RESUMO
Background: Geometrical alterations in the coronary resistance artery network and the potential involvement of Tenascin C (TNC) extracellular matrix protein were investigated in diabetic and control mice. Methods: Diabetes was induced by streptozotocin (STZ) injections (n = 7-11 animals in each group) in Tenascin C KO (TNC KO) mice and their Wild type (A/J) littermates. After 16-18 weeks the heart was removed and the whole subsurface network of the left coronary artery was prepared (down to branches of 40 µ m outer diameter), in situ pressure-perfused and studied using video-microscopy. Outer and inner diameters, wall thicknesses and bifurcation angles were measured on whole network pictures reconstructed into collages at 1.7 µ m pixel resolutions. Results: Diabetes induced abnormal morphological alterations including trifurcations, sharp bends of larger branches, and branches directed retrogradely (p < 0.001 by the χ 2 test). Networks of TNC KO mice tended to form early divisions producing parallelly running larger branches (p < 0.001 by the χ 2 probe). Networks of coronary resistance arteries were substantially more abundant in 100-180 µ m components, appearing in 2-5 mm flow distance from orifice in diabetes. This was accompanied by thickening of the wall of larger arterioles ( > 220 µ m) and thinning of the wall of smaller (100-140 µ m) arterioles (p < 0.001). Blood flow should cover larger distances in diabetic networks, but interestingly STZ-induced diabetes did not generate further geometrical changes in TNC KO mice. Conclusions: Diabetes promotes hypertrophic and hypotrophic vascular remodeling and induces vasculogenesis at well defined, specific positions of the coronary vasculature. TNC plays a pivotal role in the formation of coronary network geometry, and TNC deletion causes parallel fragmentation preventing diabetes-induced abnormal vascular morphologies.
RESUMO
Comparing SARS-CoV-2 infection-induced gene expression signatures to drug treatment-induced gene expression signatures is a promising bioinformatic tool to repurpose existing drugs against SARS-CoV-2. The general hypothesis of signature-based drug repurposing is that drugs with inverse similarity to a disease signature can reverse disease phenotype and thus be effective against it. However, in the case of viral infection diseases, like SARS-CoV-2, infected cells also activate adaptive, antiviral pathways, so that the relationship between effective drug and disease signature can be more ambiguous. To address this question, we analysed gene expression data from in vitro SARS-CoV-2 infected cell lines, and gene expression signatures of drugs showing anti-SARS-CoV-2 activity. Our extensive functional genomic analysis showed that both infection and treatment with in vitro effective drugs leads to activation of antiviral pathways like NFkB and JAK-STAT. Based on the similarity-and not inverse similarity-between drug and infection-induced gene expression signatures, we were able to predict the in vitro antiviral activity of drugs. We also identified SREBF1/2, key regulators of lipid metabolising enzymes, as the most activated transcription factors by several in vitro effective antiviral drugs. Using a fluorescently labeled cholesterol sensor, we showed that these drugs decrease the cholesterol levels of plasma-membrane. Supplementing drug-treated cells with cholesterol reversed the in vitro antiviral effect, suggesting the depleting plasma-membrane cholesterol plays a key role in virus inhibitory mechanism. Our results can help to more effectively repurpose approved drugs against SARS-CoV-2, and also highlights key mechanisms behind their antiviral effect.
Assuntos
Tratamento Farmacológico da COVID-19 , SARS-CoV-2 , Antivirais/farmacologia , Antivirais/uso terapêutico , Membrana Celular , Colesterol , Reposicionamento de Medicamentos/métodos , HumanosRESUMO
Angiotensin II (AngII) is a vasoactive peptide hormone, which, under pathological conditions, contributes to the development of cardiovascular diseases. Oxysterols, including 25-hydroxycholesterol (25-HC), the product of cholesterol-25-hydroxylase (CH25H), also have detrimental effects on vascular health by affecting vascular smooth muscle cells (VSMCs). We investigated AngII-induced gene expression changes in VSMCs to explore whether AngII stimulus and 25-HC production have a connection in the vasculature. RNA-sequencing revealed that Ch25h is significantly upregulated in response to AngII stimulus. The Ch25h mRNA levels were elevated robustly (~50-fold) 1 h after AngII (100 nM) stimulation compared to baseline levels. Using inhibitors, we specified that the AngII-induced Ch25h upregulation is type 1 angiotensin II receptor- and Gq/11 activity-dependent. Furthermore, p38 MAPK has a crucial role in the upregulation of Ch25h. We performed LC-MS/MS to identify 25-HC in the supernatant of AngII-stimulated VSMCs. In the supernatants, 25-HC concentration peaked 4 h after AngII stimulation. Our findings provide insight into the pathways mediating AngII-induced Ch25h upregulation. Our study elucidates a connection between AngII stimulus and 25-HC production in primary rat VSMCs. These results potentially lead to the identification and understanding of new mechanisms in the pathogenesis of vascular impairments.
Assuntos
Angiotensina II , Músculo Liso Vascular , Esteroide Hidroxilases , Animais , Ratos , Angiotensina II/metabolismo , Células Cultivadas , Cromatografia Líquida , Expressão Gênica , Músculo Liso Vascular/enzimologia , Miócitos de Músculo Liso/metabolismo , Espectrometria de Massas em Tandem , Esteroide Hidroxilases/genéticaRESUMO
Both the endocannabinoid system (ECS) and estrogens have significant roles in cardiovascular control processes. Cannabinoid type 1 receptors (CB1Rs) mediate acute vasodilator and hypotensive effects, although their role in cardiovascular pathological conditions is still controversial. Estrogens exert cardiovascular protection in females. We aimed to study the impact of ECS on vascular functions. Experiments were performed on CB1R knockout (CB1R KO) and wild-type (WT) female mice. Plasma estrogen metabolite levels were determined. Abdominal aortas were isolated for myography and histology. Vascular effects of phenylephrine (Phe), angiotensin II, acetylcholine (Ach) and estradiol (E2) were obtained and repeated with inhibitors of nitric oxide synthase (NOS, Nω-nitro-L-arginine) and of cyclooxygenase (COX, indomethacin). Histological stainings (hematoxylin-eosin, resorcin-fuchsin) and immunostainings for endothelial NOS (eNOS), COX-2, estrogen receptors (ER-α, ER-ß) were performed. Conjugated E2 levels were higher in CB1R KO compared to WT mice. Vasorelaxation responses to Ach and E2 were increased in CB1R KO mice, attenuated by NOS-inhibition. COX-inhibition decreased Phe-contractions, while it increased Ach-relaxation in the WT group but not in the CB1R KO. Effects of indomethacin on E2-relaxation in CB1R KO became opposite to that observed in WT. Histology revealed lower intima/media thickness and COX-2 density, higher eNOS and lower ER-ß density in CB1R KO than in WT mice. CB1R KO female mice are characterized by increased vasorelaxation associated with increased utilization of endothelial NO and a decreased impact of constrictor prostanoids. Our results indicate that the absence or inhibition of CB1Rs may have beneficial vascular effects.
Assuntos
Receptores de Canabinoides , Remodelação Vascular , Animais , Feminino , Camundongos , Acetilcolina/metabolismo , Aorta Abdominal/metabolismo , Ciclo-Oxigenase 2/metabolismo , Receptor beta de Estrogênio/metabolismo , Estrogênios/metabolismo , Indometacina/farmacologia , Camundongos Knockout , Óxido Nítrico Sintase Tipo III/metabolismo , Receptores de Canabinoides/metabolismo , VasodilataçãoRESUMO
Reliable measurement of ligand binding to cell surface receptors is of outstanding biological and pharmacological importance. Resonance energy transfer-based assays are powerful approaches to achieve this goal, but the currently available methods are hindered by the necessity of receptor tagging, which can potentially alter ligand binding properties. Therefore, we developed a tag-free system to measure ligandâreceptor interactions in live cells using the Gaussia luciferase (GLuc) as a bioluminescence resonance energy transfer donor. GLuc is as small as the commonly applied Nanoluciferase but has enhanced brightness, and its proper substrate is the frequently used coelenterazine. In our assay, bystander bioluminescence resonance energy transfer is detected between a GLuc-based extracellular surface biosensor and fluorescent ligands bound to their unmodified receptors. The broad spectrum of applications includes equilibrium and kinetic ligand binding measurements for both labeled and competitive unlabeled ligands, and the assay can be utilized for different classes of plasma membrane receptors. Furthermore, the assay is suitable for high-throughput screening, as evidenced by the identification of novel α1 adrenergic receptor ligands. Our data demonstrate that GLuc-based biosensors provide a simple, sensitive, and cost-efficient platform for drug characterization and development.
Assuntos
Técnicas de Transferência de Energia por Ressonância de Bioluminescência/métodos , Luciferases/química , Luciferases/metabolismo , Bioensaio , Membrana Celular/metabolismo , Transferência de Energia , Células HEK293 , Humanos , Cinética , Ligantes , Ligação Proteica , Transporte Proteico , Receptores Acoplados a Proteínas G/metabolismo , Transdução de SinaisRESUMO
Cells deficient in mitochondrial fusion have been shown to have defects linked to the exchange of inner membrane and matrix components. Because outer-mitochondrial membrane (OMM) constituents insert directly from the cytoplasm, a role for fusion in their intermitochondrial transfer was unanticipated. Here, we show that fibroblasts lacking the GTPases responsible for OMM fusion, mitofusins 1 and 2 (MFN1 and MFN2), display more heterogeneous distribution of OMM proteins. Proteins with different modes of OMM association display varying degrees of heterogeneity in Mfn1/2(-/-) cells and different kinetics of transfer during fusion in fusion-competent cells. Proapoptotic Bak exhibits marked heterogeneity, which is normalized upon expression of MFN2. Bak is critical for Bid-induced OMM permeabilization and cytochrome c release, and Mfn1/2(-/-) cells show dysregulation of Bid-dependent apoptotic signaling. Bid sensitivity of Bak-deficient mitochondria is regained upon fusion with Bak-containing mitochondria. Thus, OMM protein distribution depends on mitochondrial fusion and is a locus of apoptotic dysfunction in conditions of fusion deficiency.
Assuntos
Dinâmica Mitocondrial , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Animais , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Linhagem Celular , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Técnicas de Inativação de Genes , Humanos , Camundongos , Fibras Musculares Esqueléticas/metabolismo , Transporte Proteico , Ratos , Canal de Ânion 2 Dependente de Voltagem/genéticaRESUMO
(1) Adeno-associated viruses (AAV) are safe and efficient gene therapy vectors with promising results in the treatment of several diseases. Extracellular vesicles (EV) are phospholipid bilayer-surrounded structures carrying several types of lipids, proteins, and nucleic acids with the ability to cross biological barriers. EV-associated AAVs might serve as new and efficient gene therapy vectors considering that they carry the benefits of both AAVs and EVs. (2) We tested vesicle-associated AAVs and vesicles mixed with AAVs on two major cell types of the central nervous system: a neural cell line (N2A) and primary astrocyte cells. (3) In contrast to previously published in vivo observations, the extracellular vesicle packaging did not improve but, in the case of primary astrocyte cells, even inhibited the infection capacity of the AAV particles. The observed effect was not due to the inhibitory effects of the vesicles themselves, since mixing the AAVs with extracellular vesicles did not change the effectiveness. (4) Our results suggest that improvement of the in vivo efficacy of the EV-associated AAV particles is not due to the enhanced interaction between the AAV and the target cells, but most likely to the improved delivery of the AAVs through tissue barriers and to the shielding of AAVs from neutralizing antibodies.
Assuntos
Astrócitos/metabolismo , Dependovirus/metabolismo , Vesículas Extracelulares/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Vetores Genéticos/genética , Humanos , Transdução Genética/métodosRESUMO
ß-Arrestins are key regulators and signal transducers of G protein-coupled receptors (GPCRs). The interaction between receptors and ß-arrestins is generally believed to require both receptor activity and phosphorylation by GPCR kinases. In this study, we investigated whether ß-arrestins are able to bind second messenger kinase-phosphorylated, but inactive receptors as well. Because heterologous phosphorylation is a common phenomenon among GPCRs, this mode of ß-arrestin activation may represent a novel mechanism of signal transduction and receptor cross-talk. Here we demonstrate that activation of protein kinase C (PKC) by phorbol myristate acetate, Gq/11-coupled GPCR, or epidermal growth factor receptor stimulation promotes ß-arrestin2 recruitment to unliganded AT1 angiotensin receptor (AT1R). We found that this interaction depends on the stability lock, a structure responsible for the sustained binding between GPCRs and ß-arrestins, formed by phosphorylated serine-threonine clusters in the receptor's C terminus and two conserved phosphate-binding lysines in the ß-arrestin2 N-domain. Using improved FlAsH-based serine-threonine clusters ß-arrestin2 conformational biosensors, we also show that the stability lock not only stabilizes the receptor-ß-arrestin interaction, but also governs the structural rearrangements within ß-arrestins. Furthermore, we found that ß-arrestin2 binds to PKC-phosphorylated AT1R in a distinct active conformation, which triggers MAPK recruitment and receptor internalization. Our results provide new insights into the activation of ß-arrestins and reveal their novel role in receptor cross-talk.
Assuntos
Proteínas Quinases Ativadas por Mitógeno/metabolismo , beta-Arrestinas/metabolismo , Angiotensina II/metabolismo , Animais , Células COS , Chlorocebus aethiops , Células HEK293 , Humanos , Immunoblotting , Microscopia Confocal , Fosforilação , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Plasma membrane (PM) localization of Ras proteins is crucial for transmitting signals upon mitogen stimulation. Post-translational lipid modification of Ras proteins plays an important role in their recruitment to the PM. Electrostatic interactions between negatively charged PM phospholipids and basic amino acids found in K-Ras4B (K-Ras) but not in H-Ras are important for permanent K-Ras localization to the PM. Here, we investigated how acute depletion of negatively charged PM polyphosphoinositides (PPIns) from the PM alters the intracellular distribution and activity of K- and H-Ras proteins. PPIns depletion from the PM was achieved either by agonist-induced activation of phospholipase C ß or with a rapamycin-inducible system in which various phosphatidylinositol phosphatases were recruited to the PM. Redistribution of the two Ras proteins was monitored with confocal microscopy or with a recently developed bioluminescence resonance energy transfer-based approach involving fusion of the Ras C-terminal targeting sequences or the entire Ras proteins to Venus fluorescent protein. We found that PM PPIns depletion caused rapid translocation of K-Ras but not H-Ras from the PM to the Golgi. PM depletion of either phosphatidylinositol 4-phosphate (PtdIns4P) or PtdIns(4,5)P2 but not PtdIns(3,4,5)P3 was sufficient to evoke K-Ras translocation. This effect was diminished by deltarasin, an inhibitor of the Ras-phosphodiesterase interaction, or by simultaneous depletion of the Golgi PtdIns4P. The PPIns depletion decreased incorporation of [3H]leucine in K-Ras-expressing cells, suggesting that Golgi-localized K-Ras is not as signaling-competent as its PM-bound form. We conclude that PPIns in the PM are important regulators of K-Ras-mediated signals.
Assuntos
Membrana Celular/metabolismo , Complexo de Golgi/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Animais , Células COS , Chlorocebus aethiops , Difosfatos/metabolismo , Células HEK293 , Humanos , Transporte ProteicoAssuntos
Aterosclerose/genética , NADPH Oxidase 5/genética , Acetilcolina/farmacologia , Animais , Aorta Torácica/efeitos dos fármacos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Dieta Aterogênica , Epinefrina/farmacologia , Masculino , NADPH Oxidase 5/deficiência , Placa Aterosclerótica/genética , Placa Aterosclerótica/patologia , Potássio/farmacologia , CoelhosRESUMO
It was tested whether intrinsic CB1R activation modifies myogenic and agonist induced contraction of intramural coronary resistance arteries of the rat. CB1R protein was detected by immuno-histochemistry and by Western blot, its mRNA by qRT-PCR in their wall. Microsurgically prepared cylindrical coronary segments (â¼100-150µm) developed myogenic contraction (â¼20% of relaxed luminal diameter), from which a substantial relaxation (â¼15%) in response to WIN55212 (a specific agonist of the CB1Rs) has been found. CB1R-mediated relaxation was blocked by O2050 and AM251 (neutral antagonist and inverse agonist of the CB1R, respectively) and was partially blocked by the NO synthase blocker Nω-nitro-L-arginine. CB1R blockade enhanced myogenic tone and augmented AngII-induced vasoconstriction (from 17.8±1.2 to 29.1±2.9%, p<0.05). Inhibition of diacylglycerol lipase by tetrahydrolipstatin, (inhibitor of endogenous 2-AG production) also augmented coronary vasoconstriction. These observations prove that vascular endocannabinoids are significant negative modulators of the myogenic and agonist-induced tone of intramural coronary arterioles acting through CB1Rs.
Assuntos
Vasos Coronários/efeitos dos fármacos , Vasos Coronários/fisiologia , Endocanabinoides/metabolismo , Receptor CB1 de Canabinoide/agonistas , Vasoconstrição/efeitos dos fármacos , Animais , Arteríolas/efeitos dos fármacos , Arteríolas/fisiologia , Canabinoides/farmacologia , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Lipase Lipoproteica/metabolismo , Masculino , Ratos , Ratos Wistar , Receptor CB1 de Canabinoide/metabolismoRESUMO
Deciphering many roles played by inositol lipids in signal transduction and membrane function demands experimental approaches that can detect their dynamic accumulation with subcellular accuracy and exquisite sensitivity. The former criterion is met by imaging of fluorescence biosensors in living cells, whereas the latter is facilitated by biochemical measurements from populations. Here, we introduce BRET-based biosensors able to detect rapid changes in inositol lipids in cell populations with both high sensitivity and subcellular resolution in a single, convenient assay. We demonstrate robust and sensitive measurements of PtdIns4P, PtdIns(4,5)P2 and PtdIns(3,4,5)P3 dynamics, as well as changes in cytoplasmic Ins(1,4,5)P3 levels. Measurements were made during either experimental activation of lipid degradation, or PI 3-kinase and phospholipase C mediated signal transduction. Our results reveal a previously unappreciated synthesis of PtdIns4P that accompanies moderate activation of phospholipase C signaling downstream of both EGF and muscarinic M3 receptor activation. This signaling-induced PtdIns4P synthesis relies on protein kinase C, and implicates a feedback mechanism in the control of inositol lipid metabolism during signal transduction.
Assuntos
Técnicas Biossensoriais , Carbacol/farmacologia , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/agonistas , Transferência Ressonante de Energia de Fluorescência , Agonistas Muscarínicos/farmacologia , Fosfatos de Fosfatidilinositol/metabolismo , Proteína Quinase C/metabolismo , Receptores Muscarínicos/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Animais , Células COS , Chlorocebus aethiops , Ativação Enzimática , Receptores ErbB/genética , Receptores ErbB/metabolismo , Retroalimentação Fisiológica , Células HEK293 , Humanos , Hidrólise , Inositol 1,4,5-Trifosfato/metabolismo , Cinética , Lipólise , Fosfatidilinositol 3-Quinase/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Receptor Muscarínico M3 , Receptores Muscarínicos/genética , Receptores Muscarínicos/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Fosfolipases Tipo C/metabolismo , Regulação para CimaRESUMO
Biased agonism on the type I angiotensin receptor (AT1-R) can achieve different outcomes via activation of G protein-dependent and -independent cellular responses. In this study, we investigated whether the biased activation of AT1-R can lead to different regulation and intracellular processing of the receptor. We analyzed ß-arrestin binding, endocytosis, and subsequent trafficking steps, such as early and late phases of recycling of AT1-R in human embryonic kidney 293 cells expressing wild-type or biased mutant receptors in response to different ligands. We used Renilla luciferase-tagged receptors and yellow fluorescent protein-tagged ß-arrestin2, Rab5, Rab7, and Rab11 proteins in bioluminescence resonance energy transfer measurements to follow the fate of the receptor after stimulation. We found that not only is the signaling of the receptor different upon using selective ligands, but the fate within the cells is also determined by the type of the stimulation. ß-arrestin binding and the internalization kinetics of the angiotensin II-stimulated AT1-R differed from those stimulated by the biased agonists. Similarly, angiotensin II-stimulated wild-type AT1-R showed differences compared with a biased mutant AT1-R (DRY/AAY AT1-R) with regards to ß-arrestin binding and endocytosis. We found that the differences in the internalization kinetics of the receptor in response to biased agonist stimulation are due to the differences in plasma membrane phosphatidylinositol 4,5-bisphosphate depletion. Moreover, the stability of the ß-arrestin binding is a major determinant of the later fate of the internalized AT1-R receptor.
Assuntos
Receptor Tipo 1 de Angiotensina/metabolismo , 1-Fosfatidilinositol 4-Quinase/antagonistas & inibidores , Angiotensina II/farmacologia , Arrestinas/genética , Arrestinas/metabolismo , Técnicas de Transferência de Energia por Ressonância de Bioluminescência , Membrana Celular/metabolismo , Endocitose/efeitos dos fármacos , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Proteínas de Fluorescência Verde/genética , Células HEK293 , Humanos , Hidrólise , Ligantes , Luciferases de Renilla/genética , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfoinositídeo Fosfolipase C/genética , Fosfoinositídeo Fosfolipase C/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Receptor Tipo 1 de Angiotensina/agonistas , Receptor Tipo 1 de Angiotensina/genética , beta-ArrestinasRESUMO
Nephrogenic syndrome of inappropriate antidiuresis (NSIAD) is a recently discovered rare disease caused by gain-of-function mutations of the V2 vasopressin receptor gene, AVPR2. To date, mutations of Phe229 and Arg137 have been identified as gain-of-function in the V2 vasopressin receptor (V2R). These receptor mutations lead to hyponatremia, which may lead to clinical symptoms in infants. Here we present a newly identified I130N substitution in exon 2 of the V2R gene in a family, causing NSIAD. This I130N mutation resulted in constitutive activity of the V2R with constitutive cyclic adenosine monophosphate (cAMP) generation in HEK293 cells. This basal activity could be blocked by the inverse agonist tolvaptan and arginine-vasopressin stimulation enhanced the cAMP production of I130N-V2R. The mutation causes a biased receptor conformation as the basal cAMP generation activity of I130N does not lead to interaction with ß-arrestin. The constitutive activity of the mutant receptor caused constitutive dynamin-dependent and ß-arrestin-independent internalization. The inhibition of basal internalization using dominant-negative dynamin resulted in an increased cell surface expression. In contrast to the constitutive internalization, agonist-induced endocytosis was ß-arrestin dependent. Thus, tolvaptan could be used for treatment of hyponatremia in patients with NSIAD who carry the I130N-V2R mutation.
Assuntos
AMP Cíclico/biossíntese , Doenças Genéticas Ligadas ao Cromossomo X/genética , Hiponatremia/genética , Síndrome de Secreção Inadequada de HAD/genética , Receptores de Vasopressinas/genética , Adulto , Antagonistas dos Receptores de Hormônios Antidiuréticos/farmacologia , Arrestinas/metabolismo , Benzazepinas/farmacologia , Membrana Celular/química , Análise Mutacional de DNA , Dinaminas/metabolismo , Endocitose/efeitos dos fármacos , Éxons , Feminino , Células HEK293 , Humanos , Hiponatremia/tratamento farmacológico , Masculino , Mutação , Linhagem , Receptores de Vasopressinas/análise , Receptores de Vasopressinas/metabolismo , Tolvaptan , beta-ArrestinasRESUMO
Receptor endocytosis plays an important role in regulating the responsiveness of cells to specific ligands. Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] has been shown to be crucial for endocytosis of some cell surface receptors, such as EGF and transferrin receptors, but its role in G-protein-coupled receptor internalization has not been investigated. By using luciferase-labeled type 1 angiotensin II (AT1R), type 2C serotonin (5HT2CR) or ß(2) adrenergic (ß2AR) receptors and fluorescently tagged proteins (ß-arrestin-2, plasma-membrane-targeted Venus, Rab5) we were able to follow the sequence of molecular interactions along the endocytic route of the receptors in HEK293 cells using the highly sensitive method of bioluminescence resonance energy transfer and confocal microscopy. To study the role of plasma membrane PtdIns(4,5)P(2) in receptor endocytosis, we used our previously developed rapamycin-inducible heterodimerization system, in which the recruitment of a 5-phosphatase domain to the plasma membrane degrades PtdIns(4,5)P(2). Here we show that ligand-induced interaction of AT1, 5HT2C and ß(2)A receptors with ß-arrestin-2 was unaffected by PtdIns(4,5)P(2) depletion. However, trafficking of the receptors to Rab5-positive early endosomes was completely abolished in the absence of PtdIns(4,5)P(2). Remarkably, removal of the receptors from the plasma membrane was reduced but not eliminated after PtdIns(4,5)P(2) depletion. Under these conditions, stimulated AT1 receptors clustered along the plasma membrane, but did not enter the cells. Our data suggest that in the absence of PtdIns(4,5)P(2), these receptors move into clathrin-coated membrane structures, but these are not cleaved efficiently and hence cannot reach the early endosomal compartment.
Assuntos
Endocitose/fisiologia , Fosfatidilinositol 4,5-Difosfato/deficiência , Receptor Tipo 1 de Angiotensina/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Receptores 5-HT2 de Serotonina/metabolismo , Arrestinas/metabolismo , Técnicas de Transferência de Energia por Ressonância de Bioluminescência , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Vesículas Revestidas por Clatrina/metabolismo , Endocitose/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Endossomos/ultraestrutura , Genes Reporter , Células HEK293 , Humanos , Luciferases , Microscopia Confocal , Sirolimo/farmacologia , beta-Arrestina 2 , beta-Arrestinas , Proteínas rab5 de Ligação ao GTP/metabolismoRESUMO
G protein-coupled receptors (GPCRs) regulate every aspect of kidney function by mediating the effects of various endogenous and exogenous substances. A key concept in GPCR function is biased signalling, whereby certain ligands may selectively activate specific pathways within the receptor's signalling repertoire. For example, different agonists may induce biased signalling by stabilizing distinct active receptor conformations - a concept that is supported by advances in structural biology. However, the processes underlying functional selectivity in receptor signalling are extremely complex, involving differences in subcellular compartmentalization and signalling dynamics. Importantly, the molecular mechanisms of spatiotemporal bias, particularly its connection to ligand binding kinetics, have been detailed for GPCRs critical to kidney function, such as the AT1 angiotensin receptor (AT1R), V2 vasopressin receptor (V2R) and the parathyroid hormone 1 receptor (PTH1R). This expanding insight into the multifaceted nature of biased signalling paves the way for innovative strategies for targeting GPCR functions; the development of novel biased agonists may represent advanced pharmacotherapeutic approaches to the treatment of kidney diseases and related systemic conditions, such as hypertension, diabetes and heart failure.
RESUMO
Phosphatidylinositol 4,5-bisphosphate (PIP2) has been shown to be critical for the endocytosis of G protein-coupled receptors (GPCRs). We have previously demonstrated that depletion of PIP2 by chemically induced plasma membrane (PM) recruitment of a 5-phosphatase domain prevents the internalization of the ß2 adrenergic receptor (ß2AR) from the PM to early endosomes. In this study, we tested the effect of hormone-induced PM PIP2 depletion on ß2AR internalization using type-1 angiotensin receptor (AT1R) or M3 muscarinic acetylcholine receptor (M3R). We followed the endocytic route of ß2ARs in HEK 293T cells using bioluminescence resonance energy transfer between the receptor and endosome marker Rab5. To compare the effect of lipid depletion by different means, we created and tested an AT1R fusion protein that is capable of both recruitment-based and hormone-induced depletion methods. The rate of PM PIP2 depletion was measured using a biosensor based on the PH domain of phospholipase Cδ1. As expected, ß2AR internalization was inhibited when PIP2 depletion was evoked by recruiting 5-phosphatase to PM-anchored AT1R. A similar inhibition occurred when wild-type AT1R was activated by adding angiotensin II. However, stimulation of the desensitization/internalization-impaired mutant AT1R (TSTS/4A) caused very little inhibition of ß2AR internalization, despite the higher rate of measurable PIP2 depletion. Interestingly, inhibition of PIP2 resynthesis with the selective PI4KA inhibitor GSK-A1 had little effect on the change in PH-domain-measured PM PIP2 levels but did significantly decrease ß2AR internalization upon either AT1R or M3R activation, indicating the importance of a locally synthetized phosphoinositide pool in the regulation of this process.
Assuntos
Endocitose , Fosfatidilinositóis , Fosfatidilinositóis/metabolismo , Membrana Celular/metabolismo , Receptores de Angiotensina/metabolismo , Hormônios/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismoRESUMO
The stabilization of different active conformations of G protein-coupled receptors is thought to underlie the varying efficacies of biased and balanced agonists. Here, profiling the activation of signal transducers by angiotensin II type 1 receptor (AT1R) agonists revealed that the extent and kinetics of ß-arrestin binding exhibited substantial ligand-dependent differences, which were lost when receptor internalization was inhibited. When AT1R endocytosis was prevented, even weak partial agonists of the ß-arrestin pathway acted as full or near-full agonists, suggesting that receptor conformation did not exclusively determine ß-arrestin recruitment. The ligand-dependent variance in ß-arrestin translocation was much larger at endosomes than at the plasma membrane, showing that ligand efficacy in the ß-arrestin pathway was spatiotemporally determined. Experimental investigations and mathematical modeling demonstrated how multiple factors concurrently shaped the effects of agonists on endosomal receptor-ß-arrestin binding and thus determined the extent of functional selectivity. Ligand dissociation rate and G protein activity had particularly strong, internalization-dependent effects on the receptor-ß-arrestin interaction. We also showed that endocytosis regulated the agonist efficacies of two other receptors with sustained ß-arrestin binding: the V2 vasopressin receptor and a mutant ß2-adrenergic receptor. In the absence of endocytosis, the agonist-dependent variance in ß-arrestin2 binding was markedly diminished. Our results suggest that endocytosis determines the spatiotemporal bias in GPCR signaling and can aid in the development of more efficacious, functionally selective compounds.
Assuntos
Endocitose , Receptor Tipo 1 de Angiotensina , Transdução de Sinais , beta-Arrestinas , Endocitose/fisiologia , Humanos , Receptor Tipo 1 de Angiotensina/metabolismo , Receptor Tipo 1 de Angiotensina/genética , beta-Arrestinas/metabolismo , beta-Arrestinas/genética , Células HEK293 , Receptores de Vasopressinas/metabolismo , Receptores de Vasopressinas/genética , Receptores Adrenérgicos beta 2/metabolismo , Receptores Adrenérgicos beta 2/genética , Endossomos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Animais , Ligantes , Ligação Proteica , Transporte ProteicoRESUMO
The binding and function of ß-arrestins are regulated by specific phosphorylation motifs present in G protein-coupled receptors (GPCRs). However, the exact arrangement of phosphorylated amino acids responsible for establishing a stable interaction remains unclear. We employ a 1D sequence convolution model trained on GPCRs with established ß-arrestin-binding properties. With this approach, amino acid motifs characteristic of GPCRs that form stable interactions with ß-arrestins can be identified, a pattern that we name "arreSTick." Intriguingly, the arreSTick pattern is also present in numerous non-receptor proteins. Using proximity biotinylation assay and mass spectrometry analysis, we demonstrate that the arreSTick motif controls the interaction between many non-receptor proteins and ß-arrestin2. The HIV-1 Tat-specific factor 1 (HTSF1 or HTATSF1), a nuclear transcription factor, contains the arreSTick pattern, and its subcellular localization is influenced by ß-arrestin2. Our findings unveil a broader role for ß-arrestins in phosphorylation-dependent interactions, extending beyond GPCRs to encompass non-receptor proteins as well.
Assuntos
Motivos de Aminoácidos , Ligação Proteica , beta-Arrestinas , Fosforilação , Humanos , beta-Arrestinas/metabolismo , Células HEK293 , beta-Arrestina 2/metabolismo , Sequência de Aminoácidos , Estabilidade ProteicaRESUMO
Initiation and termination of signaling of the type I angiotensin receptor (AT(1)-R) can lead to dynamic changes in its localization in plasma membrane microdomains. Several markers were recently developed to investigate membrane microdomains. Here, we used several YFP-labeled fusion constructs (i.e. raft or non-raft plasma membrane markers) to analyze the agonist-induced changes in compartmentalization of AT(1)-R, including internalization or lateral movement between plasma membrane compartments in response to stimulation using bioluminescence resonance energy transfer measurements. Our data demonstrate that angiotensin II (AngII) stimulus changes the microdomain localization of wild type or mutated (DRY â AAY or TSTS â AAAA) AT(1)-Rs co-expressed with the fluorescent probes in HEK293 cells. The comparison of the trafficking of AT(1)-R upon AngII stimulus with those of [Sar(1),Ile(8)]AngII or [Sar(1),Ile(4),Ile(8)]AngII stimulus revealed different types of changes, depending on the nature of the ligand. The observed changes in receptor compartmentalization of the AT(1)-R are strikingly different from those of 5HT-2C and EGF receptors, which demonstrate the usefulness of the bioluminescence resonance energy transfer-based measurements in the investigation of receptor trafficking in the plasma membrane in living cell experiments.