Succinate receptor 1 signaling mutually depends on subcellular localization and cellular metabolism.

Succinate is a pivotal tricarboxylic acid cycle metabolite but also specifically activates the G- and G-coupled succinate receptor 1 (SUCNR1). Contradictory roles of succinate and succinate-SUCNR1 signaling include reports about its anti- or pro-inflammatory effects. The link between cellular metabolism and localization-dependent SUCNR1 signaling qualifies as a potential cause for the reported conflicts.

Integrated NMR-crystallography-computational approach for molecular recognition studies of human Gαi3 protein by a small molecule inhibitor.

The small molecule IGGi-11 targets Gαi subunits of heterotrimeric guanine nucleotide-binding proteins. Gα subunits are activated by G-protein-coupled receptors in response to extracellular stimuli by accelerating the exchange of GDP for GTP, but they are also activated by intracellular proteins like GIV, of which elevated levels correlate with increased cell migration and cancer metastasis. IGGi-11 disrupts the interaction of Gαi proteins with GIV and inhibits pro-invasive traits of metastatic breast cancer cells without interfering with GPCR signaling.

Protocol to investigate G protein-coupled receptor signaling kinetics and concentration-dependent responses using ONE-GO biosensors.

ONE vector G protein optical (ONE-GO) biosensors are versatile tools to measure the activity of G protein-coupled receptors (GPCRs) in cells. The availability of ONE-GO biosensors for ten active Gα subunits representative of all four G protein families (G, G, G, and G) permits the study of virtually any GPCR. Here, we present a protocol to implement ONE-GO biosensors in cell lines to investigate GPCR signaling kinetics and concentration-dependent responses.

Protocol for detecting endogenous GPCR activity in primary cell cultures using ONE-GO biosensors.

ONE vector G protein Optical (ONE-GO) biosensors can measure the activity of endogenously expressed G protein-coupled receptors (GPCRs) in primary cells. By detecting G proteins that belong to all four families (G, G, G, G) across cell types, these biosensors provide high experimental versatility. We first describe steps to express ONE-GO biosensors in primary cells using lentiviral transduction.

Smooth operator(s): dialing up and down neurotransmitter responses by G-protein regulators.

G-protein-coupled receptors (GPCRs) are essential mediators of neuromodulation and prominent pharmacological targets. While activation of heterotrimeric G-proteins (Gαβɣ) by GPCRs is essential in this process, much less is known about the postreceptor mechanisms that influence G-protein activity. Neurons express G-protein regulators that shape the amplitude and kinetics of GPCR-mediated synaptic responses.

Get Ready to Sharpen Your Tools: A Short Guide to Heterotrimeric G Protein Activity Biosensors.

G protein-coupled receptors (GPCRs) are the largest class of transmembrane receptors encoded in the human genome, and they initiate cellular responses triggered by a plethora of extracellular stimuli ranging from neurotransmitters and hormones to photons. Upon stimulation, GPCRs activate heterotrimeric G proteins (Gγ) in the cytoplasm, which then convey signals to their effectors to elicit cellular responses. Given the broad biological and biomedical relevance of GPCRs and G proteins in physiology and disease, there is great interest in developing and optimizing approaches to measure their signaling activity with high accuracy and across experimental systems pertinent to their functions in cellular communication.

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.

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

Heterotrimeric G proteins (Gαβγ) 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.

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 sensory neurons, URX and BAG, require conserved AJ proteins for dendrite morphogenesis.

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.

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).

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 (Gαβγ) 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.

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).

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 (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.

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 (Gαβγ). 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.

Tails don't tell the whole story.

Before engaging G protein-coupled receptors (GPCRs) to transduce extracellular signals, heterotrimeric G proteins (Gαβγ) 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 (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.

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

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.

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

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.

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.

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 Gβγ, with different downstream effectors, but how non-receptor GEFs affect the levels of these two species in cells is not known.

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.

Naturally occurring hotspot cancer mutations in Gα 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: G, G, G, and G 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 G, G, or G proteins have also been identified in patient tumor samples. In contrast, patient tumor-associated G mutations characterized to date lead to inactivation rather than activation.