scholarly journals Allosteric modulation of GPCR-induced β-arrestin trafficking and signaling by a synthetic intrabody

2022 ◽  
Author(s):  
Mithu Baidya ◽  
Madhu Chaturvedi ◽  
Hemlata Dwivedi-Agnihotri ◽  
Ashutosh Ranjan ◽  
Dominic Devost ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Phosphorylation Site ◽  
Endosomal Trafficking ◽  
Kinase Activation ◽  
Primary Determinant ◽  
Mechanistic Basis ◽  
Dynamics Simulations ◽  
Targeted Drug Discovery ◽  
G Protein Coupled ◽  
Current Paradigm

Agonist-induced phosphorylation of G protein-coupled receptors (GPCRs) is a primary determinant of β-arrestin (βarr) recruitment and trafficking. For several GPCRs, such as the vasopressin type II receptor (V2R), which exhibit high affinity for βarrs, agonist-stimulation first drives the translocation of βarrs to the plasma membrane, followed by endosomal trafficking. We previously found that mutation of a single phosphorylation site in V2R (i.e., V2RT360A) results in near-complete loss of βarr translocation to endosomes although βarrs are robustly recruited to the plasma membrane. Here, we show that a synthetic intrabody referred to as intrabody30 (Ib30), which selectively recognizes an active-like βarr1 conformation, rescues endosomal translocation of βarr1 for V2RT360A. In addition, Ib30 also rescues agonist-induced ERK1/2 MAP kinase activation for V2RT360A to levels similar to that of the wild-type V2R. Molecular dynamics simulations reveal that Ib30 binding promotes active-like conformation in βarr1 with respect to the inter-domain rotation. Interestingly, we also observe that Ib30 enhances the interaction of βarr1 with β2-adaptin, which provides a mechanistic basis for the ability of Ib30 to promote endosomal trafficking of βarr1. Taken together, our data provide a novel mechanism to positively modulate the receptor-transducer-effector axis for GPCRs using intrabodies, which can potentially be integrated in the current paradigm of GPCR-targeted drug discovery.

scholarly journals Conformational heterogeneity of the calmodulin binding interface

2016 ◽  
Vol 7 (1) ◽  
Author(s):  
Diwakar Shukla ◽  
Ariana Peck ◽  
Vijay S. Pande
Keyword(s):  
Exchange Process ◽  
Pharmacological Intervention ◽  
Calmodulin Binding ◽  
Signalling Molecules ◽  
Binding Interface ◽  
Local Unfolding ◽  
Mechanistic Basis ◽  
State Models ◽  
Dynamics Simulations ◽  
G Protein Coupled

Abstract Calmodulin (CaM) is a ubiquitous Ca2+ sensor and a crucial signalling hub in many pathways aberrantly activated in disease. However, the mechanistic basis of its ability to bind diverse signalling molecules including G-protein-coupled receptors, ion channels and kinases remains poorly understood. Here we harness the high resolution of molecular dynamics simulations and the analytical power of Markov state models to dissect the molecular underpinnings of CaM binding diversity. Our computational model indicates that in the absence of Ca2+, sub-states in the folded ensemble of CaM’s C-terminal domain present chemically and sterically distinct topologies that may facilitate conformational selection. Furthermore, we find that local unfolding is off-pathway for the exchange process relevant for peptide binding, in contrast to prior hypotheses that unfolding might account for binding diversity. Finally, our model predicts a novel binding interface that is well-populated in the Ca2+-bound regime and, thus, a candidate for pharmacological intervention.

scholarly journals Revising Endosomal Trafficking under Insulin Receptor Activation

2021 ◽  
Vol 22 (13) ◽  
pp. 6978
Author(s):  
Maria J. Iraburu ◽  
Tommy Garner ◽  
Cristina Montiel-Duarte
Keyword(s):  
Plasma Membrane ◽  
Tyrosine Kinase ◽  
Insulin Receptor ◽  
Molecular Mechanisms ◽  
Receptor Activation ◽  
Small Gtpases ◽  
Early Endosome ◽  
Tyrosine Kinase Receptors ◽  
Endosomal Trafficking ◽  
Cell Functions

The endocytosis of ligand-bound receptors and their eventual recycling to the plasma membrane (PM) are processes that have an influence on signalling activity and therefore on many cell functions, including migration and proliferation. Like other tyrosine kinase receptors (TKR), the insulin receptor (INSR) has been shown to be endocytosed by clathrin-dependent and -independent mechanisms. Once at the early endosome (EE), the sorting of the receptor, either to the late endosome (LE) for degradation or back to the PM through slow or fast recycling pathways, will determine the intensity and duration of insulin effects. Both the endocytic and the endosomic pathways are regulated by many proteins, the Arf and Rab families of small GTPases being some of the most relevant. Here, we argue for a specific role for the slow recycling route, whilst we review the main molecular mechanisms involved in INSR endocytosis, sorting and recycling, as well as their possible role in cell functions.

scholarly journals Structural and mechanistic basis for translation inhibition by macrolide and ketolide antibiotics

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Bertrand Beckert ◽  
Elodie C. Leroy ◽  
Shanmugapriya Sothiselvam ◽  
Lars V. Bock ◽  
Maxim S. Svetlov ◽  
...  
Keyword(s):  
Antibiotic Resistance Genes ◽  
Structural Basis ◽  
Sequence Motifs ◽  
Specific Sequence ◽  
Bacterial Ribosome ◽  
Translational Arrest ◽  
Exit Tunnel ◽  
Mechanistic Basis ◽  
Dynamics Simulations ◽  
Context Specific

AbstractMacrolides and ketolides comprise a family of clinically important antibiotics that inhibit protein synthesis by binding within the exit tunnel of the bacterial ribosome. While these antibiotics are known to interrupt translation at specific sequence motifs, with ketolides predominantly stalling at Arg/Lys-X-Arg/Lys motifs and macrolides displaying a broader specificity, a structural basis for their context-specific action has been lacking. Here, we present structures of ribosomes arrested during the synthesis of an Arg-Leu-Arg sequence by the macrolide erythromycin (ERY) and the ketolide telithromycin (TEL). Together with deep mutagenesis and molecular dynamics simulations, the structures reveal how ERY and TEL interplay with the Arg-Leu-Arg motif to induce translational arrest and illuminate the basis for the less stringent sequence-specific action of ERY over TEL. Because programmed stalling at the Arg/Lys-X-Arg/Lys motifs is used to activate expression of antibiotic resistance genes, our study also provides important insights for future development of improved macrolide antibiotics.

A role for Pyk2 and Src in linking G-protein-coupled receptors with MAP kinase activation

Nature ◽  
1996 ◽  
Vol 383 (6600) ◽  
pp. 547-550 ◽  
Author(s):  
Ivan Dikic ◽  
George Tokiwa ◽  
Sima Lev ◽  
Sara A. Courtneidge ◽  
Joseph Schlessinger
Keyword(s):  
Map Kinase ◽  
G Protein ◽  
G Protein Coupled Receptors ◽  
Kinase Activation ◽  
G Protein Coupled

scholarly journals The MADD-3 LAMMER Kinase Interacts with a p38 MAP Kinase Pathway to Regulate the Display of the EVA-1 Guidance Receptor in Caenorhabditis elegans

PLoS Genetics ◽  
2016 ◽  
Vol 12 (4) ◽  
pp. e1006010 ◽  
Author(s):  
Serena A. D’Souza ◽  
Luckshi Rajendran ◽  
Rachel Bagg ◽  
Louis Barbier ◽  
Derek M. van Pel ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Caenorhabditis Elegans ◽  
Map Kinase ◽  
Motor Neurons ◽  
Leading Edge ◽  
P38 Map Kinase ◽  
Endosomal Trafficking ◽  
Guidance Cue ◽  
Map Kinase Pathway ◽  
Kinase Pathway

The proper display of transmembrane receptors on the leading edge of migrating cells and cell extensions is essential for their response to guidance cues. We previously discovered that MADD-4, which is an ADAMTSL secreted by motor neurons in Caenorhabditis elegans, interacts with an UNC-40/EVA-1 co-receptor complex on muscles to attract plasma membrane extensions called muscle arms. In nematodes, the muscle arm termini harbor the post-synaptic elements of the neuromuscular junction. Through a forward genetic screen for mutants with disrupted muscle arm extension, we discovered that a LAMMER kinase, which we call MADD-3, is required for the proper display of the EVA-1 receptor on the muscle’s plasma membrane. Without MADD-3, EVA-1 levels decrease concomitantly with a reduction of the late-endosomal marker RAB-7. Through a genetic suppressor screen, we found that the levels of EVA-1 and RAB-7 can be restored in madd-3 mutants by eliminating the function of a p38 MAP kinase pathway. We also found that EVA-1 and RAB-7 will accumulate in madd-3 mutants upon disrupting CUP-5, which is a mucolipin ortholog required for proper lysosome function. Together, our data suggests that the MADD-3 LAMMER kinase antagonizes the p38-mediated endosomal trafficking of EVA-1 to the lysosome. In this way, MADD-3 ensures that sufficient levels of EVA-1 are present to guide muscle arm extension towards the source of the MADD-4 guidance cue.

Abstract 626: Interaction Between The Ang-(1-7) Receptor Mas And The Bradykinin B2 Receptor: Functional Implications

Hypertension ◽  
2014 ◽  
Vol 64 (suppl_1) ◽  
Author(s):  
Bruno Cerrato ◽  
Oscar Carretero ◽  
Hernán Grecco ◽  
Mariela M Gironacci
Keyword(s):  
Plasma Membrane ◽  
Binding Assays ◽  
Transfected Cells ◽  
Oligomer Formation ◽  
Early Endosomes ◽  
Functional Implications ◽  
Functional Consequences ◽  
Ligand Stimulation ◽  
G Protein Coupled ◽  
Fluorescence Energy

G protein-coupled receptors (R) exist as homo- or hetero-oligomers, which is essential for receptor function. Since BK actions were blocked by a Mas R antagonist or that Ang-(1-7) responses disappeared when the BK receptor B2 was blocked, we hypothesized that Mas and B2 Rs on the plasma membrane may interact through hetero-oligomer formation. Our aim was to investigate the existence of heteromerization between Mas and B2 Rs by the fluorescence energy transfer (FRET) technique and the functional consequences of this oligomer formation. HEK293T cells were transfected with the coding sequence for Mas R fused to YFP and B2 R fused to CFP. After 48 h cells were incubated in the absence and presence of 1 μM Ang-(1-7) or BK during 15 min and interaction between Mas and B2 R was evaluated by FRET. Functional consequences of this interaction were determined by ligand binding assays. A positive FRET was observed in cells cotransfected with MasR-YFP and B2R-CFP, suggesting that both Mas and B2 Rs interact by a hetero-oligomer formation in a constitutive manner. This hetero-oligomer was not altered by the agonist because FRET was not modified when the cells were stimulated with BK or Ang-(1-7). Ang-(1-7) or BK induced internalization of this hetero-oligomer into early endosomes since MasR-YFP or B2R-CFP colocalized with Rab-5, an early endosome marker, after ligand stimulation. When MasR-YFP plus B2R-CFP transfected cells were stimulated with Ang-(1-7) there was a decrease of 82±6% in Mas R and 58±4% in B2 R present in the plasma membrane. Conversely, when MasR-YFP plus B2R-CFP transfected cells were stimulated with BK there was a decrease of 91±4% in B2 R and 53±3% in Mas R in the plasma membrane. This result clearly demonstrates that in co-expressing cells of both receptors the selective stimulation of one of the GPCRs promotes co-internalization of both receptors. We conclude that Mas and B2 Rs constitutively interact through an hetero-oligomer formation at the plasma membrane which may explain the cross-talk between Ang-(1-7) and BK. This hetero-oligomer is internalized upon stimulation with either Ang-(1-7) or BK, leading to a decrease in the number of Rs present in the membrane.

scholarly journals Comment on "A G Protein Coupled Receptor Is a Plasma Membrane Receptor for the Plant Hormone Abscisic Acid"

Science ◽  
2007 ◽  
Vol 318 (5852) ◽  
pp. 914c-914c ◽  
Author(s):  
C. A. Johnston ◽  
B. R. Temple ◽  
J.-G. Chen ◽  
Y. Gao ◽  
E. N. Moriyama ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Abscisic Acid ◽  
G Protein ◽  
Plant Hormone ◽  
Membrane Receptor ◽  
G Protein Coupled Receptor ◽  
Plasma Membrane Receptor ◽  
G Protein Coupled
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