Heterotrimeric G protein signaling without GPCRs: The Gα-binding-and-activating (GBA) motif.

Heterotrimeric G proteins (Gαßγ) are molecular switches that relay signals from 7-transmembrane receptors located at the cell surface to the cytoplasm. The function of these receptors is so intimately linked to heterotrimeric G proteins that they are named G protein-coupled receptors (GPCRs), showcasing the interdependent nature of this archetypical receptor-transducer axis of transmembrane signaling in eukaryotes. It is generally assumed that activation of heterotrimeric G protein signaling occurs exclusively by the action of GPCRs, but this idea has been challenged by the discovery of alternative mechanisms by which G proteins can propagate signals in the cell. This review will focus on a general principle of G protein signaling that operates without the direct involvement of GPCRs. The mechanism of G protein signaling reviewed here is mediated by a class of G protein regulators defined by containing an evolutionarily conserved sequence named the Gα-binding-and-activating (GBA) motif. Using the best characterized proteins with a GBA motif as examples, Gα-interacting vesicle-associated protein (GIV)/Girdin and dishevelled-associating protein with a high frequency of leucine residues (DAPLE), this review will cover (i) the mechanisms by which extracellular cues not relayed by GPCRs promote the coupling of GBA motif-containing regulators with G proteins, (ii) the structural and molecular basis for how GBA motifs interact with Gα subunits to facilitate signaling, (iii) the relevance of this mechanism in different cellular and pathological processes, including cancer and birth defects, and (iv) strategies to manipulate GBA-G protein coupling for experimental therapeutics purposes, including the development of rationally engineered proteins and chemical probes.

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

G protein-coupled receptors (GPCRs) are the largest family of druggable proteins encoded 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 Optical (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 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.

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

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 Optical (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.

SAX-7/L1CAM acts with the adherens junction proteins MAGI-1, HMR-1/Cadherin, and AFD-1/Afadin to promote glial-mediated dendrite extension.

Adherens junctions (AJs) are a fundamental organizing structure for multicellular life. Although AJs are studied mainly in epithelia, their core function - stabilizing cell contacts by coupling adhesion molecules to the cytoskeleton - is important in diverse tissues. We find that two C. elegans sensory neurons, URX and BAG, require conserved AJ proteins for dendrite morphogenesis. We previously showed that URX and BAG dendrites attach to the embryonic nose via the adhesion molecule SAX-7/L1CAM, acting both in neurons and glia, and then extend by stretch during embryo elongation. Here, we find that a PDZ-binding motif (PB) in the SAX-7 cytoplasmic tail acts with other interaction motifs to promote dendrite extension. Using pull-down assays, we find that the SAX-7 PB binds the multi-PDZ scaffolding protein MAGI-1, which bridges it to the cadherin-catenin complex protein HMP-2/ß-catenin. Using cell-specific rescue and depletion, we find that both MAGI-1 and HMR-1/Cadherin act in glia to non-autonomously promote dendrite extension. Double mutant analysis indicates that each protein can act independently of SAX-7, suggesting a multivalent adhesion complex. The SAX-7 PB motif also binds AFD-1/Afadin, loss of which further enhances sax-7 BAG dendrite defects. As MAGI-1, HMR-1, and AFD-1 are all found in epithelial AJs, we propose that an AJ-like complex in glia promotes dendrite extension.

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

It is well established that G-protein-coupled receptors (GPCRs) stimulated by neurotransmitters are critical for neuromodulation. Much less is known about how heterotrimeric G-protein (Gαßγ) 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 pain and seizure susceptibility. However, the molecular basis of this mechanism remains ill-defined because the structural determinants of GINIP responsible for binding and regulating G proteins are not known. Here, we combined hydrogen-deuterium exchange mass spectrometry, computational structure predictions, biochemistry, and cell-based biophysical assays to demonstrate an effector-like binding mode of GINIP to Gαi. Specific amino acids of GINIP's PHD domain first loop are essential for G-protein binding and subsequent regulation of Gαi-GTP and Gßγ 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.

Synaptic plasticity via receptor tyrosine kinase/G-protein-coupled receptor crosstalk.

Cellular signaling involves a large repertoire of membrane receptors operating in overlapping spatiotemporal regimes and targeting many common intracellular effectors. However, both the molecular mechanisms and the physiological roles of crosstalk between receptors, especially those from different superfamilies, are poorly understood. We find that the receptor tyrosine kinase (RTK) TrkB and the G-protein-coupled receptor (GPCR) metabotropic glutamate receptor 5 (mGluR5) together mediate hippocampal synaptic plasticity in response to brain-derived neurotrophic factor (BDNF). Activated TrkB enhances constitutive mGluR5 activity to initiate a mode switch that drives BDNF-dependent sustained, oscillatory Ca2+ signaling and enhanced MAP kinase activation. This crosstalk is mediated, in part, by synergy between Gßγ, released by TrkB, and Gαq-GTP, released by mGluR5, to enable physiologically relevant RTK/GPCR crosstalk.

Synaptic plasticity via receptor tyrosine kinase/G protein-coupled receptor crosstalk.

Cellular signaling involves a large repertoire of membrane receptors operating in overlapping spatiotemporal regimes and targeting many common intracellular effectors. However, both the molecular mechanisms and physiological roles of crosstalk between receptors, especially those from different superfamilies, are poorly understood. We find that the receptor tyrosine kinase (RTK), TrkB, and the G protein-coupled receptor (GPCR), metabotropic glutamate receptor 5 (mGluR5), together mediate a novel form of hippocampal synaptic plasticity in response to brain-derived neurotrophic factor (BDNF). Activated TrkB enhances constitutive mGluR5 activity to initiate a mode-switch that drives BDNF-dependent sustained, oscillatory Ca 2+ signaling and enhanced MAP kinase activation. This crosstalk is mediated, in part, by synergy between Gßγ, released by TrkB, and Gα q -GTP, released by mGluR5, to enable a previously unidentified form of physiologically relevant RTK/GPCR crosstalk.

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

G-protein-coupled receptors (GPCRs) mediate neuromodulation through the activation of heterotrimeric G proteins (Gαßγ). Classical models depict that G protein activation leads to a one-to-one formation of Gα-GTP and Gßγ 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 Gßγ responses remain unknown. Here, we reveal a paradigm of G protein regulation whereby the neuronal protein GINIP (Gα inhibitory interacting protein) biases inhibitory GPCR responses to favor Gßγ 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 Gßγ signaling is enhanced. We show that this mechanism is essential to prevent the imbalances of neurotransmission that underlie increased seizure susceptibility in mice. Our findings reveal an additional layer of regulation within a quintessential mechanism of signal transduction that sets the tone of neurotransmission.

Detecting GPCR Signals With Optical Biosensors of Gα-GTP in Cell Lines and Primary Cell Cultures.

G protein-coupled receptors (GPCRs) are the largest class of transmembrane receptors and mediate a wide variety of physiological processes. GPCRs respond to a plethora of extracellular ligands and initiate signaling pathways inside cells via heterotrimeric G proteins (Gαßγ). Because of the critical role GPCRs play in regulating biological processes and as pharmacological targets, the availability of tools to measure their signaling activity are of high interest. Live-cell biosensors that detect the activity of G proteins in response to GPCR stimulation have emerged as a powerful approach to investigate GPCR/G protein signaling. Here, we detail methods to monitor G protein activity through direct measurement of GTP-bound Gα subunits using optical biosensors based on bioluminescence resonance energy transfer (BRET). More specifically, this article describes the use of two types of complementary biosensors. The first protocol explains how to use a multicomponent BRET biosensor that relies on expression of exogenous G proteins in cell lines. This protocol yields robust responses that are compatible with endpoint measurements of dose-dependent ligand effects or with kinetic measurements of subsecond resolution. The second protocol describes the implementation of unimolecular biosensors that detect the activation of endogenous G proteins in cell lines expressing exogenous GPCRs or in primary cells upon stimulation of endogenous GPCRs. Overall, using the biosensors as described in this article will help users characterize the mechanisms of action of many pharmacological agents and natural ligands that modulate GPCR and G protein signaling with high precision. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Using bimolecular BRET biosensors to monitor Gα-GTP formation of tagged Gα in live cells Alternate Protocol 1: Measuring GPCR dose-dependent Gα-GTP responses in endpoint format Basic Protocol 2: Using unimolecular BRET biosensors to study endogenous G protein activity Alternate Protocol 2: Using unimolecular BRET biosensors to study endogenous G protein activity in mouse cortical neurons.

Tails don't tell the whole story.

Before engaging G protein-coupled receptors (GPCRs) to transduce extracellular signals, heterotrimeric G proteins (Gαßγ) must fold properly with the help of chaperones. In this issue of Structure, Papasergi-Scott et al. (2023) unveil the molecular basis for how mammalian Ric-8 chaperones achieve selectivity for their respective Gα subunit clients.

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

It is well-established that activation of heterotrimeric G-proteins (Gαßγ) 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 Gßγ 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.

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

Activation of heterotrimeric G-proteins (Gαßγ) 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.

Activation of heterotrimeric G-proteins (Gαßγ) 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 smallmolecule 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.

DAPLE orchestrates apical actomyosin assembly from junctional polarity complexes.

Establishment of apicobasal polarity and the organization of the cytoskeleton must operate coordinately to ensure proper epithelial cell shape and function. However, the precise molecular mechanisms by which polarity complexes directly instruct the cytoskeletal machinery to determine cell shape are poorly understood. Here, we define a mechanism by which the PAR polarity complex (PAR3-PAR6-aPKC) at apical cell junctions leads to efficient assembly of the apical actomyosin network to maintain epithelial cell morphology. We found that the PAR polarity complex recruits the protein DAPLE to apical cell junctions, which in turn triggers a two-pronged mechanism that converges upon assembly of apical actomyosin. More specifically, DAPLE directly recruits the actin-stabilizing protein CD2AP to apical junctions and, concomitantly, activates heterotrimeric G protein signaling in a GPCR-independent manner to favor RhoA-myosin activation. These observations establish DAPLE as a direct molecular link between junctional polarity complexes and the formation of apical cytoskeletal assemblies that support epithelial cell shape.

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

It has become evident that activation of heterotrimeric G-proteins by cytoplasmic proteins that are not G-protein-coupled receptors (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 Gßγ, 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/Dexras1, and Ric-8A) revealed high divergence in their contribution to generating Gα-GTP and free Gßγ 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.

Receptor tyrosine kinases activate heterotrimeric G proteins via phosphorylation within the interdomain cleft of Gαi.

The molecular mechanisms by which receptor tyrosine kinases (RTKs) and heterotrimeric G proteins, two major signaling hubs in eukaryotes, independently relay signals across the plasma membrane have been extensively characterized. How these hubs cross-talk has been a long-standing question, but answers remain elusive. Using linear ion-trap mass spectrometry in combination with biochemical, cellular, and computational approaches, we unravel a mechanism of activation of heterotrimeric G proteins by RTKs and chart the key steps that mediate such activation. Upon growth factor stimulation, the guanine-nucleotide exchange modulator dissociates Gαi•ßγ trimers, scaffolds monomeric Gαi with RTKs, and facilitates the phosphorylation on two tyrosines located within the interdomain cleft of Gαi. Phosphorylation triggers the activation of Gαi and inhibits second messengers (cAMP). Tumor-associated mutants reveal how constitutive activation of this pathway impacts cell's decision to "go" vs. "grow." These insights define a tyrosine-based G protein signaling paradigm and reveal its importance in eukaryotes.

Naturally occurring hotspot cancer mutations in Gα13 promote oncogenic signaling.

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. Up-regulation 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-dependent (SRE.L) transcriptional activity. We further showed that this mechanism relies on the same RhoGEF-RhoGTPase cascade components that are up-regulated in bladder cancers. Moreover, Gα13 Arg-200 mutants induced oncogenic transformation in vitro as determined by focus 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.

Optogenetic activation of heterotrimeric G-proteins by LOV2GIVe, a rationally engineered modular protein.

Heterotrimeric G-proteins are signal transducers involved in mediating the action of many natural extracellular stimuli and many therapeutic agents. Non-invasive approaches to manipulate the activity of G-proteins with high precision are crucial to understand their regulation in space and time. Here, we developed LOV2GIVe, an engineered modular protein that allows the activation of heterotrimeric G-proteins with blue light. This optogenetic construct relies on a versatile design that differs from tools previously developed for similar purposes, that is metazoan opsins, which are light-activated G-protein-coupled receptors (GPCRs). Instead, LOV2GIVe consists of the fusion of a G-protein activating peptide derived from a non-GPCR regulator of G-proteins to a small plant protein domain, such that light uncages the G-protein activating module. Targeting LOV2GIVe to cell membranes allowed for light-dependent activation of Gi proteins in different experimental systems. In summary, LOV2GIVe expands the armamentarium and versatility of tools available to manipulate heterotrimeric G-protein activity.

Revealing the Activity of Trimeric G-proteins in Live Cells with a Versatile Biosensor Design.

Heterotrimeric G-proteins (Gαßγ) are the main transducers of signals from GPCRs, mediating the action of countless natural stimuli and therapeutic agents. However, there are currently no robust approaches to directly measure the activity of endogenous G-proteins in cells. Here, we describe a suite of optical biosensors that detect endogenous active G-proteins with sub-second resolution in live cells. Using a modular design principle, we developed genetically encoded, unimolecular biosensors for endogenous Gα-GTP and free Gßγ: the two active species of heterotrimeric G-proteins. This design was leveraged to generate biosensors with specificity for different heterotrimeric G-proteins or for other G-proteins, such as Rho GTPases. Versatility was further validated by implementing the biosensors in multiple contexts, from characterizing cancer-associated G-protein mutants to neurotransmitter signaling in primary neurons. Overall, the versatile biosensor design introduced here enables studying the activity of endogenous G-proteins in live cells with high fidelity, temporal resolution, and convenience.