Optogenetic activation of heterotrimeric Gi proteins by LOV2GIVe— a rationally engineered modular protein

Garcia‐Marcos, Mikel; Parag-Sharma, Kshitij; Marivin, Arthur; Maziarz, Marcin; Luebbers, Alex; Nguyen, Lien T.

doi:10.1101/2020.08.17.253781

Cited by 3 publications

(3 citation statements)

References 47 publications

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“…2, top). The amplitude of the responses by the two non-receptor GEFs was comparable to that observed upon stimulation of the M4 muscarinic receptor (M4R), a prototypical Gi-activating GPCR, with an agonist concentration that elicits maximal activation in this assay format (Garcia-Marcos et al, 2020). In contrast, formation of Gαi-GTP upon rapamycin stimulation was only detected in cells expressing AGS1 but not in cells expressing GIV or Ric-8A (Fig.…”

Section: Non-receptor Gefs Display Different Abilities To Promote Gα-supporting

confidence: 54%

Complementary biosensors reveal different G-protein signaling modes triggered by GPCRs and non-receptor activators

Garcia‐Marcos

2021

eLife

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It has become evident that activation of heterotrimeric G-proteins by cytoplasmic proteins that are not GPCRs plays a role in physiology and disease. Despite sharing the same biochemical Guanine-nucleotide Exchange Factor (GEF) activity as GPCRs in vitro, the mechanisms by which these cytoplasmic proteins trigger G-protein-dependent signaling in cells have not been elucidated. Heterotrimeric G-proteins can give rise to two active signaling species, Gα-GTP and dissociated Gβγ, with different downstream effectors, but how non-receptor GEFs affect the levels of these two species in cells is not known. Here, a systematic comparison of GPCRs and three unrelated non-receptor proteins with GEF activity in vitro (GIV/Girdin, AGS1, and Ric-8A) revealed high divergence in their contribution to generating Gα-GTP and free Gβγ in cells directly measured with live-cell biosensors. These findings demonstrate fundamental differences in how receptor and non-receptor G-protein activators promote signaling in cells despite sharing similar biochemical activities in vitro.

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Section: Non-receptor Gefs Display Different Abilities To Promote Gα-supporting

confidence: 54%

Complementary biosensors reveal different G-protein signaling modes triggered by GPCRs and non-receptor activators

Garcia‐Marcos

2021

eLife

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“…Similarly, an LICcompatible GINIP amplicon was inserted into the pLIC-GST plasmid kindly provided by J. Sondek (University of North Carolina at Chapel Hill) (Cabrita et al, 2006) to generate pLIC-GST-GINIP. Plasmids for the bacterial expression of His-Gαi3 (rat), GST-Gαi3, His-Gαi1, His-Gαi2, and His-Gαo have been described previously (Garcia-Marcos et al, 2009;Garcia-Marcos et al, 2020;Marivin et al, 2020). The plasmid pET24a-Gαi3 used for the bacterial expression of human His-Gαi3 was a kind gift of I. Shimada (de Opakua et al, 2017).…”

Section: Plasmidsmentioning

confidence: 99%

Fine-tuning GPCR-mediated neuromodulation by biasing signaling through different G-protein subunits

Park

Luebbers

Dao

et al. 2023

Preprint

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GPCRs mediate neuromodulation through activation of heterotrimeric G-proteins (Gαβγ). Classical models depict that G-protein activation leads to a one-to-one formation of Gα-GTP and Gβγ species. Each of these species propagates signaling by independently acting on effectors, but the mechanisms by which response fidelity is ensured by coordinating Gα and Gβγ responses remain unknown. Here, we reveal a paradigm of G-protein regulation whereby the neuronal protein GINIP biases inhibitory GPCR responses to favor Gβγ over Gα signaling. Tight binding of GINIP to Gαi-GTP precludes its association with effectors (adenylyl cyclase) and, simultaneously, with Regulator-of-G-protein-Signaling (RGS) proteins that accelerate deactivation. As a consequence, Gαi-GTP signaling is dampened whereas Gβγ signaling is enhanced. We show that this mechanism is essential to prevent imbalances of neurotransmission that underlie increased seizure susceptibilityin vivo. Our findings reveal an additional layer of regulation within a quintessential mechanism of signal transduction that sets the tone of neurotransmission.

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“…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).…”

Section: Experimental Procedures Plasmidsmentioning

confidence: 99%

Dissecting the molecular basis for the modulation of neurotransmitter GPCR signaling by GINIP

Luebbers

Zhou

Eyles

et al. 2023

Preprint

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It is well-established that activation of heterotrimeric G-proteins (Gαβγ) by G-protein-coupled receptors (GPCRs) stimulated by neurotransmitters is a key mechanism underlying neuromodulation. Much less is known about how G-protein regulation after receptor-mediated activation contributes to neuromodulation. Recent evidence indicates that the neuronal protein GINIP shapes GPCR inhibitory neuromodulation via a unique mechanism of G-protein regulation that controls neurological processes like pain and seizure susceptibility. However, the molecular basis of this mechanism remains ill-defined because the structural determinants of GINIP responsible for binding Gαi subunits and regulating G-protein signaling are not known. Here, we combined hydrogen-deuterium exchange mass-spectrometry, protein folding predictions, bioluminescence resonance energy transfer assays, and biochemical experiments to identify the first loop of the PHD domain of GINIP as an obligatory requirement for Gαi binding. Surprisingly, our results support a model in which GINIP undergoes a long-range conformational change to accommodate Gαi binding to this loop. Using cell-based assays, we demonstrate that specific amino acids in the first loop of the PHD domain are essential for the regulation of Gαi-GTP and free Gβγ signaling upon neurotransmitter GPCR stimulation. In summary, these findings shed light onto the molecular basis for a post-receptor mechanism of G-protein regulation that fine-tunes inhibitory neuromodulation.

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Optogenetic activation of heterotrimeric Gi proteins by LOV2GIVe— a rationally engineered modular protein

Cited by 3 publications

References 47 publications

Complementary biosensors reveal different G-protein signaling modes triggered by GPCRs and non-receptor activators

Complementary biosensors reveal different G-protein signaling modes triggered by GPCRs and non-receptor activators

Fine-tuning GPCR-mediated neuromodulation by biasing signaling through different G-protein subunits

Dissecting the molecular basis for the modulation of neurotransmitter GPCR signaling by GINIP

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