RESUMO
Melanopsin is a photopigment belonging to the G Protein-Coupled Receptor (GPCR) family expressed in a subset of intrinsically photosensitive retinal ganglion cells (ipRGCs) and responsible for a variety of processes. The bistability and, thus, the possibility to function under low retinal availability would make melanopsin a powerful optogenetic tool. Here, we aim to utilize mouse melanopsin to trigger macrophage migration by its subcellular optical activation with localized blue light, while simultaneously imaging the migration with red light. To reduce melanopsin's red light sensitivity, we employ a combination of in silico structure prediction and automated quantum mechanics/molecular mechanics modeling to predict minimally invasive mutations to shift its absorption spectrum towards the shorter wavelength region of the visible spectrum without compromising the signaling efficiency. The results demonstrate that it is possible to achieve melanopsin mutants that resist red light-induced activation but are activated by blue light and display properties indicating preserved bistability. Using the A333T mutant, we show that the blue light-induced subcellular melanopsin activation triggers localized PIP3 generation and macrophage migration, which we imaged using red light, demonstrating the optogenetic utility of minimally engineered melanopsins.
Assuntos
Opsinas de Bastonetes , Transdução de Sinais , Animais , Opsinas de Bastonetes/metabolismo , Opsinas de Bastonetes/genética , Opsinas de Bastonetes/química , Camundongos , Movimento Celular , Simulação por Computador , Macrófagos/metabolismo , Optogenética/métodos , Luz , MutaçãoRESUMO
Cells experience time-varying and spatially heterogeneous chemokine signals in vivo, activating cell surface proteins including G protein-coupled receptors (GPCRs). The Gαq pathway activation by GPCRs is a major signaling axis with broad physiological and pathological significance. Compared with other Gα members, GαqGTP activates many crucial effectors, including PLCß (Phospholipase Cß) and Rho GEFs (Rho guanine nucleotide exchange factors). PLCß regulates many key processes, such as hematopoiesis, synaptogenesis, and cell cycle, and is therefore implicated in terminal-debilitating diseases, including cancer, epilepsy, Huntington's Disease, and Alzheimer's Disease. However, due to a lack of genetic and pharmacological tools, examining how the dynamic regulation of PLCß signaling controls cellular physiology has been difficult. Since activated PLCß induces several abrupt cellular changes, including cell morphology, examining how the other pathways downstream of Gq-GPCRs contribute to the overall signaling has also been difficult. Here we show the engineering, validation, and application of a highly selective and efficient optogenetic inhibitor (Opto-dHTH) to completely disrupt GαqGTP-PLCß interactions reversibly in user-defined cellular-subcellular regions on optical command. Using this newly gained PLCß signaling control, our data indicate that the molecular competition between RhoGEFs and PLCß for GαqGTP determines the potency of Gq-GPCR-governed directional cell migration.
Assuntos
Transdução de Sinais , Fosfolipase C beta/genética , Fosfolipase C beta/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Prenylation is an irreversible post-translational modification that supports membrane interactions of proteins involved in various cellular processes, including migration, proliferation, and survival. Dysregulation of prenylation contributes to multiple disorders, including cancers and vascular and neurodegenerative diseases. Prenyltransferases tether isoprenoid lipids to proteins via a thioether linkage during prenylation. Pharmacological inhibition of the lipid synthesis pathway by statins is a therapeutic approach to control hyperlipidemia. Building on our previous finding that statins inhibit membrane association of G protein γ (Gγ) in a subtype-dependent manner, we investigated the molecular reasoning for this differential inhibition. We examined the prenylation of carboxy-terminus (Ct) mutated Gγ in cells exposed to Fluvastatin and prenyl transferase inhibitors and monitored the subcellular localization of fluorescently tagged Gγ subunits and their mutants using live-cell confocal imaging. Reversible optogenetic unmasking-masking of Ct residues was used to probe their contribution to prenylation and membrane interactions of the prenylated proteins. Our findings suggest that specific Ct residues regulate membrane interactions of the Gγ polypeptide, statin sensitivity, and extent of prenylation. Our results also show a few hydrophobic and charged residues at the Ct are crucial determinants of a protein's prenylation ability, especially under suboptimal conditions. Given the cell and tissue-specific expression of different Gγ subtypes, our findings indicate a plausible mechanism allowing for statins to differentially perturb heterotrimeric G protein signaling in cells depending on their Gγ-subtype composition. Our results may also provide molecular reasoning for repurposing statins as Ras oncogene inhibitors and the failure of using prenyltransferase inhibitors in cancer treatment.
Assuntos
Proteínas Heterotriméricas de Ligação ao GTP , Prenilação de Proteína , Humanos , Motivos de Aminoácidos , Resistência a Medicamentos/genética , Células HeLa , Proteínas Heterotriméricas de Ligação ao GTP/química , Proteínas Heterotriméricas de Ligação ao GTP/genética , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Modelos Moleculares , Mutação , Prenilação de Proteína/efeitos dos fármacos , Estrutura Terciária de Proteína , Transporte Proteico/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
Cells experience time-varying and spatially heterogeneous chemokine signals in vivo, activating cell surface proteins, including G protein-coupled receptors (GPCRs). The Gαq pathway activation by GPCRs is a major signaling axis with a broad physiological and pathological significance. Compared to other Gα members, GαqGTP activates many crucial effectors, including PLCß (Phospholipase Cß) and Rho GEFs (Rho guanine nucleotide exchange factors). PLCß regulates many key processes, such as hematopoiesis, synaptogenesis, and cell cycle, and is therefore implicated in terminal - debilitating diseases, including cancer, epilepsy, Huntington's Disease, and Alzheimer's Disease. However, due to a lack of genetic and pharmacological tools, examining how the dynamic regulation of PLCß signaling controls cellular physiology has been difficult. Since activated PLCß induces several abrupt cellular changes, including cell morphology, examining how the other pathways downstream of Gq-GPCRs contribute to the overall signaling has also been difficult. Here we show the engineering, validation, and application of a highly selective and efficient optogenetic inhibitor (Opto-dHTH) to completely disrupt GαqGTP-PLCß interactions reversibly in user-defined cellular-subcellular regions on optical command. Using this newly gained PLCß signaling control, our data indicate that the molecular competition between RhoGEFs and PLCß for GαqGTP determines the potency of Gq-GPCR-governed directional cell migration.
RESUMO
Prenylation is a universal and irreversible post-translational modification that supports membrane interactions of proteins involved in various cellular processes, including migration, proliferation, and survival. Thus, dysregulation of prenylation contributes to multiple disorders, including cancers, vascular diseases, and neurodegenerative diseases. During prenylation, prenyltransferase enzymes tether metabolically produced isoprenoid lipids to proteins via a thioether linkage. Pharmacological inhibition of the lipid synthesis pathway by statins has long been a therapeutic approach to control hyperlipidemia. Building on our previous finding that statins inhibit membrane association of G protein γ (Gγ) in a subtype-dependent manner, we investigated the molecular reasoning for this differential. We examined the prenylation efficacy of carboxy terminus (Ct) mutated Gγ in cells exposed to Fluvastatin and prenyl transferase inhibitors and monitored the subcellular localization of fluorescently tagged Gγ subunits and their mutants using live-cell confocal imaging. Reversible optogenetic unmasking-masking of Ct residues was used to probe their contribution to the prenylation process and membrane interactions of the prenylated proteins. Our findings suggest that specific Ct residues regulate membrane interactions of the Gγ polypeptide statin sensitivity, and prenylation efficacy. Our results also show that a few hydrophobic and charged residues at the Ct are crucial determinants of a protein's prenylation ability, especially under suboptimal conditions. Given the cell and tissue-specific expression of different Gγ subtypes, our findings explain how and why statins differentially perturb heterotrimeric G protein signaling in specific cells and tissues. Our results may provide molecular reasoning for repurposing statins as Ras oncogene inhibitors and the failure of using prenyltransferase inhibitors in cancer treatment.
RESUMO
Phospholipase C (PLC) enzymes convert the membrane phospholipid phosphatidylinositol-4,5-bisphosphate (PIP2) into inositol-1,4,5-triphosphate (IP3) and diacylglycerol (DAG). IP3 and DAG regulate numerous downstream pathways, eliciting diverse and profound cellular changes and physiological responses. In the six PLC subfamilies in higher eukaryotes, PLCß is intensively studied due to its prominent role in regulating crucial cellular events underlying many processes including cardiovascular and neuronal signaling, and associated pathological conditions. In addition to GαqGTP, Gßγ generated upon G protein heterotrimer dissociation also regulates PLCß activity. Here, we not only review how Gßγ directly activates PLCß, and also extensively modulates Gαq-mediated PLCß activity, but also provide a structure-function overview of PLC family members. Given that Gαq and PLCß are oncogenes, and Gßγ shows unique cell-tissue-organ specific expression profiles, Gγ subtype-dependent signaling efficacies, and distinct subcellular activities, this review proposes that Gßγ is a major regulator of Gαq-dependent and independent PLCß signaling.
Assuntos
Proteínas de Ligação ao GTP , Transdução de Sinais , Fosfolipase C beta/genética , Fosfolipase C beta/metabolismo , Proteínas de Ligação ao GTP/metabolismo , FosfolipídeosRESUMO
Phosphatidylinositol (3,4,5) trisphosphate (PIP3) is a plasma membrane-bound signaling phospholipid involved in many cellular signaling pathways that control crucial cellular processes and behaviors, including cytoskeleton remodeling, metabolism, chemotaxis, and apoptosis. Therefore, defective PIP3 signaling is implicated in various diseases, including cancer, diabetes, obesity, and cardiovascular diseases. Upon activation by G protein-coupled receptors (GPCRs) or receptor tyrosine kinases (RTKs), phosphoinositide-3-kinases (PI3Ks) phosphorylate phosphatidylinositol (4,5) bisphosphate (PIP2), generating PIP3. Though the mechanisms are unclear, PIP3 produced upon GPCR activation attenuates within minutes, indicating a tight temporal regulation. Our data show that subcellular redistributions of G proteins govern this PIP3 attenuation when GPCRs are activated globally, while localized GPCR activation induces sustained subcellular PIP3. Interestingly the observed PIP3 attenuation was Gγ subtype-dependent. Considering distinct cell-tissue-specific Gγ expression profiles, our findings not only demonstrate how the GPCR-induced PIP3 response is regulated depending on the GPCR activity gradient across a cell, but also show how diversely cells respond to spatial and temporal variability of external stimuli.
Assuntos
Receptores Acoplados a Proteínas G , Transdução de Sinais , Transdução de Sinais/fisiologia , Receptores Acoplados a Proteínas G/metabolismo , Membrana Celular/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Fosfatidilinositóis/metabolismoRESUMO
Heterotrimeric G proteins (αßγ subunits) that are activated by G protein-coupled receptors (GPCRs) mediate the biological responses of eukaryotic cells to extracellular signals. The α subunits and the tightly bound ßγ subunit complex of G proteins have been extensively studied and shown to control the activity of effector molecules. In contrast, the potential roles of the large family of γ subunits have been less studied. In this review, we focus on present knowledge about these proteins. Induced loss of individual γ subunit types in animal and plant models result in strikingly distinct phenotypes indicating that γ subtypes play important and specific roles. Consistent with these findings, downregulation or upregulation of particular γ subunit types result in various types of cancers. Clues about the mechanistic basis of γ subunit function have emerged from imaging the dynamic behavior of G protein subunits in living cells. This shows that in the basal state, G proteins are not constrained to the plasma membrane but shuttle between membranes and on receptor activation ßγ complexes translocate reversibly to internal membranes. The translocation kinetics of ßγ complexes varies widely and is determined by the membrane affinity of the associated γ subtype. On translocating, some ßγ complexes act on effectors in internal membranes. The variation in translocation kinetics determines differential sensitivity and adaptation of cells to external signals. Membrane affinity of γ subunits is thus a parsimonious and elegant mechanism that controls information flow to internal cell membranes while modulating signaling responses.
Assuntos
Subunidades beta da Proteína de Ligação ao GTP , Subunidades gama da Proteína de Ligação ao GTP , Receptores Acoplados a Proteínas G , Animais , Membrana Celular/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/genética , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/genética , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Transdução de Sinais , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , HumanosRESUMO
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.
Assuntos
Microcistinas , Animais , Cromatografia Líquida , Humanos , Espectrometria de Massas , Microcistinas/análise , Microcistinas/toxicidadeRESUMO
The chemical- and photo- toxicity of chromophore retinal on cells have long been debated. Although we recently showed that retinal and blue light exposure interrupt cellular signaling, a comprehensive study examining molecular underpinnings of this perturbation and its consequences to cellular fate is lacking. Here, we report molecular evidence for blue light excited-retinal induced oxidative damage of polyunsaturated lipid anchors in membrane-interacting signaling molecules and DNA damage in cells using live-cell imaging and in vitro experimentation. The incurred molecular damage irreversibly disrupted subcellular localization of these molecules, a crucial criterion for their signaling. We further show retinal accumulation in lipid-bilayers of cell membranes could enhance the lifetime of retinal in cells. Comparative response-signatures suggest that retinal triggers reactions upon photoexcitation similar to photodynamic therapy agents and generate reactive oxygen species in cells. Additionally, data also shows that exposing retinal-containing cells to sunlight induces substantial cytotoxicity. Collectively, our results explain a likely in vivo mechanism and reaction conditions under which bio-available retinal in physiological light conditions damages cells.
Assuntos
Luz/efeitos adversos , Retinaldeído/toxicidade , Dano ao DNA , Células HeLa , Humanos , Peroxidação de Lipídeos , Estresse Oxidativo , Processos Fotoquímicos , Espécies Reativas de Oxigênio/metabolismoRESUMO
G protein αq-coupled receptors (Gq-GPCRs) primarily signal through GαqGTP mediated phospholipase Cß (PLCß) stimulation and the subsequent hydrolysis of phosphatidylinositol 4, 5 bisphosphate (PIP2). Though Gq-heterotrimer activation results in both GαqGTP and Gßγ, unlike Gi/o-receptors, it is unclear if Gq-coupled receptors employ Gßγ as a major signal transducer. Compared to Gi/o- and Gs-coupled receptors, we observed that most cell types exhibit a limited free Gßγ generation upon Gq-pathway and Gαq/11 heterotrimer activation. We show that cells transfected with Gαq or endogenously expressing more than average-levels of Gαq/11 compared to Gαs and Gαi exhibit a distinct signaling regime primarily characterized by recovery-resistant PIP2 hydrolysis. Interestingly, the elevated Gq-expression is also associated with enhanced free Gßγ generation and signaling. Furthermore, the gene GNAQ, which encodes for Gαq, has recently been identified as a cancer driver gene. We also show that GNAQ is overexpressed in tumor samples of patients with Kidney Chromophobe (KICH) and Kidney renal papillary (KIRP) cell carcinomas in a matched tumor-normal sample analysis, which demonstrates the clinical significance of Gαq expression. Overall, our data indicates that cells usually express low Gαq levels, likely safeguarding cells from excessive calcium as wells as from Gßγ signaling.
Assuntos
Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Transdução de Sinais , Cálcio/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Expressão Gênica , Células HeLa , Humanos , Hidrólise , Fosfolipase C beta/metabolismo , TransfecçãoRESUMO
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.
Assuntos
Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Receptores Acoplados a Proteínas G/metabolismo , Animais , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Colesterol/metabolismo , Células HeLa , Humanos , Ácido Mevalônico/farmacologia , Camundongos , Prenilação de Proteína/efeitos dos fármacos , Células RAW 264.7 , Transdução de Sinais/efeitos dos fármacosRESUMO
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.
Assuntos
Sinalização do Cálcio , Membrana Celular/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Macrófagos/metabolismo , Modelos Biológicos , Animais , Membrana Celular/química , Movimento Celular , Biologia Computacional , Subunidades beta da Proteína de Ligação ao GTP/antagonistas & inibidores , Subunidades beta da Proteína de Ligação ao GTP/química , Subunidades beta da Proteína de Ligação ao GTP/genética , Subunidades gama da Proteína de Ligação ao GTP/antagonistas & inibidores , Subunidades gama da Proteína de Ligação ao GTP/química , Subunidades gama da Proteína de Ligação ao GTP/genética , Meia-Vida , Células HeLa , Humanos , Cinética , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Ativação de Macrófagos , Macrófagos/citologia , Macrófagos/imunologia , Camundongos , Domínios e Motivos de Interação entre Proteínas , Prenilação de Proteína , Transporte Proteico , Células RAW 264.7 , Interferência de RNA , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismoRESUMO
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.
Assuntos
Bioensaio/métodos , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Imagem Óptica/métodos , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/efeitos dos fármacos , Bioensaio/instrumentação , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Membrana Celular/metabolismo , Células HeLa , Humanos , Cinética , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Imagem Molecular/instrumentação , Imagem Molecular/métodos , Imagem Óptica/instrumentação , Fatores de TempoRESUMO
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.
Assuntos
Movimento Celular , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Macrófagos/metabolismo , Actomiosina/genética , Actomiosina/metabolismo , Animais , Cálcio/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/genética , Subunidades gama da Proteína de Ligação ao GTP/genética , Células HeLa , Humanos , Camundongos , Fosfolipase C beta/genética , Fosfolipase C beta/metabolismo , Células RAW 264.7 , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
G protein-coupled receptors (GPCRs) are targets for designing a large fraction of the drugs in the pharmaceutical industry. For GPCR-targeting drug screening using cell-based assays, measurement of cytosolic calcium has been widely used to obtain dose-response profiles. However, it remains challenging to obtain drug-specific features due to cell-to-cell heterogeneity in drug-cell responses obtained from live cell imaging. Here, we present a framework combining live cell imaging of a cell population and a feature extraction method for classification of responses of drugs targeting GPCRs CXCR4 and α2AR. We measured the calcium dynamics using confocal microscopy and compared the responses for SDF-1α and norepinephrine. The results clearly show that the clustering patterns of responses for the two GPCRs are significantly different. Additionally, we show that different drugs targeting the same GPCR induce different calcium response signatures. We also implemented principal component analysis and k means for feature extraction and used nondominated (ND) sorting for ranking a group of drugs at various doses. The presented approach can be used to model a cell population as a mixture of subpopulations. It also offers specific advantages, such as higher spatial resolution, classification of responses, and ranking of drugs, potentially providing a platform for high-content drug screening.
Assuntos
Cálcio/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Linhagem Celular Tumoral , Citosol/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Células HeLa , Humanos , Microscopia Confocal/métodos , Análise de Componente Principal/métodosRESUMO
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.