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 A Novel Fluorescence Resonance Energy Transfer Assay Demonstrates that the Human Immunodeficiency Virus Type 1 Pr55Gag I Domain Mediates Gag-Gag Interactions

2004 ◽  
Vol 78 (3) ◽  
pp. 1230-1242 ◽  
Author(s):  
Aaron Derdowski ◽  
Lingmei Ding ◽  
Paul Spearman
Keyword(s):  
Human Immunodeficiency Virus ◽  
Plasma Membrane ◽  
Energy Transfer ◽  
Protein Interactions ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Protein Protein Interactions ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) assembly takes place at the plasma membrane of cells and is directed by the Pr55Gag polyprotein (Gag). One of the essential steps in the assembly process is the multimerization of Gag. We have developed a novel fluorescence resonance energy transfer (FRET) assay for the detection of protein-protein interactions between Gag molecules. We demonstrate that Gag multimerization takes place primarily on cellular membranes, with the majority of these interactions occurring on the plasma membrane. However, distinct sites of Gag-Gag interaction are also present at punctate intracellular locations. The I domain is a functional assembly domain within the nucleocapsid region of Gag that affects particle density, the subcellular localization of Gag, and the formation of detergent-resistant Gag protein complexes. Results from this study provide evidence that the I domain mediates Gag-Gag interactions. Using Gag-fluorescent protein fusion constructs that were previously shown to define the minimal I domain within HIV-1 Pr55Gag, we show by FRET techniques that protein-protein interactions are greatly diminished when Gag proteins lacking the I domain are expressed. Gag-Tsg101 interactions are also seen in living cells and result in a shift of Tsg101 to the plasma membrane. The results within this study provide direct evidence that the I domain mediates protein-protein interactions between Gag molecules. Furthermore, this study establishes FRET as a powerful tool for the detection of protein-protein interactions involved in retrovirus 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 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 A biosensor for MAPK-dependent Lin28 signaling

2018 ◽  
Vol 29 (10) ◽  
pp. 1157-1167 ◽  
Author(s):  
Laurel M. Oldach ◽  
Kirill Gorshkov ◽  
William T. Mills ◽  
Jin Zhang ◽  
Mollie K. Meffert
Keyword(s):  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Mapk Signaling ◽  
Mitogen Activated Protein Kinase ◽  
Dynamic Monitoring ◽  
Protein Levels ◽  
Differentiated Cells

Intracellular levels of the RNA-binding protein and pluripotency factor, Lin28a, are tightly controlled to govern cellular and organismal growth. Lin28a is extensively regulated at the posttranscriptional level, and can undergo mitogen-activated protein kinase (MAPK)–mediated elevation from low basal levels in differentiated cells by phosphorylation-dependent stabilizing interaction with the RNA-silencing factor HIV TAR RNA-binding protein (TRBP). However, molecular and spatiotemporal details of this critical control mechanism remained unknown. In this work, we dissect the interacting regions of Lin28a and TRBP proteins and develop biosensors to visualize this interaction. We identify truncated domains of Lin28a and of TRBP that are sufficient to support coassociation and mutual elevation of protein levels, and a requirement for MAPK-dependent phosphorylation of TRBP at putative Erk-target serine 152, as well as Lin28a serine 200 phosphorylation, in mediating the increase of Lin28a protein by TRBP. The phosphorylation-dependent association of Lin28a and TRBP truncated constructs is leveraged to develop fluorescence resonance energy transfer (FRET)-based sensors for dynamic monitoring of Lin28a and TRBP interaction. We demonstrate the response of bimolecular and unimolecular FRET sensors to growth factor stimulation in living cells, with coimaging of Erk activation to achieve further understanding of the role of MAPK signaling in Lin28a regulation.

scholarly journals Aldosterone-induced brain MAPK signaling and sympathetic excitation are angiotensin II type-1 receptor dependent

2012 ◽  
Vol 302 (3) ◽  
pp. H742-H751 ◽  
Author(s):  
Zhi-Hua Zhang ◽  
Yang Yu ◽  
Shun-Guang Wei ◽  
Robert B. Felder
Keyword(s):  
Angiotensin Ii ◽  
Time Course ◽  
Mapk Signaling ◽  
Mitogen Activated Protein Kinase ◽  
Ang Ii ◽  
Small Interfering Rnas ◽  
Mineralocorticoid Receptor Antagonist ◽  
Peripheral Tissues ◽  
Intracerebroventricular Infusion

Angiotensin II (ANG II)-induced mitogen-activated protein kinase (MAPK) signaling upregulates angiotensin II type-1 receptors (AT1R) in hypothalamic paraventricular nucleus (PVN) and contributes to AT1R-mediated sympathetic excitation in heart failure. Aldosterone has similar effects to increase AT1R expression in the PVN and sympathetic drive. The present study was undertaken to determine whether aldosterone also activates the sympathetic nervous system via MAPK signaling and, if so, whether its effect is independent of ANG II and AT1R. In anesthetized rats, a 4-h intravenous infusion of aldosterone induced increases ( P < 0.05) in phosphorylated (p-) p44/42 MAPK in PVN, PVN neuronal excitation, renal sympathetic nerve activity (RSNA), mean blood pressure (MBP), and heart rate (HR). Intracerebroventricular or bilateral PVN microinjection of the p44/42 MAPK inhibitor PD-98059 reduced the aldosterone-induced RSNA, HR, and MBP responses. Intracerebroventricular pretreatment (5 days earlier) with pooled small interfering RNAs targeting p44/42 MAPK reduced total and p-p44/42 MAPK, aldosterone-induced c-Fos expression in the PVN, and the aldosterone-induced increases in RSNA, HR, and MBP. Intracerebroventricular infusion of either the mineralocorticoid receptor antagonist RU-28318 or the AT1R antagonist losartan blocked aldosterone-induced phosphorylation of p44/42 MAPK and prevented the increases in RSNA, HR, and MBP. These data suggest that aldosterone-induced sympathetic excitation depends upon that AT1R-induced MAPK signaling in the brain. The short time course of this interaction suggests a nongenomic mechanism, perhaps via an aldosterone-induced transactivation of the AT1R as described in peripheral tissues.

scholarly journals APPL1 mediates adiponectin-stimulated p38 MAPK activation by scaffolding the TAK1-MKK3-p38 MAPK pathway

2011 ◽  
Vol 300 (1) ◽  
pp. E103-E110 ◽  
Author(s):  
Xiaoban Xin ◽  
Lijun Zhou ◽  
Caleb M. Reyes ◽  
Feng Liu ◽  
Lily Q. Dong
Keyword(s):  
Protein Kinase ◽  
P38 Mapk ◽  
Mapk Pathway ◽  
Adaptor Protein ◽  
Mapk Signaling ◽  
Mitogen Activated Protein Kinase ◽  
Scaffolding Protein ◽  
Mapk Activation ◽  
P38 Mapk Pathway ◽  
Mitogen Activated Protein

The adaptor protein APPL1 mediates the stimulatory effect of adiponectin on p38 mitogen-activated protein kinase (MAPK) signaling, yet the underlying mechanism remains unclear. Here we show that, in C2C12 cells, overexpression or suppression of APPL1 enhanced or suppressed, respectively, adiponectin-stimulated p38 MAPK upstream kinase cascade, consisting of transforming growth factor-β-activated kinase 1 (TAK1) and mitogen-activated protein kinase kinase 3 (MKK3). In vitro affinity binding and coimmunoprecipitation experiments revealed that TAK1 and MKK3 bind to different regions of APPL1, suggesting that APPL1 functions as a scaffolding protein to facilitate adiponectin-stimulated p38 MAPK activation. Interestingly, suppressing APPL1 had no effect on TNFα-stimulated p38 MAPK phosphorylation in C2C12 myotubes, indicating that the stimulatory effect of APPL1 on p38 MAPK activation is selective. Taken together, our study demonstrated that the TAK1-MKK3 cascade mediates adiponectin signaling and uncovers a scaffolding role of APPL1 in regulating the TAK1-MKK3-p38 MAPK pathway, specifically in response to adiponectin stimulation.

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