scholarly journals Modulation of A2aR Oligomerisation by Conformational State and PIP2 Interactions Revealed by MD Simulations and Markov Models

2020 ◽  
Author(s):  
Wanling Song ◽  
Anna L. Duncan ◽  
Mark S.P. Sansom
Keyword(s):  
Protein Interactions ◽  
Large Scale ◽  
Markov Models ◽  
Transmembrane Helix ◽  
Receptor Activation ◽  
Membrane Lipid ◽  
Intracellular Loop ◽  
Gpcr Activation ◽  
State Models

AbstractG protein-coupled receptors (GPCRs) play key roles in cellular signalling. GPCRs are suggested to form dimers and higher order oligomers in response to activation. However, we do not fully understand GPCR activation at larger scales and in an in vivo context. We have characterised oligomeric configurations of the adenosine 2a receptor (A2aR) by combining large-scale molecular dynamics simulations with Markov state models. Receptor activation results in enhanced oligomerisation, more diverse oligomer populations, and a more connected oligomerisation network. The active state conformation of the A2aR shifts protein-protein association interfaces to those involving intracellular loop ICL3 and transmembrane helix TM6. Binding of PIP2 to A2aR stabilises protein-protein interactions via PIP2-mediated association interfaces. These results indicate that A2aR oligomerisation is responsive to the local membrane lipid environment. This in turn suggests a modulatory effect on A2aR whereby a given oligomerisation profile favours the dynamic formation of specific supra-molecular signalling complexes.

scholarly journals Activation pathway of a G protein-coupled receptor uncovers conformational intermediates as targets for allosteric drug design

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Shaoyong Lu ◽  
Xinheng He ◽  
Zhao Yang ◽  
Zongtao Chai ◽  
Shuhua Zhou ◽  
...  
Keyword(s):  
G Protein ◽  
Large Scale ◽  
Receptor Activation ◽  
Site Directed Mutagenesis ◽  
Therapeutic Drug ◽  
Activation Pathway ◽  
Transition Mechanism ◽  
Gpcr Activation ◽  
State Models ◽  
G Protein Coupled

AbstractG protein-coupled receptors (GPCRs) are the most common proteins targeted by approved drugs. A complete mechanistic elucidation of large-scale conformational transitions underlying the activation mechanisms of GPCRs is of critical importance for therapeutic drug development. Here, we apply a combined computational and experimental framework integrating extensive molecular dynamics simulations, Markov state models, site-directed mutagenesis, and conformational biosensors to investigate the conformational landscape of the angiotensin II (AngII) type 1 receptor (AT1 receptor) — a prototypical class A GPCR—activation. Our findings suggest a synergistic transition mechanism for AT1 receptor activation. A key intermediate state is identified in the activation pathway, which possesses a cryptic binding site within the intracellular region of the receptor. Mutation of this cryptic site prevents activation of the downstream G protein signaling and β-arrestin-mediated pathways by the endogenous AngII octapeptide agonist, suggesting an allosteric regulatory mechanism. Together, these findings provide a deeper understanding of AT1 receptor activation at an atomic level and suggest avenues for the design of allosteric AT1 receptor modulators with a broad range of applications in GPCR biology, biophysics, and medicinal chemistry.

scholarly journals General, Divergent Platform for Diastereoselective Synthesis of trans-Cyclooctenes with High Reactivity and Favorable Physiochemical Properties

2021 ◽  
Author(s):  
Jessica E. Pigga ◽  
Julia Rosenberger ◽  
Andrew Jemas ◽  
Samantha Boyd ◽  
Olga Dmitrenko ◽  
...  
Keyword(s):  
Physicochemical Properties ◽  
Large Scale ◽  
High Reactivity ◽  
Diels Alder ◽  
Synthetic Methods ◽  
High Yield ◽  
Physiochemical Properties ◽  
New Class ◽  
State Models

<p><i>trans</i>-Cyclooctenes (TCOs) are essential partners for the fastest known bioorthogonal reactions, but current synthetic methods are limited by poor diastereoselectivity. Especially hard to access are hydrophilic TCOs with favorable physicochemical properties for live cell or <i>in vivo </i>experiments. Described is a new class of TCOs, ‘a-TCOs’, that is prepared in high yield via stereocontrolled 1,2-additions of nucleophiles to trans-cyclooct-4-enone, which itself was prepared on large scale in two steps from 1,5-cyclooctadiene. Computational transition state models rationalize the diastereoselectivity of 1,2-additions to deliver a-TCO products, which were also shown to be more reactive than standard TCOs and less hydrophobic than even a <i>trans</i>-oxocene analog. Illustrating the favorable physicochemical properties of a-TCOs, a fluorescent TAMRA derivative in live HeLa cells was shown to be cell-permeable through intracellular Diels-Alder chemistry and to washout more rapidly than other TCOs.</p>

scholarly journals General, Divergent Platform for Diastereoselective Synthesis of trans-Cyclooctenes with High Reactivity and Favorable Physiochemical Properties

2021 ◽  
Author(s):  
Jessica E. Pigga ◽  
Julia Rosenberger ◽  
Andrew Jemas ◽  
Samantha Boyd ◽  
Olga Dmitrenko ◽  
...  
Keyword(s):  
Physicochemical Properties ◽  
Large Scale ◽  
High Reactivity ◽  
Diels Alder ◽  
Synthetic Methods ◽  
High Yield ◽  
Physiochemical Properties ◽  
New Class ◽  
State Models

<p><i>trans</i>-Cyclooctenes (TCOs) are essential partners for the fastest known bioorthogonal reactions, but current synthetic methods are limited by poor diastereoselectivity. Especially hard to access are hydrophilic TCOs with favorable physicochemical properties for live cell or <i>in vivo </i>experiments. Described is a new class of TCOs, ‘a-TCOs’, that is prepared in high yield via stereocontrolled 1,2-additions of nucleophiles to trans-cyclooct-4-enone, which itself was prepared on large scale in two steps from 1,5-cyclooctadiene. Computational transition state models rationalize the diastereoselectivity of 1,2-additions to deliver a-TCO products, which were also shown to be more reactive than standard TCOs and less hydrophobic than even a <i>trans</i>-oxocene analog. Illustrating the favorable physicochemical properties of a-TCOs, a fluorescent TAMRA derivative in live HeLa cells was shown to be cell-permeable through intracellular Diels-Alder chemistry and to washout more rapidly than other TCOs.</p>

Mapping the Protease Activated Receptor 4 (PAR4) Homodimer Interface.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 773-773
Author(s):  
Marvin T Nieman
Keyword(s):  
Conflicts Of Interest ◽  
Specific Interaction ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Receptor Activation ◽  
Dependent Manner ◽  
Intracellular Loop ◽  
Concentration Dependent Manner ◽  
Platelet Surface

Abstract Abstract 773 Thrombin activates platelets by binding and cleaving protease activated receptors 1 and 4 (PAR1 and PAR4). PAR1 and PAR4 communicate with each other to lower the concentration of thrombin required for PAR4 activation (Nieman Biochemistry, 2008). In addition, PAR1 and PAR4 form homo and heterodimers. However, where these receptors interact has not been defined and it is not known if dimerization influences receptor activation, downstream signaling, or both. Since PAR4 activation is important on human and mouse platelets, we sought to characterize the interaction site between PAR4 homodimers. Using bioluminescence resonance energy transfer (BRET), we mapped the PAR4 homodimer interface. The PAR4 homodimers show a specific interaction as indicated by a hyperbolic BRET signal in response to increasing PAR4-GFP expression with a fixed concentration of PAR4-Rluc. The threshold maximum BRET signal was disrupted in a concentration-dependent manner by unlabeled PAR4. In contrast, the unrelated G-protein coupled receptor, rhodopsin, was unable to disrupt the BRET signal indicating that the disruption of the PAR4 homodimer is a specific interaction. We have mapped the region required for PAR4 homodimer formation using chimeras between rhodopsin and PAR4. PAR4 does not interact with rhodopsin in BRET assays. Using a library of rho-PAR4 chimeras that have the junction at the beginning of transmembrane (TM) 2, 3, 4, 5, 6 or 7, we determined where dimer formation is restored. When the junction is placed at the beginning of TM4 or TM5, the chimera does not interact with PAR4-WT. In contrast, when the junction is moved to the end of TM2, the BRET signal is restored. These results indicate that the region on PAR4 required for homodimer formation encompasses a 63 amino acid region that includes the first extracellular loop, TM3 and the second intracellular loop. These studies establish techniques that may be used to define the interactions between other GPCRs found on the platelet surface. These receptor-receptor interactions may be another level of regulation of agonist activity and platelet function in vivo and may provide novel targets for anti-platelet therapies. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The Role of ICL1 and H8 in Class B1 GPCRs; Implications for Receptor Activlation

2022 ◽  
Vol 12 ◽  
Author(s):  
Ian Winfield ◽  
Kerry Barkan ◽  
Sarah Routledge ◽  
Nathan J. Robertson ◽  
Matthew Harris ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
G Protein ◽  
Conformational Changes ◽  
Transmembrane Helix ◽  
Receptor Activation ◽  
Cgrp Receptor ◽  
Intracellular Loop ◽  
Glucagon Receptor ◽  
G Protein Coupled

The first intracellular loop (ICL1) of G protein-coupled receptors (GPCRs) has received little attention, although there is evidence that, with the 8th helix (H8), it is involved in early conformational changes following receptor activation as well as contacting the G protein β subunit. In class B1 GPCRs, the distal part of ICL1 contains a conserved R12.48KLRCxR2.46b motif that extends into the base of the second transmembrane helix; this is weakly conserved as a [R/H]12.48KL[R/H] motif in class A GPCRs. In the current study, the role of ICL1 and H8 in signaling through cAMP, iCa2+ and ERK1/2 has been examined in two class B1 GPCRs, using mutagenesis and molecular dynamics. Mutations throughout ICL1 can either enhance or disrupt cAMP production by CGRP at the CGRP receptor. Alanine mutagenesis identified subtle differences with regard elevation of iCa2+, with the distal end of the loop being particularly sensitive. ERK1/2 activation displayed little sensitivity to ICL1 mutation. A broadly similar pattern was observed with the glucagon receptor, although there were differences in significance of individual residues. Extending the study revealed that at the CRF1 receptor, an insertion in ICL1 switched signaling bias between iCa2+ and cAMP. Molecular dynamics suggested that changes in ICL1 altered the conformation of ICL2 and the H8/TM7 junction (ICL4). For H8, alanine mutagenesis showed the importance of E3908.49b for all three signal transduction pathways, for the CGRP receptor, but mutations of other residues largely just altered ERK1/2 activation. Thus, ICL1 may modulate GPCR bias via interactions with ICL2, ICL4 and the Gβ subunit.

scholarly journals A dopamine-induced gene expression signature regulates neuronal function and cocaine response

2020 ◽  
Vol 6 (26) ◽  
pp. eaba4221 ◽  
Author(s):  
Katherine E. Savell ◽  
Jennifer J. Tuscher ◽  
Morgan E. Zipperly ◽  
Corey G. Duke ◽  
Robert A. Phillips ◽  
...  
Keyword(s):  
Gene Expression ◽  
Large Scale ◽  
Drugs Of Abuse ◽  
Transcriptional Response ◽  
Gene Expression Signature ◽  
Receptor Activation ◽  
Early Gene ◽  
Behavioral Adaptations

Drugs of abuse elevate dopamine levels in the nucleus accumbens (NAc) and alter transcriptional programs believed to promote long-lasting synaptic and behavioral adaptations. Here, we leveraged single-nucleus RNA-sequencing to generate a comprehensive molecular atlas of cell subtypes in the NAc, defining both sex-specific and cell type–specific responses to acute cocaine experience in a rat model system. Using this transcriptional map, we identified an immediate early gene expression program that is up-regulated following cocaine experience in vivo and dopamine receptor activation in vitro. Multiplexed induction of this gene program with a large-scale CRISPR-dCas9 activation strategy initiated a secondary synapse-centric transcriptional profile, altered striatal physiology in vitro, and enhanced cocaine sensitization in vivo. Together, these results define the transcriptional response to cocaine with cellular precision and demonstrate that drug-responsive gene programs can potentiate both physiological and behavioral adaptations to drugs of abuse.

scholarly journals Probing the β2 Adrenoceptor Binding Site with Catechol Reveals Differences in Binding and Activation by Agonists and Partial Agonists

2005 ◽  
Vol 280 (23) ◽  
pp. 22165-22171 ◽  
Author(s):  
Gayathri Swaminath ◽  
Xavier Deupi ◽  
Tae Weon Lee ◽  
Wen Zhu ◽  
Foon Sun Thian ◽  
...  
Keyword(s):  
Binding Site ◽  
Aromatic Ring ◽  
Partial Agonist ◽  
Transmembrane Helix ◽  
Receptor Activation ◽  
Molecular Probe ◽  
Toggle Switch ◽  
Partial Agonists ◽  
Gpcr Activation ◽  
Multistep Process

The β2 adrenergic receptor (β2AR) is a prototypical family A G protein-coupled receptor (GPCR) and an excellent model system for studying the mechanism of GPCR activation. The β2AR agonist binding site is well characterized, and there is a wealth of structurally related ligands with functionally diverse properties. In the present study, we use catechol (1,2-benzenediol, a structural component of catecholamine agonists) as a molecular probe to identify mechanistic differences between β2AR activation by catecholamine agonists, such as isoproterenol, and by the structurally related non-catechol partial agonist salbutamol. Using biophysical and pharmacologic approaches, we show that the aromatic ring of salbutamol binds to a different site on the β2AR than the aromatic ring of catecholamines. This difference is important in receptor activation as it has been hypothesized that the aromatic ring of catecholamines plays a role in triggering receptor activation through interactions with a conserved cluster of aromatic residues in the sixth transmembrane segment by a rotamer toggle switch mechanism. Our experiments indicate that the aromatic ring of salbutamol does not activate this mechanism either directly or indirectly. Moreover, the non-catechol ring of partial agonists does not interact optimally with serine residues in the fifth transmembrane helix that have been shown to play an important role in activation by catecholamines. These results demonstrate unexpected differences in binding and activation by structurally similar agonists and partial agonists. Moreover, they provide evidence that activation of a GPCR is a multistep process that can be dissected into its component parts using agonist fragments.

scholarly journals In Vivo Dynamics of Swi6 in Yeast: Evidence for a Stochastic Model of Heterochromatin

2004 ◽  
Vol 24 (8) ◽  
pp. 3157-3167 ◽  
Author(s):  
Thierry Cheutin ◽  
Stanislaw A. Gorski ◽  
Karen M. May ◽  
Prim B. Singh ◽  
Tom Misteli
Keyword(s):  
Stochastic Model ◽  
Fission Yeast ◽  
Protein Interactions ◽  
Mammalian Cells ◽  
Large Scale ◽  
Quantitative Estimation ◽  
Pericentric Heterochromatin ◽  
Heterochromatin Protein ◽  
Structural Heterochromatin

ABSTRACT The mechanism for transcriptional silencing of pericentric heterochromatin is conserved from fission yeast to mammals. Silenced genome regions are marked by epigenetic methylation of histone H3, which serves as a binding site for structural heterochromatin proteins. In the fission yeast Schizosaccharomyces pombe, the major structural heterochromatin protein is Swi6. To gain insight into Swi6 function in vivo, we have studied its dynamics in the nucleus of living yeast. We demonstrate that, in contrast to mammalian cells, yeast heterochromatin domains undergo rapid, large-scale motions within the nucleus. Similar to the situation in mammalian cells, Swi6 does not permanently associate with these chromatin domains but binds only transiently to euchromatin and heterochromatin. Swi6 binding dynamics are dependent on growth status and on the silencing factors Clr4 and Rik1, but not Clr1, Clr2, or Clr3. By comparing the kinetics of mutant Swi6 proteins in swi6− and swi6+ strains, we demonstrate that homotypic protein-protein interactions via the chromoshadow domain stabilize Swi6 binding to chromatin in vivo. Kinetic modeling allowed quantitative estimation of residence times and indicated the existence of at least two kinetically distinct populations of Swi6 in heterochromatin. The observed dynamics of Swi6 binding are consistent with a stochastic model of heterochromatin and indicate evolutionary conservation of heterochromatin protein binding properties from mammals to yeast.

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