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 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 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.

scholarly journals A Model of N-Terminal Cyclin T1 Based on FRET Experiments

2005 ◽  
Vol 6 (2) ◽  
pp. 73-79
Author(s):  
Sergio Pantano ◽  
Alessandro Marcello ◽  
Arianna Sabò ◽  
Aldo Ferrari ◽  
Vittorio Pellegrini ◽  
...  
Keyword(s):  
Structural Model ◽  
Resonance Energy Transfer ◽  
Elongation Factor ◽  
Resonance Energy ◽  
Structural Data ◽  
Site Directed Mutagenesis ◽  
Cyclin T1 ◽  
Promising Target ◽  
Immunodeficiency Virus ◽  
Hiv 1

Human Cyclin T1 is the cyclin partner of kinase CDK9 in the positive transcription elongation factor b (P-TEFb). P-TEFb is recruited by Tat, the transactivator of the human immunodeficiency virus type 1 (HIV-1), to the viral promoter by direct interactions between Tat, Cyclin T1 and thecis-acting transactivation-responsive region (TAR) present at the 5′-end of each viral mRNA. At present, no structural data for Cyclin T1 are available. Here, we build a structural model of an N-terminus portion of Cyclin T1 (aa 27–263) based on the X-ray structure of Cyclin H. The model is compared with site directed mutagenesis data from the literature and validated by fluorescence resonance energy transfer (FRET) using Tat as a probe in living cells. This model provides a first step towards the structural characterization of the CDK9–CycT1–Tat-TAR complex, which is crucial for HIV-1 replication and may constitute a promising target for pharmaceutical intervention.

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 Allosteric modulation of the SARS-CoV-2 spike conformation

2021 ◽  
Author(s):  
Marco A Diaz-Salinas ◽  
Qi Li ◽  
Monir Ejemel ◽  
Yang Wang ◽  
James B Munro
Keyword(s):  
Single Molecule ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Structural Data ◽  
Correlation Spectroscopy ◽  
Host Cells ◽  
Parallel Solution ◽  
Real Time Analysis ◽  
Multiple Conformations

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects host cells through binding to angiotensin-converting enzyme 2 (ACE2), which is mediated by the receptor-binding domain (RBD) of the viral spike (S) glycoprotein. Structural data and real-time analysis of conformational dynamics have shown that S can adopt multiple conformations, which mediate the exposure of the ACE2-binding site in the RBD. Here, using single-molecule Förster resonance energy transfer (smFRET) imaging we report the effects of ACE2 and antibody binding on the conformational dynamics of S from the Wuhan-1 strain and the B.1 variant (D614G). We found that antibodies that target diverse epitopes, including those distal to the RBD, stabilize the RBD in a position competent for ACE2 binding. Parallel solution-based binding experiments using fluorescence correlation spectroscopy (FCS) indicated antibody-mediated enhancement of ACE2 binding. These findings inform on novel strategies for therapeutic antibody cocktails.

scholarly journals Ryanodine Receptor Regulation by Intramolecular Interaction between Cytoplasmic and Transmembrane Domains

2004 ◽  
Vol 15 (6) ◽  
pp. 2627-2638 ◽  
Author(s):  
Christopher H. George ◽  
Hala Jundi ◽  
N. Lowri Thomas ◽  
Mark Scoote ◽  
Nicola Walters ◽  
...  
Keyword(s):  
Intramolecular Interaction ◽  
Ryanodine Receptors ◽  
Specific Interaction ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Transmembrane Domains ◽  
Cytoplasmic Domains ◽  
Dynamic Interactions ◽  
Intracellular Ca ◽  
The Impact

Ryanodine receptors (RyR) function as Ca2+ channels that regulate Ca2+ release from intracellular stores to control a diverse array of cellular processes. The massive cytoplasmic domain of RyR is believed to be responsible for regulating channel function. We investigated interaction between the transmembrane Ca2+-releasing pore and a panel of cytoplasmic domains of the human cardiac RyR in living cells. Expression of eGFP-tagged RyR constructs encoding distinct transmembrane topological models profoundly altered intracellular Ca2+ handling and was refractory to modulation by ryanodine, FKBP12.6 and caffeine. The impact of coexpressing dsRed-tagged cytoplasmic domains of RyR2 on intracellular Ca2+ phenotype was assessed using confocal microscopy coupled with parallel determination of in situ protein: protein interaction using fluorescence resonance energy transfer (FRET). Dynamic interactions between RyR cytoplasmic and transmembrane domains were mediated by amino acids 3722-4610 (Interacting or “I”-domain) which critically modulated intracellular Ca2+ handling and restored RyR sensitivity to caffeine activation. These results provide compelling evidence that specific interaction between cytoplasmic and transmembrane domains is an important mechanism in the intrinsic modulation of RyR Ca2+ release channels.

scholarly journals Calmodulin Binds and Stabilizes the Regulatory Enzyme, CTP:Phosphocholine Cytidylyltransferase

2007 ◽  
Vol 282 (46) ◽  
pp. 33494-33506 ◽  
Author(s):  
Bill. B. Chen ◽  
Rama K. Mallampalli
Keyword(s):  
Animal Cell ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Site Directed Mutagenesis ◽  
Molecular Signature ◽  
Adenoviral Gene Transfer ◽  
Ctp:Phosphocholine Cytidylyltransferase ◽  
Interfering Rna

CTP:phosphocholine cytidylyltransferase (CCTα) is a proteolytically sensitive enzyme essential for production of phosphatidylcholine, the major phospholipid of animal cell membranes. The molecular signals that govern CCTα protein stability are unknown. An NH2-terminal PEST sequence within CCTα did not serve as a degradation signal for the proteinase, calpain. Calmodulin (CaM) stabilized CCTα from calpain proteolysis. Adenoviral gene transfer of CaM in cells protected CCTα, whereas CaM small interfering RNA accentuated CCTα degradation by calpains. CaM bound CCTα as revealed by fluorescence resonance energy transfer and two-hybrid analysis. Mapping and site-directed mutagenesis of CCTα uncovered a motif (LQERVDKVK) harboring a vital recognition site, Gln243, whereby CaM directly binds to the enzyme. Mutagenesis of CCTα Gln243 not only resulted in loss of CaM binding but also led to complete calpain resistance in vitro and in vivo. Thus, calpains and CaM both access CCTα using a structurally similar molecular signature that profoundly affects CCTα levels. These data suggest that CaM, by antagonizing calpain, serves as a novel binding partner for CCTα that stabilizes the enzyme under proinflammatory stress.

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