding yeast N-myristoyltransferase (NMT) (Mumby and Linder, 1994) was a gift from Maurine Linder (Cornell University). ...

DAPLE protein inhibits nucleotide exchange on Gαs and Gαq via the same motif that activates Gαi

Journal of Biological Chemistry