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1.
Biochem Soc Trans ; 49(4): 1555-1565, 2021 08 27.
Artículo en Inglés | MEDLINE | ID: mdl-34436548

RESUMEN

Many receptors are able to undergo heteromerisation, leading to the formation of receptor complexes that may have pharmacological profiles distinct from those of the individual receptors. As a consequence of this, receptor heteromers can be classed as new drug targets, with the potential for achieving greater specificity and selectivity over targeting their constituent receptors. We have developed the Receptor-Heteromer Investigation Technology (Receptor-HIT), which enables the detection of receptor heteromers using a proximity-based reporter system such as bioluminescence resonance energy transfer (BRET). Receptor-HIT detects heteromers in live cells and in real time, by utilising ligand-induced signals that arise from altered interactions with specific biomolecules, such as ligands or proteins. Furthermore, monitoring the interaction between the receptors and the specific biomolecules generates functional information about the heteromer that can be pharmacologically quantified. This review will discuss various applications of Receptor-HIT, including its use with different classes of receptors (e.g. G protein-coupled receptors (GPCRs), receptor tyrosine kinases (RTKs) and others), its use to monitor receptor interactions both intracellularly and extracellularly, and also its use with genome-edited endogenous proteins.


Asunto(s)
Receptores Acoplados a Proteínas G/efectos de los fármacos , Transferencia de Energía , Humanos , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores Acoplados a Proteínas G/química
2.
Int J Mol Sci ; 22(3)2021 Jan 22.
Artículo en Inglés | MEDLINE | ID: mdl-33499147

RESUMEN

Receptor heteromerization is the formation of a complex involving at least two different receptors with pharmacology that is distinct from that exhibited by its constituent receptor units. Detection of these complexes and monitoring their pharmacology is crucial for understanding how receptors function. The Receptor-Heteromer Investigation Technology (Receptor-HIT) utilizes ligand-dependent modulation of interactions between receptors and specific biomolecules for the detection and profiling of heteromer complexes. Previously, the interacting biomolecules used in Receptor-HIT assays have been intracellular proteins, however in this study we have for the first time used bioluminescence resonance energy transfer (BRET) with fluorescently-labeled ligands to investigate heteromerization of receptors on the cell surface. Using the Receptor-HIT ligand binding assay with NanoBRET, we have successfully investigated heteromers between the angiotensin II type 1 (AT1) receptor and the ß2 adrenergic receptor (AT1-ß2AR heteromer), as well as between the AT1 and angiotensin II type 2 receptor (AT1-AT2 heteromer).


Asunto(s)
Transferencia de Energía por Resonancia de Bioluminiscencia/métodos , Ligandos , Nanotecnología/métodos , Receptores de Angiotensina/metabolismo , Unión Competitiva , Compuestos de Boro/química , Membrana Celular/metabolismo , AMP Cíclico/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Células HEK293 , Humanos , Cinética , Unión Proteica , Multimerización de Proteína , Receptor de Angiotensina Tipo 1/metabolismo , Receptor de Angiotensina Tipo 2/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Transducción de Señal
3.
J Am Soc Nephrol ; 30(11): 2191-2207, 2019 11.
Artículo en Inglés | MEDLINE | ID: mdl-31511361

RESUMEN

BACKGROUND: Recombinant human relaxin-2 (serelaxin), which has organ-protective actions mediated via its cognate G protein-coupled receptor relaxin family peptide receptor 1 (RXFP1), has emerged as a potential agent to treat fibrosis. Studies have shown that serelaxin requires the angiotensin II (AngII) type 2 receptor (AT2R) to ameliorate renal fibrogenesis in vitro and in vivo. Whether its antifibrotic actions are affected by modulation of the AngII type 1 receptor (AT1R), which is expressed on myofibroblasts along with RXFP1 and AT2R, is unknown. METHODS: We examined the signal transduction mechanisms of serelaxin when applied to primary rat renal and human cardiac myofibroblasts in vitro, and in three models of renal- or cardiomyopathy-induced fibrosis in vivo. RESULTS: The AT1R blockers irbesartan and candesartan abrogated antifibrotic signal transduction of serelaxin via RXFP1 in vitro and in vivo. Candesartan also ameliorated serelaxin's antifibrotic actions in the left ventricle of mice with cardiomyopathy, indicating that candesartan's inhibitory effects were not confined to the kidney. We also demonstrated in a transfected cell system that serelaxin did not directly bind to AT1Rs but that constitutive AT1R-RXFP1 interactions could form. To potentially explain these findings, we also demonstrated that renal and cardiac myofibroblasts expressed all three receptors and that antagonists acting at each receptor directly or allosterically blocked the antifibrotic effects of either serelaxin or an AT2R agonist (compound 21). CONCLUSIONS: These findings have significant implications for the concomitant use of RXFP1 or AT2R agonists with AT1R blockers, and suggest that functional interactions between the three receptors on myofibroblasts may represent new targets for controlling fibrosis progression.


Asunto(s)
Riñón/patología , Miocardio/patología , Miofibroblastos/fisiología , Receptor de Angiotensina Tipo 1/fisiología , Receptor de Angiotensina Tipo 2/fisiología , Receptores Acoplados a Proteínas G/fisiología , Receptores de Péptidos/fisiología , Bloqueadores del Receptor Tipo 1 de Angiotensina II/uso terapéutico , Animales , Bencimidazoles/uso terapéutico , Compuestos de Bifenilo/uso terapéutico , Células Cultivadas , Fibrosis , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratas , Ratas Sprague-Dawley , Receptor de Angiotensina Tipo 2/agonistas , Receptores Acoplados a Proteínas G/agonistas , Receptores de Péptidos/agonistas , Proteínas Recombinantes , Relaxina/fisiología , Tetrazoles/uso terapéutico
4.
Int J Mol Sci ; 22(1)2020 Dec 28.
Artículo en Inglés | MEDLINE | ID: mdl-33379211

RESUMEN

Hemorphins are known for their role in the control of blood pressure. Recently, we revealed the positive modulation of the angiotensin II (AngII) type 1 receptor (AT1R) by LVV-hemorphin-7 (LVV-H7) in human embryonic kidney (HEK293) cells. Here, we examined the molecular binding behavior of LVV-H7 on AT1R and its effect on AngII binding using a nanoluciferase-based bioluminescence resonance energy transfer (NanoBRET) assay in HEK293FT cells, as well as molecular docking and molecular dynamics (MD) studies. Saturation and real-time kinetics supported the positive effect of LVV-H7 on the binding of AngII. While the competitive antagonist olmesartan competed with AngII binding, LVV-H7 slightly, but significantly, decreased AngII's kD by 2.6 fold with no effect on its Bmax. Molecular docking and MD simulations indicated that the binding of LVV-H7 in the intracellular region of AT1R allosterically potentiates AngII binding. LVV-H7 targets residues on intracellular loops 2 and 3 of AT1R, which are known binding sites of allosteric modulators in other GPCRs. Our data demonstrate the allosteric effect of LVV-H7 on AngII binding, which is consistent with the positive modulation of AT1R activity and signaling previously reported. This further supports the pharmacological targeting of AT1R by hemorphins, with implications in vascular and renal physiology.


Asunto(s)
Angiotensina II/metabolismo , Hemoglobinas/metabolismo , Fragmentos de Péptidos/metabolismo , Receptor de Angiotensina Tipo 1/metabolismo , Células HEK293 , Humanos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular
5.
Nat Methods ; 12(7): 661-663, 2015 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-26030448

RESUMEN

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.


Asunto(s)
Transferencia de Energía por Resonancia de Bioluminiscencia/métodos , Receptores Acoplados a Proteínas G/metabolismo , Fluorescencia , Células HEK293 , Humanos , Ligandos , Receptores Adrenérgicos beta 2/metabolismo
6.
Front Endocrinol (Lausanne) ; 13: 931573, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36111299

RESUMEN

G protein-coupled receptors (GPCRs) are capable of interacting to form higher order structures such as homomers and heteromers. Heteromerisation in particular has implications for receptor function, with research showing receptors can attain unique expression, ligand binding, signalling and intracellular trafficking upon heteromerisation. As such, GPCR heteromers represent novel drug targets with extensive therapeutic potential. Changes to ligand affinity, efficacy and G protein coupling have all been described, with alterations to these pharmacological aspects now well accepted as common traits for heteromeric complexes. Changes in internalisation and trafficking kinetics, as well as ß-arrestin interactions are also becoming more apparent, however, few studies to date have explicitly looked at the implications these factors have upon the signalling profile of a heteromer. Development of ligands to target GPCR heteromers both experimentally and therapeutically has been mostly concentrated on bivalent ligands due to difficulties in identifying and developing heteromer-specific ligands. Improving our understanding of the pharmacology and physiology of GPCR heteromers will enable further development of heteromer-specific ligands with potential to provide therapeutics with increased efficacy and decreased side effects.


Asunto(s)
Receptores Acoplados a Proteínas G , Transducción de Señal , Ligandos , Multimerización de Proteína/fisiología , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/fisiología , beta-Arrestinas/metabolismo
7.
Front Cell Neurosci ; 16: 812359, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35496914

RESUMEN

The orexin system comprises two G protein-coupled receptors, OX1 and OX2 receptors (OX1R and OX2R, respectively), along with two endogenous agonists cleaved from a common precursor (prepro-orexin), orexin-A (OX-A) and orexin-B (OX-B). For the receptors, a complex array of signaling behaviors has been reported. In particular, it becomes obvious that orexin receptor coupling is very diverse and can be tissue-, cell- and context-dependent. Here, the early signal transduction interactions of the orexin receptors will be discussed in depth, with particular emphasis on the direct G protein interactions of each receptor. In doing so, it is evident that ligands, additional receptor-protein interactions and cellular environment all play important roles in the G protein coupling profiles of the orexin receptors. This has potential implications for our understanding of the orexin system's function in vivo in both central and peripheral environments, as well as the development of novel agonists, antagonists and possibly allosteric modulators targeting the orexin system.

8.
Front Endocrinol (Lausanne) ; 13: 848816, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35721749

RESUMEN

The angiotensin type 2 (AT2) receptor and the bradykinin type 2 (B2) receptor are G protein-coupled receptors (GPCRs) that have major roles in the cardiovascular system. The two receptors are known to functionally interact at various levels, and there is some evidence that the observed crosstalk may occur as a result of heteromerization. We investigated evidence for heteromerization of the AT2 receptor and the B2 receptor in HEK293FT cells using various bioluminescence resonance energy transfer (BRET)-proximity based assays, including the Receptor Heteromer Investigation Technology (Receptor-HIT) and the NanoBRET ligand-binding assay. The Receptor-HIT assay showed that Gαq, GRK2 and ß-arrestin2 recruitment proximal to AT2 receptors only occurred upon B2 receptor coexpression and activation, all of which is indicative of AT2-B2 receptor heteromerization. Additionally, we also observed specific coupling of the B2 receptor with the Gαz protein, and this was found only in cells coexpressing both receptors and stimulated with bradykinin. The recruitment of Gαz, Gαq, GRK2 and ß-arrestin2 was inhibited by B2 receptor but not AT2 receptor antagonism, indicating the importance of B2 receptor activation within AT2-B2 heteromers. The close proximity between the AT2 receptor and B2 receptor at the cell surface was also demonstrated with the NanoBRET ligand-binding assay. Together, our data demonstrate functional interaction between the AT2 receptor and B2 receptor in HEK293FT cells, resulting in novel pharmacology for both receptors with regard to Gαq/GRK2/ß-arrestin2 recruitment (AT2 receptor) and Gαz protein coupling (B2 receptor). Our study has revealed a new mechanism for the enigmatic and poorly characterized AT2 receptor to be functionally active within cells, further illustrating the role of heteromerization in the diversity of GPCR pharmacology and signaling.


Asunto(s)
Receptor de Angiotensina Tipo 2 , Receptor de Bradiquinina B2 , Bradiquinina/farmacología , Ligandos , Receptor de Angiotensina Tipo 2/fisiología , Receptor de Bradiquinina B2/fisiología , Receptores Acoplados a Proteínas G , Arrestina beta 2
9.
Biochem Pharmacol ; 188: 114521, 2021 06.
Artículo en Inglés | MEDLINE | ID: mdl-33741329

RESUMEN

Transactivation of the epidermal growth factor receptor (EGFR) by the angiotensin II (AngII) type 1 (AT1) receptor is involved in AT1 receptor-dependent growth effects and cardiovascular pathologies, however the mechanisms underpinning this transactivation are yet to be fully elucidated. Recently, a potential intermediate of this process was identified following the discovery that a kinase called TRIO was involved in AngII/AT1 receptor-mediated transactivation of EGFR. To investigate the mechanisms by which TRIO acts as an intermediate in AngII/AT1 receptor-mediated EGFR transactivation we used bioluminescence resonance energy transfer (BRET) assays to investigate proximity between the AT1 receptor, EGFR, TRIO and other proteins of interest. We found that AngII/AT1 receptor activation caused a Gαq-dependent increase in proximity of TRIO with Gγ2 and the AT1-EGFR heteromer, as well as trafficking of TRIO towards the Kras plasma membrane marker and into early, late and recycling endosomes. In contrast, we found that AngII/AT1 receptor activation caused a Gαq-independent increase in proximity of TRIO with Grb2, GRK2 and PKCζ, as well as trafficking of TRIO up to the plasma membrane from the Golgi. Furthermore, we confirmed the proximity between the AT1 receptor and the EGFR using the Receptor-Heteromer Investigation Technology, which showed AngII-induced recruitment of Grb2, GRK2, PKCζ, Gγ2 and TRIO to the EGFR upon AT1 coexpression. In summary, our results provide further evidence for the existence of the AT1-EGFR heteromer and reveal potential mechanisms by which TRIO contributes to the transactivation process.


Asunto(s)
Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Receptor de Angiotensina Tipo 2/metabolismo , Transducción de Señal/fisiología , Angiotensina II/farmacología , Relación Dosis-Respuesta a Droga , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/agonistas , Receptores ErbB/metabolismo , Células HEK293 , Humanos , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/fisiología , Receptor de Angiotensina Tipo 2/agonistas , Transducción de Señal/efectos de los fármacos , Activación Transcripcional/efectos de los fármacos , Activación Transcripcional/fisiología
10.
Nat Commun ; 12(1): 1920, 2021 03 26.
Artículo en Inglés | MEDLINE | ID: mdl-33772001

RESUMEN

Adipogenesis associated Mth938 domain containing (AAMDC) represents an uncharacterized oncogene amplified in aggressive estrogen receptor-positive breast cancers. We uncover that AAMDC regulates the expression of several metabolic enzymes involved in the one-carbon folate and methionine cycles, and lipid metabolism. We show that AAMDC controls PI3K-AKT-mTOR signaling, regulating the translation of ATF4 and MYC and modulating the transcriptional activity of AAMDC-dependent promoters. High AAMDC expression is associated with sensitization to dactolisib and everolimus, and these PI3K-mTOR inhibitors exhibit synergistic interactions with anti-estrogens in IntClust2 models. Ectopic AAMDC expression is sufficient to activate AKT signaling, resulting in estrogen-independent tumor growth. Thus, AAMDC-overexpressing tumors may be sensitive to PI3K-mTORC1 blockers in combination with anti-estrogens. Lastly, we provide evidence that AAMDC can interact with the RabGTPase-activating protein RabGAP1L, and that AAMDC, RabGAP1L, and Rab7a colocalize in endolysosomes. The discovery of the RabGAP1L-AAMDC assembly platform provides insights for the design of selective blockers to target malignancies having the AAMDC amplification.


Asunto(s)
Neoplasias de la Mama/metabolismo , Proteínas de Ciclo Celular/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Antineoplásicos/farmacología , Neoplasias de la Mama/genética , Proteínas de Ciclo Celular/genética , Everolimus/farmacología , Femenino , Proteínas Activadoras de GTPasa/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Imidazoles/farmacología , Proteínas del Tejido Nervioso/metabolismo , Oncogenes/genética , Unión Proteica , Quinolinas/farmacología , Receptores de Estrógenos/metabolismo , Transducción de Señal/efectos de los fármacos
11.
Artículo en Inglés | MEDLINE | ID: mdl-30972335

RESUMEN

Bioluminescence resonance energy transfer (BRET) is a biophysical technique used to monitor proximity within live cells. BRET exploits the naturally occurring phenomenon of dipole-dipole energy transfer from a donor enzyme (luciferase) to an acceptor fluorophore following enzyme-mediated oxidation of a substrate. This results in production of a quantifiable signal that denotes proximity between proteins and/or molecules tagged with complementary luciferase and fluorophore partners. BRET assays have been used to observe an array of biological functions including ligand binding, intracellular signaling, receptor-receptor proximity, and receptor trafficking, however, BRET assays can theoretically be used to monitor the proximity of any protein or molecule for which appropriate fusion constructs and/or fluorophore conjugates can be produced. Over the years, new luciferases and approaches have been developed that have increased the potential applications for BRET assays. In particular, the development of the small, bright and stable Nanoluciferase (NanoLuc; Nluc) and its use in NanoBRET has vastly broadened the potential applications of BRET assays. These advances have exciting potential to produce new experimental methods to monitor protein-protein interactions (PPIs), protein-ligand interactions, and/or molecular proximity. In addition to NanoBRET, Nluc has also been exploited to produce NanoBiT technology, which further broadens the scope of BRET to monitor biological function when NanoBiT is combined with an acceptor. BRET has proved to be a powerful tool for monitoring proximity and interaction, and these recent advances further strengthen its utility for a range of applications.

12.
Cell Signal ; 54: 27-34, 2019 02.
Artículo en Inglés | MEDLINE | ID: mdl-30471466

RESUMEN

Bioluminescence resonance energy transfer (BRET) is a versatile tool used to investigate membrane receptor signalling and function. We have recently developed a homogenous NanoBRET ligand binding assay to monitor interactions between G protein-coupled receptors and fluorescent ligands. However, this assay requires the exogenous expression of a receptor fused to the nanoluciferase (Nluc) and is thus not applicable to natively-expressed receptors. To overcome this limitation in HEK293 cells, we have utilised CRISPR/Cas9 genome engineering to insert Nluc in-frame with the endogenous ADORA2B locus this resulted in HEK293 cells expressing adenosine A2B receptors under endogenous promotion tagged on their N-terminus with Nluc. As expected, we found relatively low levels of endogenous (gene-edited) Nluc/A2B receptor expression compared to cells transiently transfected with expression vectors coding for Nluc/A2B. However, in cells expressing gene-edited Nluc/A2B receptors we observed clear saturable ligand binding of a non-specific fluorescent adenosine receptor antagonist XAC-X-BY630 (Kd = 21.4 nM). Additionally, at gene-edited Nluc/A2B receptors we derived pharmacological parameters of ligand binding; Kd as well as Kon and Koff for binding of XAC-X-BY630 by NanoBRET association kinetic binding assays. Lastly, cells expressing gene-edited Nluc/A2B were used to determine the pKi of unlabelled adenosine receptor ligands in competition ligand binding assays. Utilising CRISPR/Cas9 genome engineering here we show that NanoBRET ligand binding assays can be performed at gene-edited receptors under endogenous promotion in live cells, therefore overcoming a fundamental limitation of NanoBRET ligand assays.


Asunto(s)
Transferencia de Energía por Resonancia de Bioluminiscencia/métodos , Receptor de Adenosina A2B/análisis , Sistemas CRISPR-Cas , Células HEK293 , Humanos , Luciferasas/química
13.
Biochem Pharmacol ; 158: 232-242, 2018 12.
Artículo en Inglés | MEDLINE | ID: mdl-30347205

RESUMEN

The type 1 angiotensin II (AngII) receptor (AT1R) transactivates the epidermal growth factor receptor (EGFR), which leads to pathological remodeling of heart, blood vessels and kidney. End-point assays are used as surrogates of EGFR activation, however these downstream readouts are not applicable to live cells, in real-time. Herein, we report the use of a bioluminescence resonance energy transfer (BRET)-based assay to assess recruitment of the EGFR adaptor protein, growth factor receptor-bound protein 2 (Grb2), to the EGFR. In a variety of cell lines, both epidermal growth factor (EGF) and AngII stimulated Grb2 recruitment to EGFR. The BRET assay was used to screen a panel of 9 G protein-coupled receptors (GPCRs) and further developed for other EGFR family members (HER2 and HER3); the AT1R was able to transactivate HER2, but not HER3. Mechanistically, AT1R-mediated ERK1/2 activation was dependent on Gq/11 and EGFR tyrosine kinase activity, whereas the recruitment of Grb2 to the EGFR was independent of Gq/11 and only partially dependent on EGFR tyrosine kinase activity. This Gq/11 independence of EGFR transactivation was confirmed using AT1R mutants and in CRISPR cell lines lacking Gq/11. EGFR transactivation was also apparently independent of ß-arrestins. Finally, we used additional BRET-based assays and confocal microscopy to provide evidence that both AngII- and EGF-stimulation promoted AT1R-EGFR heteromerization. In summary, we report an alternative approach to monitoring AT1R-EGFR transactivation in live cells, which provides a more direct and proximal view of this process, including the potential for complexes between the AT1R and EGFR.


Asunto(s)
Transferencia de Energía por Resonancia de Bioluminiscencia/métodos , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Receptor de Angiotensina Tipo 1/metabolismo , Activación Transcripcional/fisiología , Animales , Células CHO , Cricetulus , Receptores ErbB/análisis , Receptores ErbB/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/análisis , Células HEK293 , Humanos , Masculino , Ratones , Células 3T3 NIH , Ratas , Ratas Sprague-Dawley , Receptor de Angiotensina Tipo 1/análisis
14.
Sci Rep ; 7(1): 3187, 2017 06 09.
Artículo en Inglés | MEDLINE | ID: mdl-28600500

RESUMEN

Bioluminescence resonance energy transfer (BRET) has been a vital tool for understanding G protein-coupled receptor (GPCR) function. It has been used to investigate GPCR-protein and/or -ligand interactions as well as GPCR oligomerisation. However the utility of BRET is limited by the requirement that the fusion proteins, and in particular the donor, need to be exogenously expressed. To address this, we have used CRISPR/Cas9-mediated homology-directed repair to generate protein-Nanoluciferase (Nluc) fusions under endogenous promotion, thus allowing investigation of proximity between the genome-edited protein and an exogenously expressed protein by BRET. Here we report BRET monitoring of GPCR-mediated ß-arrestin2 recruitment and internalisation where the donor luciferase was under endogenous promotion, in live cells and in real time. We have investigated the utility of CRISPR/Cas9 genome editing to create genome-edited fusion proteins that can be used as BRET donors and propose that this strategy can be used to overcome the need for exogenous donor expression.


Asunto(s)
Sistemas CRISPR-Cas/genética , Transferencia de Energía/genética , Luciferasas/química , beta-Arrestinas/genética , Edición Génica , Humanos , Ligandos , Luciferasas/genética , Nanopartículas/química , Unión Proteica , Proteínas/química , Proteínas/genética , Transducción de Señal/genética , beta-Arrestinas/química
16.
Methods Mol Biol ; 1335: 191-204, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26260602

RESUMEN

Bioluminescence resonance energy transfer (BRET) is a well-established technique for investigating G protein-coupled receptor (GPCR) pharmacology. BRET enables the monitoring of molecular proximity through the use of heterologously expressed proteins of interest and/or fluorophore-labeled ligands. Fusion to a donor luciferase enzyme or an acceptor fluorophore and subsequent detection of resonance energy transfer indicate the close proximity of the molecules of interest. As BRET is readily applied to the study of numerous GPCR signaling and regulatory paths, it is an ideal technique for investigating the pharmacology of biased ligands and receptors.


Asunto(s)
Transferencia de Energía por Resonancia de Bioluminiscencia/métodos , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Animales , Células COS , Chlorocebus aethiops , Células HEK293 , Humanos , Proteínas Recombinantes de Fusión/metabolismo
17.
PLoS One ; 10(3): e0119803, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25807547

RESUMEN

Understanding functional interactions between G protein-coupled receptors is of great physiological and pathophysiological importance. Heteromerization provides one important potential mechanism for such interaction between different signalling pathways via macromolecular complex formation. Previous studies suggested a functional interplay between angiotensin II receptor type 1 (AT1) and Chemokine (C-C motif) Receptor 2 (CCR2). However the molecular mechanisms are not understood. We investigated AT1-CCR2 functional interaction in vitro using bioluminescence resonance energy transfer in HEK293 cells and in vivo using subtotal-nephrectomized rats as a well-established model for chronic kidney disease. Our data revealed functional heteromers of these receptors resulting in CCR2-Gαi1 coupling being sensitive to AT1 activation, as well as apparent enhanced ß-arrestin2 recruitment with agonist co-stimulation that is synergistically reversed by combined antagonist treatment. Moreover, we present in vivo findings where combined treatment with AT1- and CCR2-selective inhibitors was synergistically beneficial in terms of decreasing proteinuria, reducing podocyte loss and preventing renal injury independent of blood pressure in the subtotal-nephrectomized rat model. Our findings further support a role for G protein-coupled receptor functional heteromerization in pathophysiology and provide insights into previous observations indicating the importance of AT1-CCR2 functional interaction in inflammation, renal and hypertensive disorders.


Asunto(s)
Receptores CCR2/metabolismo , Insuficiencia Renal Crónica/metabolismo , Animales , Modelos Animales de Enfermedad , Células HEK293 , Humanos , Fosfatos de Inositol/metabolismo , Riñón/metabolismo , Nefrectomía , Ratas , Receptor de Angiotensina Tipo 1/metabolismo
18.
Artículo en Inglés | MEDLINE | ID: mdl-22936924

RESUMEN

Receptor heteromerization has the potential to alter every facet of receptor functioning, leading to new pharmacological profiles with increased signaling diversity and regulation from that of the monomeric receptor, or indeed receptor homomer. An understanding of the molecular consequences of receptor heteromerization will provide new insights into the physiology and pathology mediated by receptors, expanding the possibilities for pharmacological discovery. Particularly advantageous approaches to investigate novel heteromer pharmacology utilize cell-based assay technologies that assess ligand-dependent functional responses specific to the receptor heteromer. Importantly, this allows for differentiation of heteromer-specific pharmacology from pharmacology associated with the co-expressed receptor monomers and homomers. The Receptor-Heteromer Investigation Technology (Receptor-HIT) successfully employs a proximity-based reporter system, such as bioluminescence resonance energy transfer (BRET), in a configuration that enables determination of such heteromer-specific pharmacology. Therefore, Receptor-HIT provides a simple, robust and versatile approach for investigating the elusive "biochemical fingerprint" of receptor heteromers.

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