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 or hormones to photons. Upon stimulation, GPCRs activate heterotrimeric G proteins (Gαßγ) 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. This review provides a historical perspective on approaches to measure GPCR-G protein signaling, from quantification of second messengers and other indirect readouts of activity, to biosensors that directly detect the activity of G proteins. The latter is the focus of a more detailed overview of the evolution of design principles for various optical biosensors of G protein activity with different experimental capabilities. We will highlight advantages and limitations of biosensors that detect different G protein activation hallmarks, like dissociation of Gα and Gßγ or nucleotide exchange on Gα, as well as their suitability to detect signaling mediated by endogenous versus exogenous signaling components, or in physiologically-relevant systems like primary cells. Overall, this review intends to provide an assessment of the state-of-the-art for biosensors that directly measure G protein activity to allow readers to make informed decisions on the selection and implementation of currently available tools. Significance Statement G protein activity biosensors have become essential and widespread tools to assess GPCR signaling and pharmacology. Yet, investigators face the challenge of choosing from a growing list of G protein activity biosensors. This review provides an overview of the features and capabilities of different optical biosensor designs for the direct detection of G protein activity in cells, with the aim of facilitating the rational selection of systems that align with the specific scientific questions and needs of investigators.

Assessing Protein-Protein Docking Protocols: Case Studies of G-Protein-Coupled Receptor Interactions.

The interactions of G-protein-coupled receptors (GPCRs) with other proteins are critical in several cellular processes but resolving their structural dynamics remains challenging. An increasing number of GPCR complexes have been experimentally resolved but others including receptor variants are yet to be characterized, necessitating computational predictions of their interactions. Although integrative approaches with multi-scale simulations would provide rigorous estimates of their conformational dynamics, protein-protein docking remains a first tool of choice of many researchers due to the availability of open-source programs and easy to use web servers with reasonable predictive power. Protein-protein docking algorithms have limited ability to consider protein flexibility, environment effects, and entropy contributions and are usually a first step towards more integrative approaches. The two critical steps of docking: the sampling and scoring algorithms have improved considerably and their performance has been validated against experimental data. In this chapter, we provide an overview and generalized protocol of a few docking protocols using GPCRs as test cases. In particular, we demonstrate the interactions of GPCRs with extracellular protein ligands and an intracellular protein effectors (G-protein) predicted from docking approaches and test their limitations. The current chapter will help researchers critically assess docking protocols and predict experimentally testable structures of GPCR complexes.

Protein-Protein Docking Approach to GPCR Oligomerization.

G-protein-coupled receptors (GPCRs), the largest family of human membrane proteins, play a crucial role in cellular control and are the target of approximately one-third of all drugs on the market. Targeting these complexes with selectivity or formulating small molecules capable of modulating receptor-receptor interactions could potentially offer novel avenues for drug discovery, fostering the development of more refined and safer pharmacotherapies. Due to the lack of experimentally derived X-ray crystallography spectra of GPCR oligomers, there is growing evidence supporting the development of new in silico approaches for predicting GPCR self-assembling structures. The significance of GPCR oligomerization, the challenges in modeling these structures, and the potential of protein-protein docking algorithms to address these challenges are discussed. The study also underscores the use of various software solutions for modeling GPCR oligomeric structures and presents practical cases where these techniques have been successfully applied.

Altered desensitization and internalization patterns of rodent versus human glucose-dependent insulinotropic polypeptide (GIP) receptors. An important drug discovery challenge.

BACKGROUND AND PURPOSE: The gut hormone glucose-dependent insulinotropic polypeptide (GIP) signals via the GIP receptor (GIPR), resulting in postprandial potentiation of glucose-stimulated insulin secretion. The translation of results from rodent studies to human studies has been challenged by the unexpected effects of GIPR-targeting compounds. We, therefore, investigated the variation between species, focusing on GIPR desensitization and the role of the receptor C-terminus. EXPERIMENTAL APPROACH: The GIPR from humans, mice, rats, pigs, dogs and cats was studied in vitro for cognate ligand affinity, G protein activation (cAMP accumulation), recruitment of beta-arrestin and internalization. Variants of the mouse, rat and human GIPRs with swapped C-terminal tails were studied in parallel. KEY RESULTS: The human GIPR is more prone to internalization than rodent GIPRs. Despite similar agonist affinities and potencies for Gαs activation, especially, the mouse GIPR shows reduced receptor desensitization, internalization and beta-arrestin recruitment. Using an enzyme-stabilized, long-acting GIP analogue, the species differences were even more pronounced. 'Tail-swapped' human, rat and mouse GIPRs were all fully functional in their Gαs coupling, and the mouse GIPR regained internalization and beta-arrestin 2 recruitment properties with the human tail. The human GIPR lost the ability to recruit beta-arrestin 2 when its own C-terminus was replaced by the rat or mouse tail. CONCLUSIONS AND IMPLICATIONS: Desensitization of the human GIPR is dependent on the C-terminal tail. The species-dependent functionality of the C-terminal tail and the different species-dependent internalization patterns, especially between human and mouse GIPRs, are important factors influencing the preclinical evaluation of GIPR-targeting therapeutic compounds.

Quantitative Monitoring of GPCR-Mediated Spatiotemporal IP3 Dynamics Using Confocal Fluorescence Microscopy.

Activation of G protein-coupled receptors upon chemoattractant stimulation induces activation of multiple signaling pathways. To fully understand how these signaling pathway coordinates to achieve directional migration of neutrophils, it is essential to determine the dynamics of the spatiotemporal activation profile of signaling components at the level of single living cells. Here, we describe a detailed methodology for monitoring and quantitatively analyzing the spatiotemporal dynamics of 1,4,5-inositol trisphosphate (IP3) in neutrophil-like HL60 cells in response to various chemoattractant fields by applying Förster resonance energy transfer (FRET) fluorescence microscopy.

International Union of Basic and Clinical Pharmacology. CXV: The Class F of G Protein-Coupled Receptors.

The class F of G protein-coupled receptors (GPCRs) consists of ten Frizzleds (FZD1-10) and Smoothened (SMO). FZDs bind and are activated by secreted lipoglycoproteins of the Wingless/Int-1 (WNT) family and SMO is indirectly activated by the Hedgehog (Hh) family of morphogens acting on the transmembrane protein Patched (PTCH). The advance of our understanding of FZDs and SMO as dynamic transmembrane receptors and molecular machines, which emerged during the past 14 years since the first class F GPCR IUPHAR nomenclature report, justifies an update. This article focuses on the advances in molecular pharmacology and structural biology providing new mechanistic insight into ligand recognition, receptor activation mechanisms, signal initiation and signal specification. Furthermore, class F GPCRs continue to develop as drug targets, and novel technologies and tools such as genetically encoded biosensors and CRISP/Cas9 edited cell systems have contributed to refined functional analysis of these receptors. Also, advances in crystal structure analysis and cryogenic electron microscopy contribute to a rapid development of our knowledge about structure-function relationships providing a great starting point for drug development. Despite the progress questions and challenges remain to fully understand the complexity of the WNT/FZD and Hh/SMO signaling systems. Significance Statement The recent years of research have brought about substantial functional and structural insight into mechanisms of activation of Frizzleds and Smoothened. While the advance furthers our mechanistic understanding of ligand recognition, receptor activation, signal specification and initiation, broader opportunities emerge that allow targeting class F GPCRs for therapy and regenerative medicine employing both biologics and small molecule compounds.

Genetic architecture of the toxicological response to eucalyptol and citronellal in Drosophila melanogaster.

The excessive and indiscriminate use of synthetic insecticides has led to environmental pollution, wildlife destruction, and adverse effects on human health, while simultaneously giving rise to resistance in insect pest populations. This adaptive trait is expressed through various mechanisms, such as changes in the cuticle, heightened activities of detoxifying enzymes, and alterations in the sites of action that reduce their affinity for insecticides. In this context, we associate variation in toxicological response with genomic variation, to identify genetic polymorphisms underlying the different steps of the insect (genotype)-response (phenotype)-insecticide (environment) interaction. Under this framework, our objective was to investigate the genetic factors involved in the toxicological response of D. melanogaster lines when exposed to citronellal and eucalyptol vapors (monoterpenes of plant origin). We quantified KT50 in adult males, representing the time necessary for half of the exposed individuals to be turned upside down (unable to walk or fly). Since the genomes of all lines used are completely sequenced, we perform a Genome Wide Association Study to analyze the genetic underpinnings of the toxicological response. Our investigation enabled the identification of 656 genetic polymorphisms and 316 candidate genes responsible for the overall phenotypic variation. Among these, 162 candidate genes (77.1%) exhibited specificity to citronellal, 45 (21.4%) were specific to eucalyptol, and 3 candidate genes (1.5%) namely CG34345, robo2, and Ac13E, were implicated in the variation for both monoterpenes. These suggest a widespread adaptability in the response to insecticides, encompassing genes influenced by monoterpenes and those orchestrating resistance to the toxicity of these compounds.

Development of a NanoBRET Assay Platform to Detect Intracellular Ligands for the Chemokine Receptors CCR6 and CXCR1.

A conserved intracellular allosteric binding site (IABS) was recently identified at several G protein-coupled receptors (GPCRs). This target site allows the binding of allosteric modulators and enables a new mode of GPCR inhibition. Herein, we report the development of a NanoBRET-based assay platform based on the fluorescent ligand LT221 (5), to detect intracellular binding to CCR6 and CXCR1, two chemokine receptors that have been pursued as promising drug targets in inflammation and immuno-oncology. Our assay platform enables cell-free as well as cellular NanoBRET-based binding studies in a nonisotopic and straightforward manner. By combining this screening platform with a previously reported CXCR2 assay, we investigated CXCR1/CXCR2/CCR6 selectivity profiles for both known and novel squaramide analogues derived from navarixin, a known intracellular CXCR1/CXCR2 antagonist and phase II clinical candidate for the treatment of pulmonary diseases. By means of these studies we identified compound 10, a previously reported tert-butyl analogue of navarixin, as a low nanomolar intracellular CCR6 antagonist. Further, our assay platform clearly indicated intracellular binding of the CCR6 antagonist PF-07054894, currently evaluated in phase I clinical trials for the treatment of ulcerative colitis, thereby providing profound evidence for the existence and the pharmacological relevance of a druggable IABS at CCR6.

Exploring orphan GPCRs in neurodegenerative diseases.

Neurodegenerative disorders represent a significant and growing health burden worldwide. Unfortunately, limited therapeutic options are currently available despite ongoing efforts. Over the past decades, research efforts have increasingly focused on understanding the molecular mechanisms underlying these devastating conditions. Orphan receptors, a class of receptors with no known endogenous ligands, emerge as promising druggable targets for diverse diseases. This review aims to direct attention to a subgroup of orphan GPCRs, in particular class A orphans that have roles in neurodegenerative disorders, including Alzheimer's disease, Parkinson's disease, Huntington's disease, and Multiple sclerosis. We highlight the diverse roles orphan receptors play in regulating critical cellular processes such as synaptic transmission, neuronal survival and neuro-inflammation. Moreover, we discuss the therapeutic potential of targeting orphan receptors for the treatment of neurodegenerative disorders, emphasizing recent advances in drug discovery and preclinical studies. Finally, we outline future directions and challenges in orphan receptor research.

A correlation study of adhesion G protein-coupled receptors as potential therapeutic targets for breast cancer.

BACKGROUND: Adhesion G protein-coupled receptors (aGPCRs), a distinctive subset of the G protein-coupled receptor (GPCR) superfamily, play crucial roles in various physiological and pathological processes, with implications in tumor development. Despite the global prevalence of breast cancer (BRCA), specific aGPCRs as potential drug targets or biomarkers remain underexplored. METHODS: UALCAN, GEPIA, Kaplan-Meier Plotter, MethSurv, cBiopportal, String, GeneMANIA, DAVID, Timer, Metascape, and qPCR were applied in this work. RESULTS: Our analysis revealed significantly increased transcriptional levels of ADGRB2, ADGRC1, ADGRC2, ADGRC3, ADGRE1, ADGRF2, ADGRF4, and ADGRL1 in BRCA primary tumors. Further analysis indicated a significant correlation between the expressions of certain aGPCRs and the pathological stage of BRCA. High expression of ADGRA1, ADGRF2, ADGRF4, ADGRG1, ADGRG2, ADGRG4, ADGRG6, and ADGRG7 was significantly correlated with poor overall survival (OS) in BRCA patients. Additionally, high expression of ADGRF2 and ADGRF4 indicated inferior recurrence-free survival (RFS) in BRCA patients. The RT-qPCR experiments also confirmed that the mRNA levels of ADGRF2 and ADGRF4 were higher in BRCA cells and tissues. Functional analysis highlighted the diverse roles of aGPCRs, encompassing GPCR signaling and metabolic energy reserves. Moreover, aGPCRs may exert influence or actively participate in the development of BRCA through their impact on immune status. CONCLUSION: aGPCRs, particularly ADGRF2 and ADGRF4, hold promise as immunotherapeutic targets and prognostic biomarkers in BRCA.

An orphan to the rescue of obesity and steatotic liver?

In a recent article, Leeson-Payne et al. demonstrate that GPR75 knock-out in mice results in lower body fat and reduced hepatic lipid accumulation, with an increase in physical activity and energy expenditure. Loss-of-function (LoF) GPR75 variants in the UK Biobank (UKBB) are associated with reduced liver steatosis, suggesting potential therapeutic implications in metabolic dysfunction-associated steatotic liver disease (MASLD).

PI3 kinase inhibitor PI828 uncouples aminergic GPCRs and Ca2+ mobilization irrespectively of its primary target.

The phosphoinositide 3-kinase (PI3K) is involved in regulation of multiple intracellular processes. Although the inhibitory analysis is generally employed for validating a physiological role of PI3K, increasing body of evidence suggests that PI3K inhibitors can exhibit PI3K-unrelated activity as well. Here we studied Ca2+ signaling initiated by aminergic agonists in a variety of different cells and analyzed effects of the PI3K inhibitor PI828 on cell responsiveness. It turned out that PI828 inhibited Ca2+ transients elicited by acetylcholine (ACh), histamine, and serotonin, but did not affect Ca2+ responses to norepinephrine and ATP. Another PI3K inhibitor wortmannin negligibly affected Ca2+ signaling initiated by any one of the tested agonists. Using the genetically encoded PIP3 sensor PH(Akt)-Venus, we confirmed that both PI828 and wortmannin effectively inhibited PI3K and ascertained that this kinase negligibly contributed to ACh transduction. These findings suggested that PI828 inhibited Ca2+ responses to aminergic agonists tested, involving an unknown cellular mechanism unrelated to the PI3K inhibition. Complementary physiological experiments provided evidence that PI828 could inhibit Ca2+ signals induced by certain agonists, by acting extracellularly, presumably, through their surface receptors. For the muscarinic M3 receptor, this possibility was verified with molecular docking and molecular dynamics. As demonstrated with these tools, wortmannin could be bound in the extracellular vestibule at the muscarinic M3 receptor but this did not preclude binding of ACh to the M3 receptor followed by its activation. In contrast, PI828 could sterically block the passage of ACh into the allosteric site, preventing activation of the muscarinic M3 receptor.

Role of orphan G-protein coupled receptors in tissue ischemia: A comprehensive review.

Ischemic events lead to many diseases and deaths worldwide. Ischemia/reperfusion (I/R) occurs due to reduced blood circulation in tissues followed by blood reflow. Reoxygenation of ischemic tissues is characterized by oxidative stress, inflammation, energy distress, and endoplasmic reticulum stress. There are still no adequate clinical protocols or pharmacological approaches to address the consequences of I/R damage. G protein-coupled receptors (GPCRs) are important therapeutic targets. They compose a large family of seven transmembrane-spanning proteins that are involved in many biological functions. Orphan GPCRs are a large subgroup of these receptors expressed in different organs. In the present review, we summarized the literature regarding the role of orphan GPCRs in I/R in different organs. We focused on the effect of these receptors on modulating cellular and molecular processes underlying ischemia including apoptosis, inflammation, and autophagy. The study showed that GPR3, GPR4, GPR17, GPR30, GPR31, GPR35, GPR37, GPR39, GPR55, GPR65, GPR68, GPR75, GPR81, and GPR91 are involved in ischemic events, mainly in the brain and heart. These receptors offer new possibilities for treating I/R injuries in the body.

Elucidating the Activation Mechanism of the Proton-sensing GPR68 Receptor.

GPR68 is a proton-sensing G-protein Coupled Receptor (GPCR) involved in a variety of physiological processes and disorders including neoplastic pathologies. While GPR68 and few other GPCRs have been shown to be activated by a decrease in the extracellular pH, the molecular mechanism of their activation remains largely unknown. In this work, we used a combined computational and in vitro approach to provide new insight into the activation mechanism of the receptor. Molecular Dynamics simulations of GPR68 were used to model the changes in residue interactions and motions triggered by pH. Global and local rearrangements consistent with partial activation were observed upon protonation of the inactive state. Selected extracellular histidine and transmembrane acidic residues were found to have significantly upshifted pKa values during the simulations, consistently with their previously hypothesised role in activation through changes in protonation state. Moreover, a novel pairing between histidine and acidic residues in the extracellular region was highlighted by both sequence analyses and simulation data and tested through site-directed mutagenesis. At last, we identified a previously unknown hydrophobic lock in the extracellular region that might stabilise the inactive conformation and regulate the transition to the active state.

Bayesian network models identify cooperative GPCR:G protein interactions that contribute to G protein coupling.

Cooperative interactions in protein-protein interfaces demonstrate the interdependency or the linked network-like behavior and their effect on the coupling of proteins. Cooperative interactions also could cause ripple or allosteric effects at a distance in protein-protein interfaces. Although they are critically important in protein-protein interfaces, it is challenging to determine which amino acid pair interactions are cooperative. In this work, we have used Bayesian network modeling, an interpretable machine learning method, combined with molecular dynamics trajectories to identify the residue pairs that show high cooperativity and their allosteric effect in the interface of G protein-coupled receptor (GPCR) complexes with Gα subunits. Our results reveal six GPCR:Gα contacts that are common to the different Gα subtypes and show strong cooperativity in the formation of interface. Both the C terminus helix5 and the core of the G protein are codependent entities and play an important role in GPCR coupling. We show that a promiscuous GPCR coupling to different Gα subtypes, makes all the GPCR:Gα contacts that are specific to each Gα subtype (Gαs, Gαi, and Gαq). This work underscores the potential of data-driven Bayesian network modeling in elucidating the intricate dependencies and selectivity determinants in GPCR:G protein complexes, offering valuable insights into the dynamic nature of these essential cellular signaling components.

Highlights and hot topics in GPCR research from 'Down Under'.

LINKED ARTICLES: This article is part of a themed issue Therapeutic Targeting of G Protein-Coupled Receptors: hot topics from the Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists 2021 Virtual Annual Scientific Meeting. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v181.14/issuetoc.

Molecular dissection of PI3Kß synergistic activation by receptor tyrosine kinases, GßGγ, and Rho-family GTPases.

Phosphoinositide 3-kinase (PI3K) beta (PI3Kß) is functionally unique in the ability to integrate signals derived from receptor tyrosine kinases (RTKs), G-protein coupled receptors, and Rho-family GTPases. The mechanism by which PI3Kß prioritizes interactions with various membrane-tethered signaling inputs, however, remains unclear. Previous experiments did not determine whether interactions with membrane-tethered proteins primarily control PI3Kß localization versus directly modulate lipid kinase activity. To address this gap in our knowledge, we established an assay to directly visualize how three distinct protein interactions regulate PI3Kß when presented to the kinase in a biologically relevant configuration on supported lipid bilayers. Using single molecule Total Internal Reflection Fluorescence (TIRF) Microscopy, we determined the mechanism controlling PI3Kß membrane localization, prioritization of signaling inputs, and lipid kinase activation. We find that auto-inhibited PI3Kß prioritizes interactions with RTK-derived tyrosine phosphorylated (pY) peptides before engaging either GßGγ or Rac1(GTP). Although pY peptides strongly localize PI3Kß to membranes, stimulation of lipid kinase activity is modest. In the presence of either pY/GßGγ or pY/Rac1(GTP), PI3Kß activity is dramatically enhanced beyond what can be explained by simply increasing membrane localization. Instead, PI3Kß is synergistically activated by pY/GßGγ and pY/Rac1 (GTP) through a mechanism consistent with allosteric regulation.

GPR56, an Adhesion GPCR with Multiple Roles in Human Diseases, Current Status and Future Perspective.

Human G protein-coupled receptor 56 (GPR56) belongs to a member of the adhesion G-protein coupled receptor (aGPCR) family and widely exists in the central nervous system and various types of tumor tissues. Recent studies have shown that abnormal expression or dysfunction of GPR56 is closely associated with many physiological and pathological processes, including brain development, neuropsychiatric disorders, cardiovascular diseases and cancer progression. In addition, GPR56 has been proven to enhance the susceptibility of some antipsychotics and anticarcinogens in response to the treatment of neuropsychological diseases and cancer. Although there have been some reports about the functions of GPR56, the underlying mechanisms implicated in these diseases have not been clarified thoroughly, especially in depression and epilepsy. Therefore, in this review, we described the molecular structure and signal transduction pathway of GPR56 and carried out a comprehensive summary of GPR56's function in the development of psychiatric disorders and cancer. Our review showed that GPR56 deficiency led to depressive-like behaviors and an increase in resistance to antipsychotic treatment. In contrast, the upregulation of GPR56 contributed to tumor cell proliferation and metastasis in malignant diseases such as glioblastoma, colorectal cancer, and ovarian cancer. Moreover, we elucidated specific signaling pathways downstream of GPR56 related to the pathogenesis of these diseases. In summary, our review provides compelling arguments for an attractive therapeutic target of GPR56 in improving the therapeutic efficiency for patients suffering from psychiatric disorders and cancer.

Multiplex Detection of Fluorescent Chemokine Binding to CXC Chemokine Receptors by NanoBRET.

NanoLuc-mediated bioluminescence resonance energy transfer (NanoBRET) has gained popularity for its ability to homogenously measure ligand binding to G protein-coupled receptors (GPCRs), including the subfamily of chemokine receptors. These receptors, such as ACKR3, CXCR4, CXCR3, play a crucial role in the regulation of the immune system, are associated with inflammatory diseases and cancer, and are seen as promising drug targets. The aim of this study was to optimize NanoBRET-based ligand binding to NLuc-ACKR3 and NLuc-CXCR4 using different fluorescently labeled chemokine CXCL12 analogs and their use in a multiplex NanoBRET binding assay of two chemokine receptors at the same time. The four fluorescent CXCL12 analogs (CXCL12-AZD488, -AZD546, -AZD594, -AZD647) showed high-affinity saturable binding to both NLuc-ACKR3 and NLuc-CXCR4, with relatively low levels of non-specific binding. Additionally, the binding of all AZDye-labeled CXCL12s to Nluc receptors was inhibited by pharmacologically relevant unlabeled chemokines and small molecules. The NanoBRET binding assay for CXCL10-AZD488 binding to Nluc-CXCR3 was also successfully established and successfully employed for the simultaneous measurement of the binding of unlabeled small molecules to NLuc-CXCR3 and NLuc-CXCR4. In conclusion, multiplexing the NanoBRET-based competition binding assay is a promising tool for testing unlabeled (small) molecules against multiple GPCRs simultaneously.