Visualization of endogenous G proteins on endosomes and other organelles.

Classical G protein-coupled receptor (GPCR) signaling takes place in response to extracellular stimuli and involves receptors and heterotrimeric G proteins located at the plasma membrane. It has recently been established that GPCR signaling can also take place from intracellular membrane compartments, including endosomes that contain internalized receptors and ligands. While the mechanisms of GPCR endocytosis are well understood, it is not clear how well internalized receptors are supplied with G proteins. To address this gap we use gene editing, confocal microscopy, and bioluminescence resonance energy transfer to study the distribution and trafficking of endogenous G proteins. We show here that constitutive endocytosis is sufficient to supply newly internalized endocytic vesicles with 20-30% of the G protein density found at the plasma membrane. We find that G proteins are present on early, late, and recycling endosomes, are abundant on lysosomes, but are virtually undetectable on the endoplasmic reticulum, mitochondria, and the medial Golgi apparatus. Receptor activation does not change heterotrimer abundance on endosomes. Our findings provide a subcellular map of endogenous G protein distribution, suggest that G proteins may be partially excluded from nascent endocytic vesicles, and are likely to have implications for GPCR signaling from endosomes and other intracellular compartments.

A short C-terminal peptide in Gγ regulates Gßγ signaling efficacy.

G protein beta-gamma (Gßγ) subunits anchor to the plasma membrane (PM) through the carboxy-terminal (CT) prenyl group in Gγ. This interaction is crucial for the PM localization and functioning of Gßγ, allowing GPCR-G protein signaling to proceed. The diverse Gγ family has 12 members, and we have recently shown that the signaling efficacies of major Gßγ effectors are Gγ-type dependent. This dependency is due to the distinct series of membrane-interacting abilities of Gγ. However, the molecular process allowing for Gßγ subunits to exhibit a discrete and diverse range of Gγ-type-dependent membrane affinities is unclear and cannot be explained using only the type of prenylation. The present work explores the unique designs of membrane-interacting CT residues in Gγ as a major source for this Gγ-type-dependent Gßγ signaling. Despite the type of prenylation, the results show signaling efficacy at the PM, and associated cell behaviors of Gßγ are governed by crucially located specific amino acids in the five to six residue preprenylation region of Gγ. The provided molecular picture of Gγ-membrane interactions may explain how cells gain Gγ-type-dependent G protein-GPCR signaling as well as how Gßγ elicits selective signaling at various subcellular compartments.

Subtype-dependent regulation of Gßγ signalling.

G protein-coupled receptors (GPCRs) transmit information to the cell interior by transducing external signals to heterotrimeric G protein subunits, Gα and Gßγ subunits, localized on the inner leaflet of the plasma membrane. Though the initial focus was mainly on Gα-mediated events, Gßγ subunits were later identified as major contributors to GPCR-G protein signalling. A broad functional array of Gßγ signalling has recently been attributed to Gß and Gγ subtype diversity, comprising 5 Gß and 12 Gγ subtypes, respectively. In addition to displaying selectivity towards each other to form the Gßγ dimer, numerous studies have identified preferences of distinct Gßγ combinations for specific GPCRs, Gα subtypes and effector molecules. Importantly, Gß and Gγ subtype-dependent regulation of downstream effectors, representing a diverse range of signalling pathways and physiological functions have been found. Here, we review the literature on the repercussions of Gß and Gγ subtype diversity on direct and indirect regulation of GPCR/G protein signalling events and their physiological outcomes. Our discussion additionally provides perspective in understanding the intricacies underlying molecular regulation of subtype-specific roles of Gßγ signalling and associated diseases.

Microcystins: Biogenesis, Toxicity, Analysis, and Control.

Microcystins are cyclic peptide toxins formed by cyanobacteria. These toxins are recognized for their association with algal blooms, posing a significant threat to ecosystems and drinking water quality. Due to the growing environmental concerns they raise, a comprehensive review on microcystins' genesis, toxicity, and analytical methods for their quantitative determination is outlined. Genes, including the mcyABC cluster, regulate microcystin biogenesis. Bioanalytical experiments have identified key environmental factors, such as temperature and nitrogen availability, that promote microcystin production. Microcystin toxicity is explored based on its modulatory effects on protein phosphatases 1 and 2A in specific tissues and organs. Additionally, biochemical mechanisms of chelation, transportation, resultant oxidative stress, and tumor promotion abilities of microcystins are also discussed. Various analytical methods to separate, detect, and quantify microcystins, including the quantitative real-time polymerase chain reaction, enzyme-linked immunosorbent assay, nuclear magnetic resonance spectroscopy, and chromatographic platforms-linked tandem mass spectrometry (LC-MS) for unequivocal structural identification, are also reviewed. Since control of microcystins in water is of great necessity, both water treatment and mechanisms of abiotic transformation and microbial degradation are also discussed.

Optical approaches for single-cell and subcellular analysis of GPCR-G protein signaling.

G protein-coupled receptors (GPCRs), G proteins, and their signaling associates are major signal transducers that control the majority of cellular signaling and regulate key biological functions including immune, neurological, cardiovascular, and metabolic processes. These pathways are targeted by over one-third of drugs on the market; however, the current understanding of their function is limited and primarily derived from cell-destructive approaches providing an ensemble of static, multi-cell information about the status and composition of molecules. Spatiotemporal behavior of molecules involved is crucial to understanding in vivo cell behaviors both in health and disease, and the advent of genetically encoded fluorescence proteins and small fluorophore-based biosensors has facilitated the mapping of dynamic signaling in cells with subcellular acuity. Since we and others have developed optogenetic methods to regulate GPCR-G protein signaling in single cells and subcellular regions using dedicated wavelengths, the desire to develop and adopt optogenetically amenable assays to measure signaling has motivated us to take a broader look at the available optical tools and approaches compatible with measuring single-cell and subcellular GPCR-G protein signaling. Here we review such key optical approaches enabling the examination of GPCR, G protein, secondary messenger, and downstream molecules such as kinase and lipid signaling in living cells. The methods reviewed employ both fluorescence and bioluminescence detection. We not only further elaborate the underlying principles of these sensors but also discuss the experimental criteria and limitations to be considered during their use in single-cell and subcellular signal mapping.

Statins Perturb Gßγ Signaling and Cell Behavior in a Gγ Subtype Dependent Manner.

Guanine nucleotide-binding proteins (G proteins) facilitate the transduction of external signals to the cell interior, regulate most eukaryotic signaling, and thus have become crucial disease drivers. G proteins largely function at the inner leaflet of the plasma membrane (PM) using covalently attached lipid anchors. Both small monomeric and heterotrimeric G proteins are primarily prenylated, either with a 15-carbon farnesyl or a 20-carbon geranylgeranyl polyunsaturated lipid. The mevalonate [3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase] pathway synthesizes lipids for G-protein prenylation. It is also the source of the precursor lipids for many biomolecules, including cholesterol. Consequently, the rate-limiting enzymes of the mevalonate pathway are major targets for cholesterol-lowering medications and anticancer drug development. Although prenylated G protein γ (Gγ) is essential for G protein-coupled receptor (GPCR)-mediated signaling, how mevalonate pathway inhibitors, statins, influence subcellular distribution of Gßγ dimer and Gαßγ heterotrimer, as well as their signaling upon GPCR activation, is poorly understood. The present study shows that clinically used statins not only significantly disrupt PM localization of Gßγ but also perturb GPCR-G protein signaling and associated cell behaviors. The results also demonstrate that the efficiency of prenylation inhibition by statins is Gγ subtype-dependent and is more effective toward farnesylated Gγ types. Since Gγ is required for Gßγ signaling and shows a cell- and tissue-specific subtype distribution, the present study can help understand the mechanisms underlying clinical outcomes of statin use in patients. This work also reveals the potential of statins as clinically usable drugs to control selected GPCR-G protein signaling.

G protein γ (Gγ) subtype dependent targeting of GRK2 to M3 receptor by Gßγ.

Interactions of cytosolic G protein coupled receptor kinase 2 (GRK2) with activated G protein coupled receptors (GPCRs) induce receptor phosphorylation and desensitization. GRK2 is recruited to active M3-muscarinic receptors (M3R) with the participation of the receptor, Gαq and Gßγ. Since we have shown that signaling efficacy of Gßγ is governed by its Gγ subtype identity, the present study examined whether recruitment of GRK2 to M3R is also Gγ subtype dependent. To capture the dynamics of GRK2-recruitment concurrently with GPCR-G protein activation, we employed live cell confocal imaging and a novel assay based on Gßγ translocation. Data show that M3R activation-induced GRK2 recruitment is Gγ subtype dependent in which Gßγ dimers with low PM-affinity Gγ9 exhibited a two-fold higher GRK2-recruitment compared to high PM affinity Gγ3 expressing cells. Since 12-mammalian Gγ types exhibit a cell and tissue specific expressions and the PM-affinity of a Gγ is linked to its subtype identity, our results indicate a mechanism by which Gγ profile of a cell controls GRK2 signaling and GPCR desensitization.

Regulation of G Protein ßγ Signaling.

Heterotrimeric guanine nucleotide-binding proteins (G proteins) deliver external signals to the cell interior, upon activation by the external signal stimulated G protein-coupled receptors (GPCRs).While the activated GPCRs control several pathways independently, activated G proteins control the vast majority of cellular and physiological functions, ranging from vision to cardiovascular homeostasis. Activated GPCRs dissociate GαGDPßγ heterotrimer into GαGTP and free Gßγ. Earlier, GαGTP was recognized as the primary signal transducer of the pathway and Gßγ as a passive signaling modality that facilitates the activity of Gα. However, Gßγ later found to regulate more number of pathways than GαGTP does. Once liberated from the heterotrimer, free Gßγ interacts and activates a diverse range of signaling regulators including kinases, lipases, GTPases, and ion channels, and it does not require any posttranslation modifications. Gßγ family consists of 48 members, which show cell- and tissue-specific expressions, and recent reports show that cells employ the subtype diversity in Gßγ to achieve desired signaling outcomes. In addition to activated GPCRs, which induce free Gßγ generation and the rate of GTP hydrolysis in Gα, which sequester Gßγ in the heterotrimer, terminating Gßγ signaling, additional regulatory mechanisms exist to regulate Gßγ activity. In this chapter, we discuss structure and function, subtype diversity and its significance in signaling regulation, effector activation, regulatory mechanisms as well as the disease relevance of Gßγ in eukaryotes.

Gγ identity dictates efficacy of Gßγ signaling and macrophage migration.

G protein ßγ subunit (Gßγ) is a major signal transducer and controls processes ranging from cell migration to gene transcription. Despite having significant subtype heterogeneity and exhibiting diverse cell- and tissue-specific expression levels, Gßγ is often considered a unified signaling entity with a defined functionality. However, the molecular and mechanistic basis of Gßγ's signaling specificity is unknown. Here, we demonstrate that Gγ subunits, bearing the sole plasma membrane (PM)-anchoring motif, control the PM affinity of Gßγ and thereby differentially modulate Gßγ effector signaling in a Gγ-specific manner. Both Gßγ signaling activity and the migration rate of macrophages are strongly dependent on the PM affinity of Gγ. We also found that the type of C-terminal prenylation and five to six pre-CaaX motif residues at the PM-interacting region of Gγ control the PM affinity of Gßγ. We further show that the overall PM affinity of the Gßγ pool of a cell type is a strong predictor of its Gßγ signaling-activation efficacy. A kinetic model encompassing multiple Gγ types and parameterized for empirical Gßγ behaviors not only recapitulated experimentally observed signaling of Gßγ, but also suggested a Gγ-dependent, active-inactive conformational switch for the PM-bound Gßγ, regulating effector signaling. Overall, our results unveil crucial aspects of signaling and cell migration regulation by Gγ type-specific PM affinities of Gßγ.

Measurement of GPCR-G protein activity in living cells.

G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors in eukaryotic genomes. They control a variety of cellular and physiological processes such as hormone secretion and heart rate, and therefore are associated with a majority of pathological conditions including cancer and heart diseases. Currently established assays to measure ligand-induced activation of GPCRs and G proteins possess limitations such as being time consuming, indirect, and expensive. Thus, an efficient method to measure GPCR-G protein activation is required to identify novel pharmacological modulators to control them and gain insights about molecular underpinnings of the associated pathways. Activation of GPCRs induces dissociation of G protein heterotrimers to form GαGTP and free Gßγ. Free Gßγ subunits have been shown to translocate reversibly from the plasma membrane to internal membranes. Gßγ translocation therefore represents the GPCR-G protein activation, and thus, imaging of this process can be used to quantify the kinetics and magnitude of the pathway activation-deactivation in real time in living cells. The objective of this chapter is to elaborate the protocols of (i) generation and optimization of the required sensor constructs; (ii) development of cell culture, transient transfection, imaging, and optogenetic procedures; (iii) imaging and data analysis methods; and (iv) stable cell line generation, pertaining to this assay to measure GPCR-G protein activation.

Two independent but synchronized Gßγ subunit-controlled pathways are essential for trailing-edge retraction during macrophage migration.

Chemokine-induced directional cell migration is a universal cellular mechanism and plays crucial roles in numerous biological processes, including embryonic development, immune system function, and tissue remodeling and regeneration. During the migration of a stationary cell, the cell polarizes, forms lamellipodia at the leading edge (LE), and triggers the concurrent retraction of the trailing edge (TE). During cell migration governed by inhibitory G protein (Gi)-coupled receptors (GPCRs), G protein ßγ (Gßγ) subunits control the LE signaling. Interestingly, TE retraction has been linked to the activation of the small GTPase Ras homolog family member A (RhoA) by the Gα12/13 pathway. However, it is not clear how the activation of Gi-coupled GPCRs at the LE orchestrates the TE retraction in RAW264.7 macrophages. Here, using an optogenetic approach involving an opsin to activate the Gi pathway in defined subcellular regions of RAW cells, we show that in addition to their LE activities, free Gßγ subunits also govern TE retraction by operating two independent, yet synchronized, pathways. The first pathway involves RhoA activation, which prevents dephosphorylation of the myosin light chain, allowing actomyosin contractility to proceed. The second pathway activates phospholipase Cß and induces myosin light chain phosphorylation to enhance actomyosin contractility through increasing cytosolic calcium. We further show that both of these pathways are essential, and inhibition of either one is sufficient to abolish the Gi-coupled GPCR-governed TE retraction and subsequent migration of RAW cells.

Reversible G Protein ßγ9 Distribution-Based Assay Reveals Molecular Underpinnings in Subcellular, Single-Cell, and Multicellular GPCR and G Protein Activity.

Current assays to measure the activation of G protein coupled receptors (GPCRs) and G proteins are time-consuming, indirect, and expensive. Therefore, an efficient method which directly measures the ability of a ligand to govern GPCR-G protein interactions can help to understand the molecular underpinnings of the associated signaling. A live cell imaging-based approach is presented here to directly measure ligand-induced GPCR and G protein activity in real time. The number of active GPCRs governs G protein heterotrimer (αßγ) dissociation, thereby controlling the concentration of free ßγ subunits. The described γ9 assay measures the GPCR activation-induced extent of the reversible ßγ9 subunit exchange between the plasma membrane (PM) and internal membranes (IMs). Confocal microscopy-based γ9 assay quantitatively determines the concentration dependency of ligands on GPCR activation. Demonstrating the high-throughput screening (HTS) adaptability, the γ9 assay performed using an imaging plate reader measures the ligand-induced GPCR activation. This suggests that the γ9 assay can be employed to screen libraries of compounds for their ability to activate GPCRs. Together with subcellular optogenetics, the spatiotemporal sensitivity of the γ9 assay permits experimental determination of the limits of spatially restricted activation of GPCRs and G proteins in subcellular regions of single cells. This assay works effectively for GPCRs coupled to αi/o and αs heterotrimers, including light-sensitive GPCRs. In addition, computational modeling of experimental data from the assay is used to decipher intricate molecular details of the GPCR-G protein activation process. Overall, the γ9 assay provides a robust strategy for quantitative as well as qualitative determination of GPCR and G protein function on a single-cell, multicell, and subcellular level. This assay not only provides information about the inner workings of the signaling pathway, but it also strengthens GPCR deorphanization as well as drug discovery efforts.