RESUMO
ß-arrestin plays a key role in G protein-coupled receptor (GPCR) signaling and desensitization. Despite recent structural advances, the mechanisms that govern receptor-ß-arrestin interactions at the plasma membrane of living cells remain elusive. Here, we combine single-molecule microscopy with molecular dynamics simulations to dissect the complex sequence of events involved in ß-arrestin interactions with both receptors and the lipid bilayer. Unexpectedly, our results reveal that ß-arrestin spontaneously inserts into the lipid bilayer and transiently interacts with receptors via lateral diffusion on the plasma membrane. Moreover, they indicate that, following receptor interaction, the plasma membrane stabilizes ß-arrestin in a longer-lived, membrane-bound state, allowing it to diffuse to clathrin-coated pits separately from the activating receptor. These results expand our current understanding of ß-arrestin function at the plasma membrane, revealing a critical role for ß-arrestin preassociation with the lipid bilayer in facilitating its interactions with receptors and subsequent activation.
Assuntos
Receptores Acoplados a Proteínas G , Transdução de Sinais , beta-Arrestinas , beta-Arrestinas/metabolismo , Membrana Celular/metabolismo , Clatrina/metabolismo , Endocitose , Bicamadas Lipídicas , Receptores Acoplados a Proteínas G/metabolismo , Simulação de Dinâmica MolecularRESUMO
The importance of cell phenotype in determining the molecular mechanisms underlying ß2 -adrenoceptor (ß2AR) function has been noted previously when comparing responses in primary cells and recombinant model cell lines. Here, we have generated haplotype-specific SNAP-tagged ß2AR human embryonic stem (ES) cell lines and applied fluorescence correlation spectroscopy (FCS) to study cell surface receptors in progenitor cells and in differentiated fibroblasts and cardiomyocytes. FCS was able to quantify SNAP-tagged ß2AR number and diffusion in both ES-derived cardiomyocytes and CRISPR/Cas9 genome-edited HEK293T cells, where the expression level was too low to detect using standard confocal microscopy. These studies demonstrate the power of FCS in investigating cell surface ß2ARs at the very low expression levels often seen in endogenously expressing cells. Furthermore, the use of ES cell technology in combination with FCS allowed us to demonstrate that cell surface ß2ARs internalize in response to formoterol-stimulation in ES progenitor cells but not following their differentiation into ES-derived fibroblasts. This indicates that the process of agonist-induced receptor internalization is strongly influenced by cell phenotype and this may have important implications for drug treatment with long-acting ß2AR agonists.
Assuntos
Células-Tronco Embrionárias/fisiologia , Fibroblastos/fisiologia , Miócitos Cardíacos/fisiologia , Receptores Adrenérgicos beta 2/metabolismo , Espectrometria de Fluorescência/métodos , Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Diferenciação Celular , Corantes Fluorescentes/química , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Células HEK293 , Humanos , Proteínas de Membrana , Propranolol/farmacologia , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/genéticaRESUMO
The glucagon-like peptide-1 receptor (GLP-1R) is a class B G protein-coupled receptor and mainstay therapeutic target for the treatment of type 2 diabetes and obesity. Recent reports have highlighted how biased agonism at the GLP-1R affects sustained glucose-stimulated insulin secretion through avoidance of desensitization and downregulation. A number of GLP-1R agonists (GLP-1RAs) feature a fatty acid moiety to prolong their pharmacokinetics via increased albumin binding, but the potential for these chemical changes to influence GLP-1R function has rarely been investigated beyond potency assessments for cAMP. Here, we directly compare the prototypical GLP-1RA exendin-4 with its C-terminally acylated analog, exendin-4-C16. We examine relative propensities of each ligand to recruit and activate G proteins and ß-arrestins, endocytic and postendocytic trafficking profiles, and interactions with model and cellular membranes in HEK293 and HEK293T cells. Both ligands had similar cAMP potency, but exendin-4-C16 showed â¼2.5-fold bias toward G protein recruitment and a â¼60% reduction in ß-arrestin-2 recruitment efficacy compared with exendin-4, as well as reduced GLP-1R endocytosis and preferential targeting toward recycling pathways. These effects were associated with reduced movement of the GLP-1R extracellular domain measured using a conformational biosensor approach and a â¼70% increase in insulin secretion in INS-1 832/3 cells. Interactions with plasma membrane lipids were enhanced by the acyl chain. Exendin-4-C16 showed extensive albumin binding and was highly effective for lowering of blood glucose in mice over at least 72 hours. Our study highlights the importance of a broad approach to the evaluation of GLP-1RA pharmacology. SIGNIFICANCE STATEMENT: Acylation is a common strategy to enhance the pharmacokinetics of peptide-based drugs. This work shows how acylation can also affect various other pharmacological parameters, including biased agonism, receptor trafficking, and interactions with the plasma membrane, which may be therapeutically important.
Assuntos
Exenatida/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Incretinas/metabolismo , Transdução de Sinais/fisiologia , Acilação/efeitos dos fármacos , Acilação/fisiologia , Animais , Exenatida/farmacologia , Células HEK293 , Humanos , Incretinas/farmacologia , Secreção de Insulina/efeitos dos fármacos , Secreção de Insulina/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Transdução de Sinais/efeitos dos fármacosRESUMO
A disintegrin and metalloprotease 10 (ADAM10) is a transmembrane protein essential for embryonic development, and its dysregulation underlies disorders such as cancer, Alzheimer's disease, and inflammation. ADAM10 is a "molecular scissor" that proteolytically cleaves the extracellular region from >100 substrates, including Notch, amyloid precursor protein, cadherins, growth factors, and chemokines. ADAM10 has been recently proposed to function as six distinct scissors with different substrates, depending on its association with one of six regulatory tetraspanins, termed TspanC8s. However, it remains unclear to what degree ADAM10 function critically depends on a TspanC8 partner, and a lack of monoclonal antibodies specific for most TspanC8s has hindered investigation of this question. To address this knowledge gap, here we designed an immunogen to generate the first monoclonal antibodies targeting Tspan15, a model TspanC8. The immunogen was created in an ADAM10-knockout mouse cell line stably overexpressing human Tspan15, because we hypothesized that expression in this cell line would expose epitopes that are normally blocked by ADAM10. Following immunization of mice, this immunogen strategy generated four Tspan15 antibodies. Using these antibodies, we show that endogenous Tspan15 and ADAM10 co-localize on the cell surface, that ADAM10 is the principal Tspan15-interacting protein, that endogenous Tspan15 expression requires ADAM10 in cell lines and primary cells, and that a synthetic ADAM10/Tspan15 fusion protein is a functional scissor. Furthermore, two of the four antibodies impaired ADAM10/Tspan15 activity. These findings suggest that Tspan15 directly interacts with ADAM10 in a functional scissor complex.
Assuntos
Proteína ADAM10/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Proteínas de Membrana/metabolismo , Complexos Multiproteicos/metabolismo , Tetraspaninas/metabolismo , Células A549 , Proteína ADAM10/genética , Secretases da Proteína Precursora do Amiloide/genética , Animais , Células HEK293 , Humanos , Células Jurkat , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Complexos Multiproteicos/genética , Tetraspaninas/genéticaRESUMO
Bioluminescence resonance energy transfer (BRET) is a well-established method for investigating protein-protein interactions. Here we present a BRET approach to monitor ligand binding to G protein-coupled receptors (GPCRs) on the surface of living cells made possible by the use of fluorescent ligands in combination with a bioluminescent protein (NanoLuc) that can be readily expressed on the N terminus of GPCRs.
Assuntos
Técnicas de Transferência de Energia por Ressonância de Bioluminescência/métodos , Receptores Acoplados a Proteínas G/metabolismo , Fluorescência , Células HEK293 , Humanos , Ligantes , Receptores Adrenérgicos beta 2/metabolismoRESUMO
Fluorescent ligands have proved to be powerful tools in the study of G protein-coupled receptors in living cells. Here we have characterized a new fluorescent ligand PSB603-BY630 that has high selectivity for the human adenosine A2B receptor (A2BR). The A2BR appears to play an important role in regulating immune responses in the tumor microenvironment. Here we have used PSB603-BY630 to monitor specific binding to A2BRs in M1- and M2-like macrophages derived from CD14+ human monocytes. PSB603-BY630 bound with high affinity (18.3 nM) to nanoluciferase-tagged A2BRs stably expressed in HEK293G cells. The ligand exhibited very high selectivity for the A2BR with negligible specific-binding detected at NLuc-A2AR, NLuc-A1R, or NLuc-A3R receptors at concentrations up to 500 nM. Competition binding studies showed the expected pharmacology at A2BR with the A2BR-selective ligands PSB603 and MRS-1706 demonstrating potent inhibition of the specific binding of 50 nM PSB603-BY630 to A2BR. Functional studies in HEK293G cells using Glosensor to monitor Gs-coupled cyclic AMP responses indicated that PSB603-BY630 acted as a negative allosteric regular of the agonist responses to BAY 60-6583. Furthermore, flow cytometry analysis confirmed that PSB603-BY630 could be used to selectively label endogenous A2BRs expressed on human macrophages. This ligand should be an important addition to the library of fluorescent ligands which are selective for the different adenosine receptor subtypes, and will enable study of the role of A2BRs on immune cells in the tumor microenvironment.
Assuntos
Corantes Fluorescentes , Macrófagos , Receptor A2B de Adenosina , Humanos , Células HEK293 , Receptor A2B de Adenosina/metabolismo , Ligantes , Corantes Fluorescentes/química , Macrófagos/metabolismo , Macrófagos/imunologia , Ligação Competitiva , Antagonistas do Receptor A2 de Adenosina/farmacologia , Agonistas do Receptor A2 de Adenosina/farmacologiaRESUMO
The study of protein function and dynamics in their native cellular environment is essential for progressing fundamental science. To overcome the requirement of genetic modification of the protein or the limitations of dissociable fluorescent ligands, ligand-directed (LD) chemistry has most recently emerged as a complementary, bioorthogonal approach for labeling native proteins. Here, we describe the rational design, development, and application of the first ligand-directed chemistry approach for labeling the A1AR in living cells. We pharmacologically demonstrate covalent labeling of A1AR expressed in living cells while the orthosteric binding site remains available. The probes were imaged using confocal microscopy and fluorescence correlation spectroscopy to study A1AR localization and dynamics in living cells. Additionally, the probes allowed visualization of the specific localization of A1ARs endogenously expressed in dorsal root ganglion (DRG) neurons. LD probes developed here hold promise for illuminating ligand-binding, receptor signaling, and trafficking of the A1AR in more physiologically relevant environments.
Assuntos
Corantes Fluorescentes , Receptor A1 de Adenosina , Ligantes , Receptor A1 de Adenosina/metabolismo , Receptor A1 de Adenosina/química , Humanos , Corantes Fluorescentes/química , Animais , Gânglios Espinais/metabolismo , Gânglios Espinais/citologia , Células HEK293 , Neurônios/metabolismoRESUMO
This study introduces (S)-Opto-prop-2, a second-generation photoswitchable ligand designed for precise modulation of ß2-adrenoceptor (ß2AR). Synthesised by incorporating an azobenzene moiety with propranolol, (S)-Opto-prop-2 exhibited a high PSScis (photostationary state for cis isomer) percentage (â¼90 %) and a favourable half-life (>10 days), facilitating diverse bioassay measurements. In vitro, the cis-isomer displayed substantially higher ß2AR binding affinity than the trans-isomer (1000-fold), making (S)-Opto-prop-2 one of the best photoswitchable GPCR (G protein-coupled receptor) ligands reported so far. Molecular docking of (S)-Opto-prop-2 in the X-ray structure of propranolol-bound ß2AR followed by site-directed mutagenesis studies, identified D1133.32, N3127.39 and F2896.51 as crucial residues that contribute to ligand-receptor interactions at the molecular level. In vivo efficacy was assessed using a rabbit ocular hypertension model, revealing that the cis isomer mimicked propranolol's effects in reducing intraocular pressure, while the trans isomer was inactive. Dynamic optical modulation of ß2AR by (S)-Opto-prop-2 was demonstrated in two different cAMP bioassays and using live-cell confocal imaging, indicating reversible and dynamic control of ß2AR activity using the new photopharmacology tool. In conclusion, (S)-Opto-prop-2 emerges as a promising photoswitchable ligand for precise and reversible ß2AR modulation with light. The new tool shows superior cis-on binding affinity, one of the largest reported differences in affinity (1000-fold) between its two configurations, in vivo efficacy, and dynamic modulation. This study contributes valuable insights into the evolving field of photopharmacology, offering a potential avenue for targeted therapy in ß2AR-associated pathologies.
Assuntos
Antagonistas de Receptores Adrenérgicos beta 2 , Receptores Adrenérgicos beta 2 , Animais , Humanos , Masculino , Coelhos , Antagonistas de Receptores Adrenérgicos beta 2/farmacologia , Antagonistas de Receptores Adrenérgicos beta 2/química , Antagonistas Adrenérgicos beta/farmacologia , Antagonistas Adrenérgicos beta/química , Compostos Azo/química , Compostos Azo/farmacologia , Células CHO , Cricetulus , Células HEK293 , Simulação de Acoplamento Molecular/métodos , Processos Fotoquímicos , Propranolol/farmacologia , Propranolol/química , Receptores Adrenérgicos beta 2/metabolismo , Receptores Adrenérgicos beta 2/químicaRESUMO
E-selectin is expressed on endothelial cells in response to inflammatory cytokines and mediates leukocyte rolling and extravasation. However, studies have been hampered by lack of experimental approaches to monitor expression in real time in living cells. Here, NanoLuc Binary Technology (NanoBiT) in conjunction with CRISPR-Cas9 genome editing was used to tag endogenous E-selectin in human umbilical vein endothelial cells (HUVECs) with the 11 amino acid nanoluciferase fragment HiBiT. Addition of the membrane-impermeable complementary fragment LgBiT allowed detection of cell surface expression. This allowed the effect of inflammatory mediators on E-selectin expression to be monitored in real time in living endothelial cells. NanoBiT combined with CRISPR-Cas9 gene editing allows sensitive monitoring of real-time changes in cell surface expression of E-selectin and offers a powerful tool for future drug discovery efforts aimed at this important inflammatory protein.
RESUMO
The C-X-C chemokine receptor type 4, or CXCR4, is a chemokine receptor found to promote cancer progression and metastasis of various cancer cell types. To investigate the pharmacology of this receptor, and to further elucidate its role in cancer, novel chemical tools are a necessity. In the present study, using classic medicinal chemistry approaches, small-molecule-based fluorescent probes were designed and synthesized based on previously reported small-molecule antagonists. Here, we report the development of three distinct chemical classes of fluorescent probes that show specific binding to the CXCR4 receptor in a novel fluorescence-based NanoBRET binding assay (pKD ranging 6.6-7.1). Due to their retained affinity at CXCR4, we furthermore report their use in competition binding experiments and confocal microscopy to investigate the pharmacology and cellular distribution of this receptor.
Assuntos
Corantes Fluorescentes , Receptores CXCR4 , Receptores CXCR4/metabolismo , Ligantes , Corantes Fluorescentes/química , Ligação Proteica , Quimiocinas/metabolismo , Quimiocina CXCL12/metabolismoRESUMO
Fluorescence correlation spectroscopy (FCS) is a quantitative spectroscopy technique which could potentially increase throughput and sensitivity of screening for ligand, substrate and inhibitor binding to membrane proteins in solution. However, the purification of membrane proteins in their active forms is complex, as the lipid bilayer provides stability and its removal often causes the protein to become conformationally unstable. This has limited the application of biophysical techniques such as FCS to study the function of membrane proteins. The recent application of native extraction techniques such as styrene maleic acid lipid particles (SMALPs) has resolved this issue and FCS has emerged as a powerful option for studying proteins extracted in this way. This review will discuss the application of FCS to study purified membrane proteins in detergent micelles, nanodiscs and SMALPs and its potential to be used routinely in membrane protein drug discovery.
Assuntos
Detergentes , Proteínas de Membrana , Fluorescência , Bicamadas Lipídicas/química , Proteínas de Membrana/química , Poliestirenos/químicaRESUMO
Interleukin-23 (IL-23) is a pro-inflammatory cytokine involved in the host defense against pathogens but is also implicated in the development of several autoimmune disorders. The IL-23 receptor has become a key target for drug discovery, but the exact mechanism of the receptor ligand interaction remains poorly understood. In this study the affinities of IL-23 for its individual receptor components (IL23R and IL12Rß1) and the heteromeric complex formed between them have been measured in living cells using NanoLuciferase-tagged full-length proteins. Here, we demonstrate that TAMRA-tagged IL-23 has a greater than 7-fold higher affinity for IL12Rß1 than IL23R. However, in the presence of both receptor subunits, IL-23 affinity is increased more than three orders of magnitude to 27 pM. Furthermore, we show that IL-23 induces a potent change in the position of the N-terminal domains of the two receptor subunits, consistent with a conformational change in the heteromeric receptor structure.
Assuntos
Técnicas de Transferência de Energia por Ressonância de Bioluminescência , Interleucina-23/imunologia , Luciferases/imunologia , Receptores de Interleucina/imunologia , Células Cultivadas , Feminino , Células HEK293 , Humanos , Interleucina-23/química , Luciferases/metabolismo , Ligação Proteica , Receptores de Interleucina/químicaRESUMO
Fluorescent ligand technologies have proved to be powerful tools to improve our understanding of ligand-receptor interactions. Here we have characterized a small focused library of nine fluorescent ligands based on the highly selective ß2 -adrenoceptor (ß2 AR) antagonist ICI 118,551. The majority of fluorescent ICI 118,551 analogs had good affinity for the ß2 AR (pKD >7.0) with good selectivity over the ß1 AR (pKD <6.0). The most potent and selective ligands being 8c (ICI 118,551-Gly-Ala-BODIPY-FL-X; ß2 AR pKD 7.48), 9c (ICI 118,551-ßAla-ßAla-BODIPY-FL-X; ß2 AR pKD 7.48), 12a (ICI 118,551-PEG-BODIPY-X-630/650; ß2 AR pKD 7.56), and 12b (ICI 118,551-PEG-BODIPY-FL; ß2 AR pKD 7.42). 9a (ICI 118,551-ßAla-ßAla-BODIPY-X-630/650) had the highest affinity at recombinant ß2 ARs (pKD 7.57), but also exhibited significant binding affinity to the ß1 AR (pKD 6.69). Nevertheless, among the red fluorescent ligands, 9a had the best imaging characteristics in recombinant HEK293 T cells and labeling was mostly confined to the cell surface. In contrast, 12a showed the highest propensity to label intracellular ß2 ARs in HEK293 T cell expressing exogenous ß2 ARs. This suggests that a combination of the polyethylene glycol (PEG) linker and the BODIPY-X-630/650 makes this ICI 118,551 derivative particularly susceptible to crossing the cell membrane to access the intracellular ß2 ARs. We have also used these ligands in combination with CRISPR/Cas9 genome-edited HEK293 T cells to undertake for the first time real-time ligand binding to native HEK293 T ß2 ARs at low native receptor expression levels. These studies provided quantitative data on ligand-binding characteristics but also allowed real-time visualization of the ligand-binding interactions in genome-edited cells using NanoBRET luminescence imaging.
Assuntos
Antagonistas de Receptores Adrenérgicos beta 2/farmacologia , Propanolaminas/farmacologia , Receptores Adrenérgicos beta 2 , Sistemas CRISPR-Cas , Fluorescência , Edição de Genes , Células HEK293 , Humanos , Ligantes , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/metabolismoRESUMO
G protein-coupled receptors (GPCRs) are the largest family of membrane receptors and major targets for FDA-approved drugs. The ability to quantify GPCR expression and ligand binding characteristics in different cell types and tissues is therefore important for drug discovery. The advent of genome editing along with developments in fluorescent ligand design offers exciting new possibilities to probe GPCRs in their native environment. This review provides an overview of the recent technical advances employed to study the localisation and ligand binding characteristics of genome-edited and endogenously expressed GPCRs.
Assuntos
Descoberta de Drogas , Edição de Genes , Receptores Acoplados a Proteínas G/genética , Regulação da Expressão Gênica/genética , Humanos , Ligantes , Transdução de Sinais/genéticaRESUMO
During normal- and patho-physiological situations, the behavior of the beta2-adrenoreceptor (ß2AR) is influenced by polymorphic variants. The functional impact of such polymorphisms has been suggested from data derived from genetic association studies, in vitro experiments with primary cells, and transgenic overexpression models. However, heterogeneous genetic background and non-physiological transgene expression levels confound interpretation, leading to conflicting mechanistic conclusions. To overcome these limitations, we used CRISPR/Cas9 gene editing technology in human pluripotent stem cells (hPSCs) to create a unique suite of four isogenic homozygous variants at amino acid positions 16(G/R) and 27(G/Q), which reside in the N terminus of the ß2AR. By producing cardiomyocytes from these hPSC lines, we determined that at a functional level ß2AR signaling dominated over ß1AR . Examining changes in beat rates and responses to isoprenaline, Gi coupling, cyclic AMP (cAMP) production, downregulation, and desensitization indicated that responses were often heightened for the GE variant, implying differential dominance of both polymorphic location and amino acid substitution. This finding was corroborated, since GE showed hypersensitivity to doxorubicin-induced cardiotoxicity relative to GQ and RQ variants. Thus, understanding the effect of ß2AR polymorphisms on cardiac response to anticancer therapy may provide a route for personalized medicine and facilitate immediate clinical impact.
RESUMO
Fluorescent antagonists offer the ability to interrogate G protein-coupled receptor pharmacology. With resonance energy transfer techniques, fluorescent antagonists can be implemented to monitor receptor-ligand interactions using assays originally designed for radiolabeled probes. The fluorescent nature of these antagonists also enables the localization and distribution of the receptors to be visualized in living cells. Here, we describe the generation of modified purinergic receptors with the NanoLuc luciferase or SNAP-tag, using the P1 adenosine A3 receptor as an example. We also describe the procedure of characterizing a novel fluorescent purinergic antagonist using ligand-mediated bioluminescence resonance energy transfer assays and confocal microscopy.
Assuntos
Técnicas de Transferência de Energia por Ressonância de Bioluminescência/métodos , Microscopia de Fluorescência/métodos , Agonistas do Receptor Purinérgico P1/metabolismo , Receptor A3 de Adenosina/metabolismo , Receptores Purinérgicos P1/metabolismo , Fluorescência , Células HEK293 , Humanos , Luciferases/metabolismo , Ligação Proteica , Multimerização Proteica , Agonistas do Receptor Purinérgico P1/química , Receptor A3 de Adenosina/química , Receptores Purinérgicos P1/química , Transdução de SinaisRESUMO
The fundamental importance of membrane proteins in cellular processes has driven a marked increase in the use of membrane mimetic approaches for studying and exploiting these proteins. Nano-encapsulation strategies which preserve the native lipid bilayer environment are particularly attractive. Consequently, the use of poly(styrene co-maleic acid) (SMA) has been widely adopted to solubilise proteins directly from cell membranes by spontaneously forming "SMA Lipid Particles" (SMALPs). G-protein-coupled receptors (GPCRs) are ubiquitous "chemical switches", are central to cell signalling throughout the evolutionary tree, form the largest family of membrane proteins in humans and are a major drug discovery target. GPCR-SMALPs that retain binding capability would be a versatile platform for a wide range of down-stream applications. Here, using the adenosine A2A receptor (A2AR) as an archetypical GPCR, we show for the first time the utility of fluorescence correlation spectroscopy (FCS) to characterise the binding capability of GPCRs following nano-encapsulation. Unbound fluorescent ligand CA200645 exhibited a monophasic autocorrelation curve (dwell time, τD = 68 ± 2 µs; diffusion coefficient, D = 287 ± 15 µm2 s-1). In the presence of A2AR-SMALP, bound ligand was also evident (τD = 625 ± 23 µs; D = 30 ± 4 µm2 s-1). Using a non-receptor control (ZipA-SMALP) plus competition binding confirmed that this slower component represented binding to the encapsulated A2AR. Consequently, the combination of GPCR-SMALP and FCS is an effective platform for the quantitative real-time characterisation of nano-encapsulated receptors, with single molecule sensitivity, that will have widespread utility for future exploitation of GPCR-SMALPs in general.
Assuntos
Ligantes , Maleatos/química , Receptores Acoplados a Proteínas G/metabolismo , Estireno/química , Materiais Biomiméticos , Fluorescência , Humanos , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Ligação Proteica , Receptor A2A de Adenosina/química , Receptor A2A de Adenosina/metabolismo , Receptores Acoplados a Proteínas G/química , Imagem Individual de Molécula , Espectrometria de FluorescênciaRESUMO
Endogenous adenosine A2B receptors (A2BAR) mediate cAMP accumulation in HEK 293 cells. Here we have used a biosensor to investigate the mechanism of action of the A2BAR antagonist PSB 603 in HEK 293 cells. The A2A agonist CGS 21680 elicited a small response in these cells (circa 20% of that obtained with NECA), suggesting that they also contain a small population of A2A receptors. The responses to NECA and adenosine were antagonised by PSB 603, but not by the selective A2AAR antagonist SCH 58261. In contrast, CGS 21680 responses were not antagonised by high concentrations of PSB 603, but were sensitive to inhibition by SCH 58261. Analysis of the effect of increasing concentrations of PSB 603 on the response to NECA indicated a non-competitive mode of action yielding a marked reduction in the NECA EMAX with no significant effect on EC50 values. Kinetics analysis of the effect of PSB 603 on the A2BAR-mediated NECA responses confirmed a saturable effect that was consistent with an allosteric mode of antagonism. The possibility that PSB 603 acts as a negative allosteric modulator of A2BAR suggests new approaches to the development of therapeutic agents to treat conditions where adenosine levels are high.