Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 192
Filtrar
1.
Methods Mol Biol ; 2041: 155-162, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31646487

RESUMO

Assays based on bioluminescence resonance energy transfer (BRET) provide a sensitive and simple method to study protein-protein interactions in live cells. Here we describe a protocol using BRET technique to investigate potential interactions between P2X subunits. This approach combined with bimolecular fluorescence complementation (BiFC) can also be employed to determine the stoichiometry of heteromeric P2X receptors.


Assuntos
Técnicas de Transferência de Energia por Ressonância de Bioluminescência/métodos , Mapeamento de Interação de Proteínas/métodos , Multimerização Proteica , Receptores Purinérgicos P2X/química , Receptores Purinérgicos P2X/metabolismo , Humanos , Medições Luminescentes , Ligação Proteica , Transdução de Sinais
2.
Methods Mol Biol ; 2041: 163-181, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31646488

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 Sinais
3.
Mol Pharmacol ; 97(1): 23-34, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31707356

RESUMO

Smoothened (SMO) is a GPCR that mediates hedgehog signaling. Hedgehog binds the transmembrane protein Patched, which in turn regulates SMO activation. Overactive SMO signaling is oncogenic and is therefore a clinically established drug target. Here we establish a nanoluciferase bioluminescence resonance energy transfer (NanoBRET)-based ligand binding assay for SMO providing a sensitive and high throughput-compatible addition to the toolbox of GPCR pharmacologists. In the NanoBRET-based binding assay, SMO is N terminally tagged with nanoluciferase (Nluc) and binding of BODIPY-cyclopamine is assessed by quantifying resonance energy transfer between receptor and ligand. The assay allowed kinetic analysis of ligand-receptor binding in living HEK293 cells, competition binding experiments using commercially available SMO ligands (SANT-1, cyclopamine-KAAD, SAG1.3 and purmorphamine), and pharmacological dissection of two BODIPY-cyclopamine binding sites. This high throughput-compatible assay is superior to commonly used SMO ligand binding assays in the separation of specific from non-specific ligand binding and, provides a suitable complement to chemical biology strategies for the discovery of novel SMO-targeting drugs. SIGNIFICANCE STATEMENT: We established a NanoBRET-based binding assay for SMO with superior sensitivity compared to fluorescence-based assays. This assay allows distinction of two separate binding sites for BODIPY-cyclopamine on the SMO transmembrane core in live cells in real time. The assay is a valuable complement for drug discovery efforts and will support a better understanding of Class F GPCR pharmacology.


Assuntos
Sítios de Ligação/genética , Bioensaio/métodos , Transdução de Sinais/efeitos dos fármacos , Receptor Smoothened/antagonistas & inibidores , Alcaloides de Veratrum/farmacologia , Técnicas de Transferência de Energia por Ressonância de Bioluminescência/métodos , Compostos de Boro/química , Cinamatos/farmacologia , Descoberta de Drogas/métodos , Técnicas de Inativação de Genes , Células HEK293 , Proteínas Hedgehog/metabolismo , Humanos , Ligantes , Luciferases/química , Morfolinas/farmacologia , Nanoestruturas/química , Purinas/farmacologia , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Alcaloides de Veratrum/química
4.
Chem Commun (Camb) ; 56(2): 281-284, 2019 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-31807738

RESUMO

Bioluminescence resonance energy transfer (BRET) is a commonly used assay system for studying protein-protein interactions and protein folding in vivo. Conventional BRET systems have solely depended on an overlap of the energy donor and acceptor spectra. In this study, we engineered a conceptually unique ligand-activatable BRET system (termed BRET9), where a full-length Artificial Luciferase variant 23 (ALuc23), acting as the energy donor, is sandwiched between a protein pair of interest, FRB and FKBP12, and linked to a fluorescent protein as the energy acceptor. A specific ligand, rapamycin, then activates inter- and intramolecular interactions of FRB and FKBP12, which develop molecular strain in the sandwiched ALuc23 to accelerate further folding. We found that this system greatly enhanced both the total bioluminescence spectrum and the BRET signal in the far-red (FR) region. We characterized the molecular construct by studying 18 different designs categorized into four groups. The best BRET system design allowed an approximately 5-fold enhancement of the bioluminescence intensities in the FR region. This new BRET system provides a robust ligand-activatable platform that efficiently reports FR bioluminescence signals in cells and living animal models.


Assuntos
Luciferases/química , Serina-Treonina Quinases TOR/metabolismo , Proteína 1A de Ligação a Tacrolimo/metabolismo , Animais , Técnicas de Transferência de Energia por Ressonância de Bioluminescência/métodos , Linhagem Celular Tumoral , Humanos , Ligantes , Limite de Detecção , Luciferases/genética , Proteínas Luminescentes/química , Camundongos Endogâmicos BALB C , Camundongos Nus , Mutação , Ligação Proteica , Sirolimo/química , Sirolimo/metabolismo
5.
Proc Natl Acad Sci U S A ; 116(48): 24093-24099, 2019 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-31712427

RESUMO

Receptor-activity-modifying proteins (RAMPs) are single transmembrane-spanning proteins which serve as molecular chaperones and allosteric modulators of G-protein-coupled receptors (GPCRs) and their signaling pathways. Although RAMPs have been previously studied in the context of their effects on Family B GPCRs, the coevolution of RAMPs with many GPCR families suggests an expanded repertoire of potential interactions. Using bioluminescence resonance energy transfer-based and cell-surface expression approaches, we comprehensively screen for RAMP interactions within the chemokine receptor family and identify robust interactions between RAMPs and nearly all chemokine receptors. Most notably, we identify robust RAMP interaction with atypical chemokine receptors (ACKRs), which function to establish chemotactic gradients for directed cell migration. Specifically, RAMP3 association with atypical chemokine receptor 3 (ACKR3) diminishes adrenomedullin (AM) ligand availability without changing G-protein coupling. Instead, RAMP3 is required for the rapid recycling of ACKR3 to the plasma membrane through Rab4-positive vesicles following either AM or SDF-1/CXCL12 binding, thereby enabling formation of dynamic spatiotemporal chemotactic gradients. Consequently, genetic deletion of either ACKR3 or RAMP3 in mice abolishes directed cell migration of retinal angiogenesis. Thus, RAMP association with chemokine receptor family members represents a molecular interaction to control receptor signaling and trafficking properties.


Assuntos
Proteína 3 Modificadora da Atividade de Receptores/fisiologia , Receptores CCR3/metabolismo , Técnicas de Transferência de Energia por Ressonância de Bioluminescência , Movimento Celular , Células HEK293 , Humanos , Lisossomos/metabolismo , Neovascularização Fisiológica , Proteína 3 Modificadora da Atividade de Receptores/genética , Proteína 3 Modificadora da Atividade de Receptores/metabolismo , Receptores CXCR/metabolismo , Transdução de Sinais
6.
Int J Mol Sci ; 20(15)2019 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-31366084

RESUMO

Duration of receptor antagonism, measured as the recovery of agonist responsiveness, is gaining attention as a method to evaluate the 'effective' target-residence for antagonists. These functional assays might be a good alternative for kinetic binding assays in competition with radiolabeled or fluorescent ligands, as they are performed on intact cells and better reflect consequences of dynamic cellular processes on duration of receptor antagonism. Here, we used a bioluminescence resonance energy transfer (BRET)-based assay that monitors heterotrimeric G protein activation via scavenging of released Venus-Gß1γ2 by NanoLuc (Nluc)-tagged membrane-associated-C-terminal fragment of G protein-coupled receptor kinase 3 (masGRK3ct-Nluc) as a tool to probe duration of G protein-coupled receptor (GPCR) antagonism. The Gαi-coupled histamine H3 receptor (H3R) was used in this study as prolonged antagonism is associated with adverse events (e.g., insomnia) and consequently, short-residence time ligands might be preferred. Due to its fast and prolonged response, this assay can be used to determine the duration of functional antagonism by measuring the recovery of agonist responsiveness upon washout of pre-bound antagonist, and to assess antagonist re-equilibration time via Schild-plot analysis. Re-equilibration of pre-incubated antagonist with agonist and receptor could be followed in time to monitor the transition from insurmountable to surmountable antagonism. The BRET-based G protein activation assay can detect differences in the recovery of H3R responsiveness and re-equilibration of pre-bound antagonists between the tested H3R antagonists. Fast dissociation kinetics were observed for marketed drug pitolisant (Wakix®) in this assay, which suggests that short residence time might be beneficial for therapeutic targeting of the H3R.


Assuntos
Técnicas de Transferência de Energia por Ressonância de Bioluminescência/métodos , Antagonistas dos Receptores Histamínicos H3/farmacologia , Receptores Histamínicos H3/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Quinase 3 de Receptor Acoplado a Proteína G/genética , Quinase 3 de Receptor Acoplado a Proteína G/metabolismo , Células HEK293 , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Ligação Proteica
7.
Pharmacol Res Perspect ; 7(4): e00513, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31384473

RESUMO

Relaxin family peptide 1 (RXFP1) is the receptor for relaxin a peptide hormone with important therapeutic potential. Like many G protein-coupled receptors (GPCRs), RXFP1 has been reported to form homodimers. Given the complex activation mechanism of RXFP1 by relaxin, we wondered whether homodimerization may be explicitly required for receptor activation, and therefore sought to determine if there is any relaxin-dependent change in RXFP1 proximity at the cell surface. Bioluminescence resonance energy transfer (BRET) between recombinantly tagged receptors is often used in GPCR proximity studies. RXFP1 targets poorly to the cell surface when overexpressed in cell lines, with the majority of the receptor proteins sequestered within the cell. Thus, any relaxin-induced changes in RXFP1 proximity at the cell surface may be obscured by BRET signal originating from intracellular compartments. We therefore, utilized the newly developed split luciferase system called HiBiT to specifically label the extracellular terminus of cell surface RXFP1 receptors in combination with mCitrine-tagged receptors, using the GABAB heterodimer as a positive control. This demonstrated that the BRET signal detected from RXFP1-RXFP1 proximity at the cell surface does not appear to be due to stable physical interactions. The fact that there is also no relaxin-mediated change in RXFP1-RXFP1 proximity at the cell surface further supports these conclusions. This work provides a basis by which cell surface GPCR proximity and expression levels can be specifically studied using a facile and homogeneous labeling technique such as HiBiT.


Assuntos
Luciferases/metabolismo , Receptores Acoplados a Proteínas-G/química , Receptores Acoplados a Proteínas-G/metabolismo , Receptores de Peptídeos/química , Receptores de Peptídeos/metabolismo , Técnicas de Transferência de Energia por Ressonância de Bioluminescência , Células HEK293 , Humanos , Multimerização Proteica , Relaxina/metabolismo , Coloração e Rotulagem
8.
Mol Pharmacol ; 96(3): 308-319, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31266815

RESUMO

The dopamine D2 receptor (D2R) mediates ligand-biased signaling with potential therapeutic implications. However, internalization, choice of endocytic routes, and degradation of the D2R in lysosomes may also participate in agonist-directed trafficking. We developed bioluminescence resonance energy transfer (BRET) assays that measure relative distances between Renilla luciferase8-tagged D2R and green fluorescent protein 2 (GFP2)-tagged K-Ras (plasma membrane marker), and between luciferase8-tagged D2R and GFP2-Rab5 (early), GFP2-Rab4 (recycling), or GFP2-Rab7 (late) endosomal markers. The BRET signal between D2R-Luc and GFP2-K-Ras was robustly diminished after receptor internalization induced by dopamine, with subsequent BRET signals increasing when luciferase8-tagged D2R approached GFP2-Rab proteins in endosomal compartments. All BRET signals were blocked by the selective D2R antagonist haloperidol and were decreased by low temperature and high sucrose blocks, two parameters interfering with internalization. Some antipsychotic drugs, such as aripiprazole, are less efficacious in internalizing D2R than most of the antiparkinsonian agents. However, antipsychotics were nearly as efficacious as antiparkinsonians in directing the D2R toward early and recycling endosomes. The Rab7 marker for the late endosome/lysosome route was also capable of discriminating between D2R compounds. We could show that some drugs engaged the D2R either to interact preferentially with arrestin-3 or to internalize. Our study revealed that D2R trafficking in cells was differentially regulated by antipsychotic and antiparkinsonian drugs. Taken together, the BRET assays reported here could further help decipher D2R ligand-induced arrestin-3 recruitment and trafficking, with potentially more selective therapeutic profiles and fewer undesired side effects.


Assuntos
Arrestinas/metabolismo , Técnicas de Transferência de Energia por Ressonância de Bioluminescência , Proteínas de Fluorescência Verde/metabolismo , Receptores de Dopamina D2/agonistas , Animais , Células CHO , Cricetulus , Antagonistas de Dopamina/farmacologia , Haloperidol/farmacologia , Lisossomos/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de Sinais , Proteínas rab de Ligação ao GTP/metabolismo
9.
Int J Mol Sci ; 20(14)2019 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-31330984

RESUMO

Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are important regulators of metabolism, making their receptors (GLP-1R and GIPR) attractive targets in the treatment of type 2 diabetes mellitus (T2DM). GLP-1R agonists are used clinically to treat T2DM but the use of GIPR agonists remains controversial. Recent studies suggest that simultaneous activation of GLP-1R and GIPR with a single peptide provides superior glycemic control with fewer adverse effects than activation of GLP-1R alone. We investigated the signaling properties of a recently reported dual-incretin receptor agonist (P18). GLP-1R, GIPR, and the closely related glucagon receptor (GCGR) were expressed in HEK-293 cells. Activation of adenylate cyclase via Gαs was monitored using a luciferase-linked reporter gene (CRE-Luc) assay. Arrestin recruitment was monitored using a bioluminescence resonance energy transfer (BRET) assay. GLP-1, GIP, and glucagon displayed exquisite selectivity for their receptors in the CRE-Luc assay. P18 activated GLP-1R with similar potency to GLP-1 and GIPR with higher potency than GIP. Interestingly, P18 was less effective than GLP-1 at recruiting arrestin to GLP-1R and was inactive at GCGR. These data suggest that P18 can act as both a dual-incretin receptor agonist, and as a G protein-biased agonist at GLP-1R.


Assuntos
Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Glucagon/metabolismo , Receptores dos Hormônios Gastrointestinais/agonistas , Receptores dos Hormônios Gastrointestinais/metabolismo , Receptores de Glucagon/metabolismo , Sequência de Aminoácidos , Arrestina/metabolismo , Arrestina/farmacologia , Técnicas de Transferência de Energia por Ressonância de Bioluminescência , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Ligantes , Peptídeos/química , Peptídeos/farmacologia , Receptores de Glucagon/antagonistas & inibidores
10.
Mol Pharmacol ; 96(2): 233-246, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31189666

RESUMO

The orphan G-protein-coupled receptor (GPCR) GPR158 is expressed in the brain, where it is involved in the osteocalcin effect on cognitive processes, and at the periphery, where it may contribute to glaucoma and cancers. GPR158 forms a complex with RGS7-ß5, leading to the regulation of neighboring GPCR-induced Go protein activity. GPR158 also interacts with αo, although no canonical Go coupling has been reported. GPR158 displays three VCPWE motifs in its C-terminal domain that are putatively involved in G-protein regulation. Here, we addressed the scaffolding function of GPR158 and its VCPWE motifs on Go. We observed that GPR158 interacted with and stabilized the amount of RGS7-ß5 through a 50-residue region downstream of its transmembrane domain and upstream of the VCPWE motifs. We show that two VCPWE motifs are involved in αo binding. Using a Gαo-ßγ bioluminescence resonance energy transfer (BRET) sensor, we found that GPR158 decreases the BRET signal as observed upon G-protein activation; however, no constitutive activity of GPR158 could be detected through the measurement of various G-protein-mediated downstream responses. We propose that the effect of GPR158 on Go is unlikely due to a canonical activation of Go, but rather to the trapping of Gαo by the VCPWE motifs, possibly leading to its dissociation from ßγ Such action of GPR158 is expected to prolong the ßγ activity, as also observed with some activators of G-protein signaling. Taken together, our data revealed a complex functional scaffolding or signaling role for GPR158 controlling Go through an original mechanism.


Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Proteínas RGS/metabolismo , Receptores Acoplados a Proteínas-G/química , Receptores Acoplados a Proteínas-G/metabolismo , Motivos de Aminoácidos , Sítios de Ligação , Técnicas de Transferência de Energia por Ressonância de Bioluminescência , Regulação da Expressão Gênica , Células HEK293 , Humanos , Mutagênese Sítio-Dirigida , Ligação Proteica , Receptores Acoplados a Proteínas-G/genética
11.
Analyst ; 144(12): 3765-3772, 2019 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-31089611

RESUMO

Investigation of the functions of insulin-secreting cells in response to glucose in single-living cells is essential for improving our knowledge on the pathogenesis of diabetes. Therefore, it is desired to develop a new convenient method that enables the direct detection of insulin secreted from single-living cells. Here, insulin-sensor-cells expressing a protein-based insulin-detecting probe immobilized on the extracellular membrane were developed to evaluate the insulin-secretion response in single-living pancreatic ß cells. The protein-based insulin-detecting probe (NαLY) was composed of a bioluminescent protein (nano-luc), the αCT segment of the insulin receptor, L1 and CR domains of the insulin receptor, and a fluorescent protein (YPet). NαLY exhibited a bioluminescence resonance energy transfer (BRET) signal in response to insulin; thus, cells of Hepa1-6 line were genetically engineered to express NαLY on the extracellular membrane. The cells were found to act as insulin-sensor-cells, exhibiting a BRET signal in response to insulin. When the insulin-sensor-cells and pancreatic ß cells (MIN6 cell line) were cocultured and stimulated with glucose, insulin-sensor-cells nearby pancreatic ß cells showed the spike-shaped BRET signal response, whereas the insulin-sensor-cells close to one pancreatic ß cell did not exhibit such signal response. However, all the insulin-sensor-cells showed a gradual increase in BRET signals, which were presumably attributed to the increase in insulin concentrations in the culture dish, confirming the function of these insulin-sensor-cells. Therefore, we demonstrated that heterogenetic insulin secretion in single-living pancreatic ß cells could be measured directly using the insulin sensor cells.


Assuntos
Técnicas de Transferência de Energia por Ressonância de Bioluminescência/métodos , Técnicas Biossensoriais/métodos , Células Secretoras de Insulina/metabolismo , Insulina/análise , Análise de Célula Única/métodos , Animais , Linhagem Celular Tumoral , Técnicas de Cocultura/métodos , Fluorescência , Glucose/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Engenharia de Proteínas/métodos , Receptor de Insulina/genética , Receptor de Insulina/metabolismo
12.
Nat Commun ; 10(1): 2008, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-31043612

RESUMO

G protein-gated inwardly rectifying potassium channel (GIRK) plays a key role in regulating neurotransmission. GIRK is opened by the direct binding of the G protein ßγ subunit (Gßγ), which is released from the heterotrimeric G protein (Gαßγ) upon the activation of G protein-coupled receptors (GPCRs). GIRK contributes to precise cellular responses by specifically and efficiently responding to the Gi/o-coupled GPCRs. However, the detailed mechanisms underlying this family-specific and efficient activation are largely unknown. Here, we investigate the structural mechanism underlying the Gi/o family-specific activation of GIRK, by combining cell-based BRET experiments and NMR analyses in a reconstituted membrane environment. We show that the interaction formed by the αA helix of Gαi/o mediates the formation of the Gαi/oßγ-GIRK complex, which is responsible for the family-specific activation of GIRK. We also present a model structure of the Gαi/oßγ-GIRK complex, which provides the molecular basis underlying the specific and efficient regulation of GIRK.


Assuntos
Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/ultraestrutura , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/ultraestrutura , Subunidades beta da Proteína de Ligação ao GTP/ultraestrutura , Subunidades gama da Proteína de Ligação ao GTP/ultraestrutura , Ativação do Canal Iônico/fisiologia , Técnicas de Transferência de Energia por Ressonância de Bioluminescência , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/isolamento & purificação , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/isolamento & purificação , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/isolamento & purificação , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura
13.
Cell Chem Biol ; 26(6): 830-841.e9, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-30956148

RESUMO

Vascular endothelial growth factor (VEGF) is an important mediator of endothelial cell proliferation and angiogenesis via its receptor VEGFR2. A common tumor associated with elevated VEGFR2 signaling is infantile hemangioma that is caused by a rapid proliferation of vascular endothelial cells. The current first-line treatment for infantile hemangioma is the ß-adrenoceptor antagonist, propranolol, although its mechanism of action is not understood. Here we have used bioluminescence resonance energy transfer and VEGFR2 genetically tagged with NanoLuc luciferase to demonstrate that oligomeric complexes involving VEGFR2 and the ß2-adrenoceptor can be generated in both cell membranes and intracellular endosomes. These complexes are induced by agonist treatment and retain their ability to couple to intracellular signaling proteins. Furthermore, coupling of ß2-adrenoceptor to ß-arrestin2 is prolonged by VEGFR2 activation. These data suggest that protein-protein interactions between VEGFR2, the ß2-adrenoceptor, and ß-arrestin2 may provide insight into their roles in health and disease.


Assuntos
Receptores Adrenérgicos beta 2/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Técnicas de Transferência de Energia por Ressonância de Bioluminescência , Células Cultivadas , Corantes Fluorescentes/química , Células HEK293 , Humanos , Ligantes , Luciferases/química , Luciferases/metabolismo , Ligação Proteica , Receptores Adrenérgicos beta 2/química , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/química , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética
14.
Methods Mol Biol ; 1947: 169-182, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30969416

RESUMO

Ligand-biased signaling could have a significant impact on drug discovery programs. As such, many approaches to screening now target a larger section of the signaling responses downstream of an individual G protein-coupled receptor (GPCR). Biosensor-based platforms have been developed to capture signaling signatures. Despite the ability to use such signaling signatures, they may still be particular to an individual cell type and thus such platforms may not be portable from cell to cell, necessitating further cell-specific biosensor development. We have developed a complementary strategy based on capturing receptor-proximal conformational profiles using intra-molecular BRET-based sensors composed of a Renilla luciferase donor engineered into the carboxy-terminus and CCPGCC motifs which bind fluorescent hairpin biarsenical dyes engineered into different positions into the receptor primary structure. Here, we discuss how these experiments can be conducted and combined with CRISPR/Cas9 genome editing to assess specific G protein-dependent and -independent events.


Assuntos
Técnicas de Transferência de Energia por Ressonância de Bioluminescência/métodos , Técnicas Biossensoriais/métodos , Sistemas CRISPR-Cas , Edição de Genes/métodos , Luciferases de Renilla/metabolismo , Receptores Acoplados a Proteínas-G/química , Receptores Acoplados a Proteínas-G/metabolismo , Fluorescência , Humanos , Ligantes , Conformação Proteica , Receptores Acoplados a Proteínas-G/genética , Transdução de Sinais
15.
Methods Mol Biol ; 1947: 183-197, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30969417

RESUMO

How G protein-coupled receptors are assembled is a matter of considerable interest owing in large part to their remarkable pharmacological importance. For determining receptor stoichiometry, resonance energy transfer-based methods offer considerable advantages insofar as they provide the necessary spatial resolution, and because measurements can be made in situ, relatively easily. This chapter describes three complementary stoichiometric assays that rely on measurements of bioluminescence resonance energy transfer. These quantitative approaches make it possible to identify true protein-protein interactions from non-specific associations that inevitably result from constraining proteins in cellular membranes. In our experience, concordant data obtained in two or more of these assays, benchmarked with suitable controls, strongly predict receptor stoichiometry.


Assuntos
Técnicas de Transferência de Energia por Ressonância de Bioluminescência/métodos , Membrana Celular/metabolismo , Luciferases de Renilla/metabolismo , Receptores Acoplados a Proteínas-G/química , Receptores Acoplados a Proteínas-G/metabolismo , Fluorescência , Células HEK293 , Humanos , Ligantes , Ligação Proteica , Conformação Proteica
16.
Methods Mol Biol ; 1947: 199-215, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30969418

RESUMO

G protein-coupled receptors (GPCRs) are the target for many drugs. Evidence continues to accumulate demonstrating that multiple receptors form homo- and heteromeric complexes, which in turn dynamically couple with G proteins, and other interacting proteins. Here, we describe a method to simultaneously determine the identity of up to four distinct constituents of GPCR complexes using a combination of sequential bioluminescence resonance energy transfer 2-fluorescence resonance energy transfer (SRET2) with bimolecular fluorescence complementation (BiFC). The method is amenable to moderate throughput screening of changes in response to ligands and time-course analysis of protein-protein oligomerization.


Assuntos
Técnicas de Transferência de Energia por Ressonância de Bioluminescência/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Imunofluorescência/métodos , Luciferases de Renilla/metabolismo , Multimerização Proteica , Receptores Acoplados a Proteínas-G/química , Receptores Acoplados a Proteínas-G/metabolismo , Fluorescência , Humanos , Microscopia de Fluorescência
17.
Methods Mol Biol ; 1957: 59-68, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30919346

RESUMO

ß-Arrestins are adaptors that regulate the signaling and trafficking of G protein-coupled receptors (GPCRs). Bioluminescence resonance energy transfer (BRET) is a sensitive and versatile method for real-time monitoring of protein-protein interactions and protein kinesis within live cells, such as the recruitment of ß-arrestins to activated receptors at the plasma membrane (PM) and the trafficking of GPCR/ß-arrestin complexes to endosomes. Trafficking of receptor/ß-arrestin complexes can be assessed by BRET through tagging ß-arrestins with the donor luciferase from Renilla reniformis (Rluc) and anchoring the acceptor green fluorescent protein from the same species (rGFP) in distinct cell compartments (e.g., PM or endosomes) to generate highly efficient bystander BRET (referred to as enhanced bystander BRET (EbBRET)) upon re-localization of ß-arrestins to these compartments following receptor activation. Here, we outline the protocol for quantitatively monitoring ß-arrestin recruitment to agonist-activated Angiotensin II type 1 receptor (AT1R) and ß2-adrenergic receptor (ß2AR) at the PM and the trafficking of receptor/ß-arrestin complexes into endosomes using EbBRET-based biosensors.


Assuntos
Técnicas de Transferência de Energia por Ressonância de Bioluminescência/métodos , Receptores Acoplados a Proteínas-G/metabolismo , beta-Arrestinas/metabolismo , Membrana Celular/metabolismo , Células HEK293 , Humanos , Cinética , Ligantes , Transporte Proteico , Fatores de Tempo , beta-Arrestinas/agonistas
18.
Methods Mol Biol ; 1957: 83-91, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30919348

RESUMO

Initially identified as monomers, G protein-coupled receptors (GPCRs) can also form functional dimers that act as distinct signalling hubs for the integration of cellular signalling. We previously found that the angiotensin II (Ang II) type 1 receptor (AT1R) and the prostaglandin F2α (PGF2α) receptor (FP), both important in the control of smooth muscle contractility, form such a functional heterodimeric complex in HEK 293 and vascular smooth muscle cells (Goupil et al., J Biol Chem 290:3137-3148, 2015; Sleno et al., J Biol Chem 292:12139-12152, 2017). In addition to canonical G protein coupling, GPCRs recruit and engage ß-arrestin-dependent pathways. Using BRET-based biosensors, we demonstrate how to assess recruitment of ß-arrestin-1 and -2 to AT1R and the AT1R/FP dimer in response to Ang II. Surprisingly, ß-arrestin-1 and -2 were recruited to the dimer, in response to PGF2α as well, even though FP alone cannot recruit either ß-arrestin-1 and -2.


Assuntos
Técnicas de Transferência de Energia por Ressonância de Bioluminescência/métodos , Multimerização Proteica , Receptores Acoplados a Proteínas-G/metabolismo , beta-Arrestinas/metabolismo , Angiotensina II/farmacologia , Dinoprosta/farmacologia , Células HEK293 , Humanos , Multimerização Proteica/efeitos dos fármacos , Receptor Tipo 1 de Angiotensina/metabolismo
19.
Methods Mol Biol ; 1957: 93-104, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30919349

RESUMO

Ubiquitination of G protein-coupled receptors (GPCRs) is an important dynamic posttranslational modification that has been linked to the intracellular trafficking of internalized GPCRs to lysosomes. Ubiquitination of GPCRs is mediated by specific E3 ubiquitin ligases that are scaffolded by the adaptor proteins called ß-arrestins. Traditionally, detection of GPCR ubiquitination is achieved by using ubiquitin antibodies to Western blot immunoprecipitates of detergent-solubilized GPCRs expressed in heterologous cells. However, studies have also shown that bioluminescence resonance energy transfer (BRET)-based techniques can reveal ubiquitination of GPCRs in intact cells and in real time. This chapter describes a step-by-step protocol to evaluate ubiquitination of GPCRs using the BRET methodology.


Assuntos
Técnicas de Transferência de Energia por Ressonância de Bioluminescência/métodos , Receptores Acoplados a Proteínas-G/metabolismo , Ubiquitinação , beta-Arrestinas/metabolismo , Análise de Dados , Células HEK293 , Humanos , Ligação Proteica , Ubiquitina/metabolismo
20.
Methods Mol Biol ; 1957: 139-158, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30919352

RESUMO

ß-Arrestins 1 and 2 (ß-arr1 and ß-arr2) are ubiquitous proteins with common and distinct functions. They were initially identified as proteins recruited to stimulated G protein-coupled receptors (GPCRs), regulating their desensitization and internalization. The discovery that ß-arrs could also interact with more than 400 non-GPCR protein partners brought to light their central roles as multifunctional scaffold proteins regulating multiple signalling pathways from the plasma membrane to the nucleus, downstream of GPCRs or independently from these receptors. Through the regulation of the activities and subcellular localization of their binding partners, ß-arrs control various cell processes such as proliferation, cytoskeletal rearrangement, cell motility, and apoptosis. Thus, the identification of ß-arrs binding partners and the characterization of their mode of interaction in cells are central to the understanding of their function. Here we provide methods to explore the molecular interaction of ß-arrs with other proteins in cellulo.


Assuntos
Mapeamento de Interação de Proteínas/métodos , beta-Arrestinas/metabolismo , Técnicas de Transferência de Energia por Ressonância de Bioluminescência , Células HEK293 , Humanos , Imunoprecipitação , Ligação Proteica , Saccharomyces cerevisiae/metabolismo , Técnicas do Sistema de Duplo-Híbrido
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA