scholarly journals Investigation of Receptor Heteromers Using NanoBRET Ligand Binding

2021 ◽  
Vol 22 (3) ◽  
pp. 1082
Author(s):  
Elizabeth K. M. Johnstone ◽  
Heng B. See ◽  
Rekhati S. Abhayawardana ◽  
Angela Song ◽  
K. Johan Rosengren ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Ligand Binding ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Receptor Heteromers ◽  
Intracellular Proteins ◽  
Β2 Adrenergic Receptor ◽  
First Time

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 β2 adrenergic receptor (AT1-β2AR heteromer), as well as between the AT1 and angiotensin II type 2 receptor (AT1-AT2 heteromer).

scholarly journals Minimal requirements for ubiquitination-mediated regulation of thyroid hormone activation

2014 ◽  
Vol 53 (2) ◽  
pp. 217-226 ◽  
Author(s):  
Péter Egri ◽  
Balázs Gereben
Keyword(s):  
Thyroid Hormone ◽  
Resonance Energy Transfer ◽  
E3 Ligase ◽  
Resonance Energy ◽  
Globular Domain ◽  
Native Form ◽  
Molecular Features ◽  
Molecular Machinery

Activation of thyroxine by outer ring deiodination is the crucial first step of thyroid hormone action. Substrate-induced ubiquitination of type 2 deiodinase (D2) is the most rapid and sensitive mechanism known to regulate thyroid hormone activation. While the molecular machinery responsible for D2 ubiquitination has been extensively studied, the combination of molecular features sufficient and required to allow D2 ubiquitination have not previously been determined. To address this question, we constructed chimeric deiodinases by introducing different combinations of D2-specific elements into type 1 deiodinase (D1), another member of the deiodinase enzyme family, which, however, does not undergo ubiquitination in its native form. Studies on the chimeric proteins expressed transiently in HEK-293T cells revealed that combined insertion of the D2-specific instability loop and the K237/K244 D2 ubiquitin carrier lysines into the corresponding positions of D1 could not ubiquitinate D1 unless the chimera was directed to the endoplasmic reticulum (ER). Fluorescence resonance energy transfer measurements demonstrated that the C-terminal globular domain of the ER-directed chimera was able to interact with the E3 ligase subunit WSB1. However, this interaction did not occur between the chimera and the TEB4 (MARCH6) E3 ligase, although a native D2 could readily interact with the N-terminus of TEB4. In conclusion, insertion of the instability loop and ubiquitin carrier lysines in combination with direction to the ER are sufficient and required to govern WSB1-mediated ubiquitination of an activating deiodinase enzyme.

scholarly journals Differential Contribution of Transmembrane Domains IV, V, VI, and VII to Human Angiotensin II Type 1 Receptor Homomer Formation

2017 ◽  
Vol 292 (8) ◽  
pp. 3341-3350 ◽  
Author(s):  
Brent M. Young ◽  
Elaine Nguyen ◽  
Matthew A. J. Chedrawe ◽  
Jan K. Rainey ◽  
Denis J. Dupré
Keyword(s):  
Crystal Structure ◽  
Angiotensin Ii ◽  
Drug Targets ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Structural Data ◽  
Transmembrane Domains ◽  
Site Directed Mutagenesis ◽  
Hydrophobic Residues

G protein-coupled receptors (GPCRs) play an important role in drug therapy and represent one of the largest families of drug targets. The angiotensin II type 1 receptor (AT1R) is notable as it has a central role in the treatment of cardiovascular disease. Blockade of AT1R signaling has been shown to alleviate hypertension and improve outcomes in patients with heart failure. Despite this, it has become apparent that our initial understanding of AT1R signaling is oversimplified. There is considerable evidence to suggest that AT1R signaling is highly modified in the presence of receptor-receptor interactions, but there is very little structural data available to explain this phenomenon even with the recent elucidation of the AT1R crystal structure. The current study investigates the involvement of transmembrane domains in AT1R homomer assembly with the goal of identifying hydrophobic interfaces that contribute to receptor-receptor affinity. A recently published crystal structure of the AT1R was used to guide site-directed mutagenesis of outward-facing hydrophobic residues within the transmembrane region of the AT1R. Bioluminescence resonance energy transfer was employed to analyze how receptor mutation affects the assembly of AT1R homomers with a specific focus on hydrophobic residues. Mutations within transmembrane domains IV, V, VI, and VII had no effect on angiotensin-mediated β-arrestin1 recruitment; however, they exhibited differential effects on the assembly of AT1R into oligomeric complexes. Our results demonstrate the importance of hydrophobic amino acids at the AT1R transmembrane interface and provide the first glimpse of the requirements for AT1R complex assembly.

scholarly journals Insights into the Interaction of LVV-Hemorphin-7 with Angiotensin II Type 1 Receptor

2020 ◽  
Vol 22 (1) ◽  
pp. 209
Author(s):  
Amanat Ali ◽  
Elizabeth K. M. Johnstone ◽  
Bincy Baby ◽  
Heng B. See ◽  
Angela Song ◽  
...  
Keyword(s):  
Molecular Docking ◽  
Angiotensin Ii ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Md Simulations ◽  
Hek293 Cells ◽  
Renal Physiology ◽  
Binding Behavior ◽  
Intracellular Loops

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.

Functional selectivity profiling of the angiotensin II type 1 receptor using pathway-wide BRET signaling sensors

2018 ◽  
Vol 11 (559) ◽  
pp. eaat1631 ◽  
Author(s):  
Yoon Namkung ◽  
Christian LeGouill ◽  
Sahil Kumar ◽  
Yubo Cao ◽  
Larissa B. Teixeira ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
G Protein ◽  
G Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Functional Selectivity ◽  
Gpcr Signaling ◽  
Downstream Signaling ◽  
Protein Levels

G protein–coupled receptors (GPCRs) are important therapeutic targets that exhibit functional selectivity (biased signaling), in which different ligands or receptor variants elicit distinct downstream signaling. Understanding all the signaling events and biases that contribute to both the beneficial and adverse effects of GPCR stimulation by given ligands is important for drug discovery. Here, we report the design, validation, and use of pathway-selective bioluminescence resonance energy transfer (BRET) biosensors that monitor the engagement and activation of signaling effectors downstream of G proteins, including protein kinase C (PKC), phospholipase C (PLC), p63RhoGEF, and Rho. Combined with G protein and β-arrestin BRET biosensors, our sensors enabled real-time monitoring of GPCR signaling at different levels in downstream pathways in both native and engineered cells. Profiling of the responses to 14 angiotensin II (AngII) type 1 receptor (AT1R) ligands enabled the clustering of compounds into different subfamilies of biased ligands and showed that, in addition to the previously reported functional selectivity between Gαq and β-arrestin, there are also biases among G protein subtypes. We also demonstrated that biases observed at the receptor and G protein levels propagated to downstream signaling pathways and that these biases could occur through the engagement of different G proteins to activate a common effector. We also used these tools to determine how naturally occurring AT1R variants affected signaling bias. This suite of BRET biosensors provides a useful resource for fingerprinting biased ligands and mutant receptors and for dissecting functional selectivity at various levels of GPCR signaling.

scholarly journals Angiotensin II type 1 receptor variants alter endosomal receptor–β-arrestin complex stability and MAPK activation

2020 ◽  
Vol 295 (38) ◽  
pp. 13169-13180
Author(s):  
Yubo Cao ◽  
Sahil Kumar ◽  
Yoon Namkung ◽  
Laurence Gagnon ◽  
Aaron Cho ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Angiotensin Ii ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Mapk Signaling ◽  
Mitogen Activated Protein Kinase ◽  
Endosomal Trafficking ◽  
Mapk Activation ◽  
Naturally Occurring

The angiotensin II (AngII) type 1 receptor (AT1R), a member of the G protein–coupled receptor (GPCR) family, signals through G proteins and β-arrestins, which act as adaptors to regulate AT1R internalization and mitogen-activated protein kinase (MAPK) ERK1/2 activation. β-arrestin–dependent ERK1/2 regulation is the subject of important studies because its spatiotemporal control remains poorly understood for many GPCRs, including AT1R. To study the link between β-arrestin–dependent trafficking and ERK1/2 signaling, we investigated three naturally occurring AT1R variants that show distinct receptor–β-arrestin interactions: A163T, T282M, and C289W. Using bioluminescence resonance energy transfer (BRET)–based and conformational fluorescein arsenical hairpin–BRET sensors coupled with high-resolution fluorescence microscopy, we show that all AT1R variants form complexes with β-arrestin2 at the plasma membrane and efficiently internalize into endosomes upon AngII stimulation. However, mutant receptors imposed distinct conformations in β-arrestin2 and differentially impacted endosomal trafficking and MAPK signaling. Notably, T282M accumulated in endosomes, but its ability to form stable complexes following internalization was reduced, markedly impairing its ability to co-traffic with β-arrestin2. We also found that despite β-arrestin2 overexpression, T282M's and C289W's residency with β-arrestin2 in endosomes was greatly reduced, leading to decreased β-arrestin–dependent ERK1/2 activation, faster recycling of receptors to the plasma membrane, and impaired AngII-mediated proliferation. Our findings reveal that naturally occurring AT1R variants alter the patterns of receptor/β-arrestin2 trafficking and suggest conformationally dependent β-arrestin–mediated MAPK activation as well as endosomal receptor–β-arrestin complex stabilization in the mitogenic response of AT1R.

scholarly journals EXPRESS: Pulmonary Hypertension Associated With Neurofibromatosis Type 2

2021 ◽  
pp. 204589402110295
Author(s):  
Hirohisa Taniguchi ◽  
Tomoya Takashima ◽  
Ly Tu ◽  
Raphaël Thuillet ◽  
Asuka Furukawa ◽  
...  
Keyword(s):  
Pulmonary Hypertension ◽  
Neurofibromatosis Type ◽  
Pulmonary Vascular Remodeling ◽  
Life Threatening ◽  
History Of ◽  
Pulmonary Arterial ◽  
First Time

Although precapillary pulmonary hypertension (PH) is a rare but severe complication of patients with neurofibromatosis type 1 (NF1), its association with NF2 remains unknown. Herein, we report a case of a 44-year-old woman who was initially diagnosed with idiopathic pulmonary arterial hypertension (IPAH) and treated with PAH-specific combination therapy. However, a careful assessment for a relevant family history of the disease and genetic testing reveal that this patient had a mutation in the NF2 gene. Using immunofluorescence and Western blotting, we demonstrated a decrease in endothelial NF2 protein in lungs from IPAH patients compared to control lungs, suggesting a potential role of NF2 in PAH development. To our knowledge, this is the first time that precapillary PH has been described in a patient with NF2. The altered endothelial NF2 expression pattern in PAH lungs should stimulate work to better understand how NF2 is contributing to the pulmonary vascular remodeling associated to these severe life-threatening conditions.

Blockade of central angiotensin II type 1 and type 2 receptors suppresses adrenalectomy-induced NaCl intake in rats

1996 ◽  
Vol 66 (1-2) ◽  
pp. 47-50 ◽  
Author(s):  
O. Galaverna ◽  
C. Polidori ◽  
R.R. Sakai ◽  
F. Liénard ◽  
S.Y. Chow ◽  
...  
Keyword(s):  
Angiotensin Ii

Angiotensin II-induced thirst, but not sodium appetite, via AT1 receptors in organum cavum prelamina terminalis

1995 ◽  
Vol 268 (6) ◽  
pp. R1401-R1405 ◽  
Author(s):  
M. el Ghissassi ◽  
S. N. Thornton ◽  
S. Nicolaidis
Keyword(s):  
Angiotensin Ii ◽  
Salt Intake ◽  
High Sensitivity ◽  
Ang Ii ◽  
Nacl Solution ◽  
At1 Receptors ◽  
Sodium Appetite ◽  
Specific Antagonist

The angiotensin receptor specificity, with respect to fluid intake, of the organum cavum prelamina terminalis (OCPLT), a recently discovered discrete forebrain structure with high sensitivity to angiotensin II (ANG II), was investigated. ANG II (10 ng) microinjected into the OCPLT significantly increased water consumption but did not induce intake of a hypertonic (3%) NaCl solution. Losartan, an ANG II type 1 (AT1) receptor-specific antagonist, produced dose-related (1-100 ng) inhibition of ANG II-induced drinking. The ANG II type 2 receptor-specific antagonist CGP-42112A was ineffective. Intake of the 3% NaCl solution in response to microinjection of either of the antagonists into the OCPLT was never observed. These findings suggest that water intake produced by microinjection of ANG II into the OCPLT is mediated by AT1 receptors uniquely and that, in contrast to other regions of the brain, these receptors do not induce salt intake when stimulated by ANG II.

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