scholarly journals Molecular Basis for G-protein-Coupled Receptor (GPCR) Activation and Biased Signalling at the Platelet Thrombin Receptor Proteinase Activated Receptor-4 (PAR4)

2019 ◽  
Author(s):  
Pierre E. Thibeault ◽  
Jordan C. LeSarge ◽  
D’Arcy Arends ◽  
Michaela Fernandes ◽  
Peter Chidiac ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
G Protein ◽  
Molecular Basis ◽  
Calcium Signalling ◽  
Cathepsin G ◽  
Mitogen Activated Protein Kinase ◽  
Proteolytic Activation ◽  
Gpcr Activation ◽  
Tethered Ligand ◽  
G Protein Coupled

AbstractProteinase Activated Receptor-4 (PAR4) is a member of the proteolytically-activated PAR family of G-Protein-coupled Receptors (GPCRs). PARs are activated following proteolytic cleavage of the receptor N-terminus by enzymes such as thrombin, trypsin, and cathepsin-G to reveal the receptor-activating motif termed the tethered ligand. The tethered ligand binds intramolecularly to the receptor and triggers receptor signalling and cellular responses. In spite of this unusual mechanism of activation, PARs are fundamentally peptide receptors and can also be activated by exogenous application of short synthetic peptides derived from the tethered ligand sequence. In order to gain a better understanding of the molecular basis for PAR4-dependent signalling, we examined signalling responses to a library of peptides derived from the canonical PAR4 activating peptide (PAR4-AP), AYPGKF-NH2. We examined peptide residues involved in activation of the Gαq/11-coupled calcium signalling pathway, β-arrestin recruitment, and mitogen-activated protein kinase pathway activation. The peptide N-methyl-alanine-YPGKF-NH2 was identified as a compound that is a poor activator of PAR4-dependent calcium signalling but was fully competent in recruiting β-arrestin-1 and -2. In order to gain a better understanding of the ligand-binding pocket, we used in silico docking to identify key residues involved in PAR4 interaction with AYPGKF-NH2. The predicted interactions were verified by site-directed mutagenesis and analysis of calcium signalling and β-arrestin-1/-2 recruitment following proteolytic activation (with thrombin) or activation with the synthetic agonist peptide (AYPGKF-NH2). We determined that a key extracellular loop-2 aspartic acid residue (Asp230) is critical for signalling following both proteolytic and peptide activation of PAR4. Finally, we investigated platelet aggregation in response to AyPGKF-NH2 (a peptide with D-tyrosine in position two) which is unable to activate calcium signalling, and AYPGRF-NH2 a peptide that is equipotent to the parental peptide AYPGKF-NH2 for calcium signalling but is more potent at recruiting β-arrestins. We found that AyPGKF-NH2 fails to activate platelets while AYPGRF-NH2 causes a platelet aggregation response that is greater than that seen with the parental peptide and is comparable to that seen with thrombin stimulation. Overall, these studies uncover molecular determinants for agonist binding and signalling through a non-canonically activated GPCR and provide a template for development of small molecule modulators of PAR4.

scholarly journals Molecular basis for activation and biased signaling at the thrombin-activated GPCR proteinase activated receptor-4 (PAR4)

2019 ◽  
Vol 295 (8) ◽  
pp. 2520-2540 ◽  
Author(s):  
Pierre E. Thibeault ◽  
Jordan C. LeSarge ◽  
D'Arcy Arends ◽  
Michaela Fernandes ◽  
Peter Chidiac ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Calcium Signaling ◽  
Molecular Basis ◽  
Mapk Pathway ◽  
Cathepsin G ◽  
Site Directed Mutagenesis ◽  
Mitogen Activated Protein Kinase ◽  
Agonist Peptide ◽  
Biased Signaling ◽  
Tethered Ligand

Proteinase-activated receptor (PAR)-4 is a member of the proteolytically-activated PAR family of G-protein–coupled receptors (GPCR) that represents an important target in the development of anti-platelet therapeutics. PARs are activated by proteolytic cleavage of their receptor N terminus by enzymes such as thrombin, trypsin, and cathepsin-G. This reveals the receptor-activating motif, termed the tethered ligand that binds intramolecularly to the receptor and triggers signaling. However, PARs are also activated by exogenous application of synthetic peptides derived from the tethered-ligand sequence. To better understand the molecular basis for PAR4-dependent signaling, we examined PAR4-signaling responses to a peptide library derived from the canonical PAR4-agonist peptide, AYPGKF-NH2, and we monitored activation of the Gαq/11-coupled calcium-signaling pathway, β-arrestin recruitment, and mitogen-activated protein kinase (MAPK) pathway activation. We identified peptides that are poor activators of PAR4-dependent calcium signaling but were fully competent in recruiting β-arrestin-1 and -2. Peptides that were unable to stimulate PAR4-dependent calcium signaling could not trigger MAPK activation. Using in silico docking and site-directed mutagenesis, we identified Asp230 in the extracellular loop-2 as being critical for PAR4 activation by both agonist peptide and the tethered ligand. Probing the consequence of biased signaling on platelet activation, we found that a peptide that cannot activate calcium signaling fails to cause platelet aggregation, whereas a peptide that is able to stimulate calcium signaling and is more potent for β-arrestin recruitment triggered greater levels of platelet aggregation compared with the canonical PAR4 agonist peptide. These findings uncover molecular determinants critical for agonist binding and biased signaling through PAR4.

scholarly journals Structural Characterization of Receptor–Receptor Interactions in the Allosteric Modulation of G Protein-Coupled Receptor (GPCR) Dimers

2021 ◽  
Vol 22 (6) ◽  
pp. 3241
Author(s):  
Raudah Lazim ◽  
Donghyuk Suh ◽  
Jai Woo Lee ◽  
Thi Ngoc Lan Vu ◽  
Sanghee Yoon ◽  
...  
Keyword(s):  
G Protein ◽  
Protein Interactions ◽  
Metabotropic Glutamate Receptor ◽  
Three Dimensional ◽  
Allosteric Modulation ◽  
Experimental Investigations ◽  
G Protein Coupled Receptor ◽  
Metabotropic Glutamate ◽  
Gpcr Activation ◽  
G Protein Coupled

G protein-coupled receptor (GPCR) oligomerization, while contentious, continues to attract the attention of researchers. Numerous experimental investigations have validated the presence of GPCR dimers, and the relevance of dimerization in the effectuation of physiological functions intensifies the attractiveness of this concept as a potential therapeutic target. GPCRs, as a single entity, have been the main source of scrutiny for drug design objectives for multiple diseases such as cancer, inflammation, cardiac, and respiratory diseases. The existence of dimers broadens the research scope of GPCR functions, revealing new signaling pathways that can be targeted for disease pathogenesis that have not previously been reported when GPCRs were only viewed in their monomeric form. This review will highlight several aspects of GPCR dimerization, which include a summary of the structural elucidation of the allosteric modulation of class C GPCR activation offered through recent solutions to the three-dimensional, full-length structures of metabotropic glutamate receptor and γ-aminobutyric acid B receptor as well as the role of dimerization in the modification of GPCR function and allostery. With the growing influence of computational methods in the study of GPCRs, we will also be reviewing recent computational tools that have been utilized to map protein–protein interactions (PPI).

scholarly journals Applications of molecular replacement to G protein-coupled receptors

2013 ◽  
Vol 69 (11) ◽  
pp. 2287-2292 ◽  
Author(s):  
Andrew C. Kruse ◽  
Aashish Manglik ◽  
Brian K. Kobilka ◽  
William I. Weis
Keyword(s):  
G Protein ◽  
Structural Biology ◽  
Structural Information ◽  
G Protein Coupled Receptors ◽  
Integral Membrane Proteins ◽  
Molecular Replacement ◽  
Gpcr Activation ◽  
Health And Disease ◽  
Almost All ◽  
G Protein Coupled

G protein-coupled receptors (GPCRs) are a large class of integral membrane proteins involved in regulating virtually every aspect of human physiology. Despite their profound importance in human health and disease, structural information regarding GPCRs has been extremely limited until recently. With the advent of a variety of new biochemical and crystallographic techniques, the structural biology of GPCRs has advanced rapidly, offering key molecular insights into GPCR activation and signal transduction. To date, almost all GPCR structures have been solved using molecular-replacement techniques. Here, the unique aspects of molecular replacement as applied to individual GPCRs and to signaling complexes of these important proteins are discussed.

G-protein-coupled-receptor activation of the smooth muscle calponin gene

2001 ◽  
Vol 357 (2) ◽  
pp. 587-592 ◽  
Author(s):  
Nickolai O. DULIN ◽  
Sergei N. ORLOV ◽  
Chad M. KITCHEN ◽  
Tatyana A. VOYNO-YASENETSKAYA ◽  
Joseph M. MIANO
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase ◽  
G Protein ◽  
Transcriptional Activation ◽  
Fold Increase ◽  
Receptor Activation ◽  
G Protein Coupled Receptors ◽  
Mitogen Activated Protein Kinase ◽  
G Protein Coupled

A hallmark of cultured smooth muscle cells (SMCs) is the rapid down-regulation of several lineage-restricted genes that define their in vivo differentiated phenotype. Identifying factors that maintain an SMC differentiated phenotype has important implications in understanding the molecular underpinnings governing SMC differentiation and their subversion to an altered phenotype in various disease settings. Here, we show that several G-protein coupled receptors [α-thrombin, lysophosphatidic acid and angiotensin II (AII)] increase the expression of smooth muscle calponin (SM-Calp) in rat and human SMC. The increase in SM-Calp protein appears to be selective for G-protein-coupled receptors as epidermal growth factor was without effect. Studies using AII showed a 30-fold increase in SM-Calp protein, which was dose- and time-dependent and mediated by the angiotensin receptor-1 (AT1 receptor). The increase in SM-Calp protein with AII was attributable to transcriptional activation of SM-Calp based on increases in steady-state SM-Calp mRNA, increases in SM-Calp promoter activity and complete abrogation of protein induction with actinomycin D. To examine the potential role of extracellular signal-regulated kinase (Erk1/2), protein kinase B, p38 mitogen-activated protein kinase and protein kinase C in AII-induced SM-Calp, inhibitors to each of the signalling pathways were used. None of these signalling molecules appears to be crucial for AII-induced SM-Calp expression, although Erk1/2 may be partially involved. These results identify SM-Calp as a target of AII-mediated signalling, and suggest that the SMC response to AII may incorporate a novel activity of SM-Calp.

scholarly journals Lipid Receptor GPR31 (G-Protein–Coupled Receptor 31) Regulates Platelet Reactivity and Thrombosis Without Affecting Hemostasis

2020 ◽  
Author(s):  
Layla Van Doren ◽  
Nga Nguyen ◽  
Christopher Garzia ◽  
Elizabeth Fletcher ◽  
Ryan Stevenson ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Carotid Artery ◽  
G Protein ◽  
Arterial Thrombosis ◽  
Platelet Reactivity ◽  
Human Platelets ◽  
Calcium Flux ◽  
G Protein Coupled Receptor ◽  
Carotid Artery Injury ◽  
G Protein Coupled

Objective: 12-LOX (12-lipoxygenase) produces a number of bioactive lipids including 12(S)-HETE that are involved in inflammation and platelet reactivity. The GPR31 (G-protein–coupled receptor 31) is the proposed receptor of 12(S)-HETE; however, it is not known whether the 12(S)-HETE-GPR31 signaling axis serves to enhance or inhibit platelet activity. Approach and Results: Using pepducin technology and biochemical approaches, we provide evidence that 12(S)-HETE-GPR31 signals through Gi to enhance PAR (protease-activated receptor)-4–mediated platelet activation and arterial thrombosis using both human platelets and mouse carotid artery injury models. 12(S)-HETE suppressed AC (adenylyl cyclase) activity through GPR31 and resulted in Rap1 and p38 activation and low but detectable calcium flux but did not induce platelet aggregation. A GPR31 third intracellular (i3) loop–derived pepducin, GPR310 (G-protein–coupled receptor 310), significantly inhibited platelet aggregation in response to thrombin, collagen, and PAR4 agonist, AYPGKF, in human and mouse platelets but relative sparing of PAR1 agonist SFLLRN in human platelets. GPR310 treatment gave a highly significant 80% protection ( P =0.0018) against ferric chloride–induced carotid artery injury in mice by extending occlusion time, without any effect on tail bleeding. PAR4-mediated dense granule secretion and calcium flux were both attenuated by GPR310. Consistent with these results, GPR310 inhibited 12(S)-HETE–mediated and PAR4-mediated Rap1-GTP and RASA3 translocation to the plasma membrane and attenuated PAR4-Akt and ERK activation. GPR310 caused a right shift in thrombin-mediated human platelet aggregation, comparable to the effects of inhibition of the Gi-coupled P2Y 12 receptor. Co-immunoprecipitation studies revealed that GPR31 and PAR4 form a heterodimeric complex in recombinant systems. Conclusions: The 12-LOX product 12(S)-HETE stimulates GPR31-Gi–signaling pathways, which enhance thrombin-PAR4 platelet activation and arterial thrombosis in human platelets and mouse models. Suppression of this bioactive lipid pathway, as exemplified by a GPR31 pepducin antagonist, may provide beneficial protective effects against platelet aggregation and arterial thrombosis with minimal effect on hemostasis.

scholarly journals An Efficient Strategy to Estimate Thermodynamics and Kinetics of G Protein-Coupled Receptor Activation Using Metadynamics and Maximum Caliber

2018 ◽  
Author(s):  
Derya Meral ◽  
Davide Provasi ◽  
Marta Filizola
Keyword(s):  
G Protein ◽  
Adaptive Sampling ◽  
Receptor Activation ◽  
Conformational Space ◽  
Kinetic Properties ◽  
G Protein Coupled Receptor ◽  
Efficient Strategy ◽  
Gpcr Activation ◽  
Kinetic Rates ◽  
G Protein Coupled

ABSTRACTComputational strategies aimed at unveiling the thermodynamic and kinetic properties of G Protein-Coupled Receptor (GPCR) activation require extensive molecular dynamics simulations of the receptor embedded in an explicit lipid-water environment. A possible method for efficiently sampling the conformational space of such a complex system is metadynamics (MetaD) with path collective variables (CV). Here, we applied well-tempered MetaD with path CVs to one of the few GPCRs for which both inactive and fully active experimental structures are available, the μ-opioid receptor (MOR), and assessed the ability of this enhanced sampling method to estimate thermodynamic properties of receptor activation in line with those obtained by more computationally expensive adaptive sampling protocols. While n-body information theory (nBIT) analysis of these simulations confirmed that MetaD can efficiently characterize ligand-induced allosteric communication across the receptor, standard MetaD cannot be used directly to derive kinetic rates because transitions are accelerated by a bias potential. Applying the principle of Maximum Caliber (MaxCal) to the free-energy landscape of morphine-bound MOR reconstructed from MetaD, we obtained Markov State Models (MSMs) that yield kinetic rates of MOR activation in agreement with those obtained by adaptive sampling. Taken together, these results suggest that the MetaD-MaxCal combination creates an efficient strategy for estimating thermodynamic and kinetic properties of GPCR activation at an affordable computational cost.

scholarly journals The molecular basis of subtype selectivity of human kinin G-protein-coupled receptors

2018 ◽  
Vol 14 (3) ◽  
pp. 284-290 ◽  
Author(s):  
Lisa Joedicke ◽  
Jiafei Mao ◽  
Georg Kuenze ◽  
Christoph Reinhart ◽  
Tejaswi Kalavacherla ◽  
...  
Keyword(s):  
G Protein ◽  
Molecular Basis ◽  
G Protein Coupled Receptors ◽  
Subtype Selectivity ◽  
G Protein Coupled

scholarly journals Structure of the Mouse Sex Peptide Pheromone ESP1 Reveals a Molecular Basis for Specific Binding to the Class C G-protein-coupled Vomeronasal Receptor

2013 ◽  
Vol 288 (22) ◽  
pp. 16064-16072 ◽  
Author(s):  
Sosuke Yoshinaga ◽  
Toru Sato ◽  
Makoto Hirakane ◽  
Kaori Esaki ◽  
Takashi Hamaguchi ◽  
...  
Keyword(s):  
G Protein ◽  
Molecular Basis ◽  
Specific Binding ◽  
Sex Peptide ◽  
Vomeronasal Receptor ◽  
Peptide Pheromone ◽  
Class C ◽  
G Protein Coupled

scholarly journals Role of GRK6 in the Regulation of Platelet Activation through Selective G Protein-Coupled Receptor (GPCR) Desensitization

2020 ◽  
Vol 21 (11) ◽  
pp. 3932 ◽  
Author(s):  
Preeti Kumari Chaudhary ◽  
Sanggu Kim ◽  
Youngheun Jee ◽  
Seung-Hun Lee ◽  
Kyung-Mee Park ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
G Protein ◽  
Platelet Activation ◽  
Functional Role ◽  
Thrombus Formation ◽  
Receptor Activation ◽  
G Protein Coupled ◽  
Gpcr Desensitization ◽  
Stimulation Of

Platelet G protein-coupled receptors (GPCRs) regulate platelet function by mediating the response to various agonists, including adenosine diphosphate (ADP), thromboxane A2, and thrombin. Although GPCR kinases (GRKs) are considered to have the crucial roles in most GPCR functions, little is known regarding the regulation of GPCR signaling and mechanisms of GPCR desensitization by GRKs in platelets. In this study, we investigated the functional role of GRK6 and the molecular basis for regulation of specific GPCR desensitization by GRK6 in platelets. We used GRK6 knockout mice to evaluate the functional role of GRK6 in platelet activation. Platelet aggregation, dense- and α-granule secretion, and fibrinogen receptor activation induced by 2-MeSADP, U46619, thrombin, and AYPGKF were significantly potentiated in GRK6−/− platelets compared to the wild-type (WT) platelets. However, collagen-related peptide (CRP)-induced platelet aggregation and secretion were not affected in GRK6−/− platelets. Interestingly, platelet aggregation induced by co-stimulation of serotonin and epinephrine which activate Gq-coupled 5HT2A and Gz-coupled α2A adrenergic receptors, respectively, was not affected in GRK6−/− platelets, suggesting that GRK6 was involved in specific GPCR regulation. In addition, platelet aggregation in response to the second challenge of ADP and AYPGKF was restored in GRK6−/− platelets whereas re-stimulation of the agonist failed to induce aggregation in WT platelets, indicating that GRK6 contributed to P2Y1, P2Y12, and PAR4 receptor desensitization. Furthermore, 2-MeSADP-induced Akt phosphorylation and AYPGKF-induced Akt, extracellular signal-related kinase (ERK), and protein kinase Cδ (PKCδ) phosphorylation were significantly potentiated in GRK6−/− platelets. Finally, GRK6−/− mice exhibited an enhanced and stable thrombus formation after FeCl3 injury to the carotid artery and shorter tail bleeding times, indicating that GRK6−/− mice were more susceptible to thrombosis and hemostasis. We conclude that GRK6 plays an important role in regulating platelet functional responses and thrombus formation through selective GPCR desensitization.

Mode of action of P2Y12 antagonists as inhibitors of platelet function

2011 ◽  
Vol 105 (01) ◽  
pp. 96-106 ◽  
Author(s):  
Jackie Glenn ◽  
Ann White ◽  
Sue Fox ◽  
Hans van Giezen ◽  
Sven Nylander ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
G Protein ◽  
Platelet Function ◽  
Adenosine Diphosphate ◽  
P2y12 Receptor ◽  
Receptor Antagonists ◽  
Receptor Agonists ◽  
P2y12 Antagonists ◽  
Induced Aggregation ◽  
G Protein Coupled

SummaryP2Y12 receptor antagonists are antithrombotic agents that inhibit platelet function by blocking the effects of adenosine diphosphate (ADP) at P2Y12 receptors. However, some P2Y12 receptor antagonists may affect platelet function through additional mechanisms. It was the objective of this study to investigate the possibility that P2Y12 antagonists inhibit platelet function through interaction with G-protein-coupled receptors other than P2Y12 receptors. We compared the effects of cangrelor, ticagrelor and the prasugrel active metabolite on platelet aggregation and on phosphorylation of vasodilator-stimulated phosphoprotein (VASP). We compared their effects with those of selective IP, EP4 and A2A agonists, which act at Gs-coupled receptors. All three P2Y12 antagonists were strong inhibitors of ADP-induced platelet aggregation but only partial inhibitors of aggregation induced by thrombin receptor activating peptide (TRAP) or the thromboxane A2 mimetic U46619. Further, after removing ADP and its metabolites using apyrase and adenosine deaminase, the P2Y12 antagonists produced only minor additional inhibition of TRAP or U46619-induced aggregation. Conversely, the Gs-coupled receptor agonists always produced strong inhibition of aggregation irrespective of whether ADP was removed. Other experiments using selective receptor agonists and antagonists provided no evidence of any of the P2Y12 antagonists acting through PAR1, TP, IP, EP4, A2A or EP3 receptors. All three P2Y12 antagonists enhanced VASPphosphorylation to a small and equal extent but the effects were much smaller than those of the IP, EP4 and A2A agonists. The effects of cangrelor, ticagrelor and prasugrel on platelet function are mediated mainly through P2Y12 receptors and not through another G-protein-coupled receptor.

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