scholarly journals A KLK4 proteinase substrate capture approach to antagonize PAR1

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Eitan Rabinovitch ◽  
Koishiro Mihara ◽  
Amiram Sananes ◽  
Marianna Zaretsky ◽  
Michael Heyne ◽  
...  
Keyword(s):  
Tumor Metastasis ◽  
Computational Design ◽  
Saturation Mutagenesis ◽  
Tissue Kallikrein ◽  
Proteolytic Activation ◽  
Erk Activation ◽  
Tumor Invasiveness ◽  
Kallikrein 4 ◽  
Key Driver ◽  
G Protein Coupled

AbstractProteinase-activated receptor-1 (PAR1), triggered by thrombin and other serine proteinases such as tissue kallikrein-4 (KLK4), is a key driver of inflammation, tumor invasiveness and tumor metastasis. The PAR1 transmembrane G-protein-coupled receptor therefore represents an attractive target for therapeutic inhibitors. We thus used a computational design to develop a new PAR1 antagonist, namely, a catalytically inactive human KLK4 that acts as a proteinase substrate-capture reagent, preventing receptor cleavage (and hence activation) by binding to and occluding the extracellular R41-S42 canonical PAR1 proteolytic activation site. On the basis of in silico site-saturation mutagenesis, we then generated KLK4S207A,L185D, a first-of-a-kind ‘decoy’ PAR1 inhibitor, by mutating the S207A and L185D residues in wild-type KLK4, which strongly binds to PAR1. KLK4S207A,L185D markedly inhibited PAR1 cleavage, and PAR1-mediated MAPK/ERK activation as well as the migration and invasiveness of melanoma cells. This ‘substrate-capturing’ KLK4 variant, engineered to bind to PAR1, illustrates proof of principle for the utility of a KLK4 ‘proteinase substrate capture’ approach to regulate proteinase-mediated PAR1 signaling.

Crystal Structures of Human Tissue Kallikrein 4: Activity Modulation by a Specific Zinc Binding Site

2006 ◽  
Vol 362 (5) ◽  
pp. 1094-1107 ◽  
Author(s):  
Mekdes Debela ◽  
Viktor Magdolen ◽  
Valerie Grimminger ◽  
Christian Sommerhoff ◽  
Albrecht Messerschmidt ◽  
...  
Keyword(s):  
Binding Site ◽  
Crystal Structures ◽  
Human Tissue ◽  
Zinc Binding ◽  
Tissue Kallikrein ◽  
Zinc Binding Site ◽  
Kallikrein 4

scholarly journals NHERF2 Specifically Interacts with LPA2 Receptor and Defines the Specificity and Efficiency of Receptor-Mediated Phospholipase C-β3 Activation

2004 ◽  
Vol 24 (11) ◽  
pp. 5069-5079 ◽  
Author(s):  
Yong-Seok Oh ◽  
Nam Won Jo ◽  
Jung Woong Choi ◽  
Hyeon Soo Kim ◽  
Sang-Won Seo ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Phospholipase C ◽  
Pdz Domain ◽  
The Other ◽  
Binding Motif ◽  
Lpa Receptor ◽  
Erk Activation ◽  
Downstream Signaling ◽  
Receptor Isoforms ◽  
G Protein Coupled

ABSTRACT Lysophosphatidic acid (LPA) activates a family of cognate G protein-coupled receptors and is involved in various pathophysiological processes. However, it is not clearly understood how these LPA receptors are specifically coupled to their downstream signaling molecules. This study found that LPA2, but not the other LPA receptor isoforms, specifically interacts with Na+/H+ exchanger regulatory factor2 (NHERF2). In addition, the interaction between them requires the C-terminal PDZ domain-binding motif of LPA2 and the second PDZ domain of NHERF2. Moreover, the stable expression of NHERF2 potentiated LPA-induced phospholipase C-β (PLC-β) activation, which was markedly attenuated by either a mutation in the PDZ-binding motif of LPA2 or by the gene silencing of NHERF2. Using its second PDZ domain, NHERF2 was found to indirectly link LPA2 to PLC-β3 to form a complex, and the other PLC-β isozymes were not included in the protein complex. Consistently, LPA2-mediated PLC-β activation was specifically inhibited by the gene silencing of PLC-β3. In addition, NHERF2 increases LPA-induced ERK activation, which is followed by cyclooxygenase-2 induction via a PLC-dependent pathway. Overall, the results suggest that a ternary complex composed of LPA2, NHERF2, and PLC-β3 may play a key role in the LPA2-mediated PLC-β signaling pathway.

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 Seeing β-arrestin in action: The role of β-arrestins in Histamine 1 Receptor signaling

2019 ◽  
Author(s):  
A Pietraszewska-Bogiel ◽  
J Goedhart
Keyword(s):  
G Protein ◽  
Live Cell ◽  
Chemical Mediator ◽  
Intracellular Location ◽  
Erk Activation ◽  
Class A ◽  
Transient Interactions ◽  
Major Chemical ◽  
G Protein Coupled

ABSTRACTß-arrestins regulate G protein-coupled receptor functions by influencing their signaling activity and intracellular location. Histamine is a major chemical mediator of allergic reactions, and its action is mainly mediated by the Gq/11- and Gi-coupled H1R. Contrary to accumulating insights into G protein-mediated signaling downstream of H1R, very little is known about the function of ß-arrestins in H1R signaling. Here, we describe dynamic, live cell measurements of ß-arrestin recruitment upon H1R activation in HEK293TN cells. Our observations classify H1R as a class A receptor, undergoing transient interactions with ß-arrestin. To investigate the relative contributions of G proteins and ß-arrestins to H1R signaling, we use specific G protein inhibitors, as well as ß-arrestin overexpression and depletion, and quantify various signaling outcomes in a panel of dynamic, live cell biosensor assays. Overall, we link ß-arrestins to desensitization of H1R-mediated signaling and show that ERK activation downstream of endogenous (HeLa, HUVEC) or transiently expressed (HEK293TN) H1R is largely Gq-mediated.

scholarly journals ERK: A Key Player in the Pathophysiology of Cardiac Hypertrophy

2019 ◽  
Vol 20 (9) ◽  
pp. 2164 ◽  
Author(s):  
Simona Gallo ◽  
Annapia Vitacolonna ◽  
Alessandro Bonzano ◽  
Paolo Comoglio ◽  
Tiziana Crepaldi
Keyword(s):  
Cardiac Hypertrophy ◽  
Tyrosine Kinases ◽  
Pressure Overload ◽  
Compensatory Mechanism ◽  
Erk Activation ◽  
Cardiac Side Effects ◽  
Beneficial Effects ◽  
Concentric Hypertrophy ◽  
Extracellular Signal ◽  
G Protein Coupled

Cardiac hypertrophy is an adaptive and compensatory mechanism preserving cardiac output during detrimental stimuli. Nevertheless, long-term stimuli incite chronic hypertrophy and may lead to heart failure. In this review, we analyze the recent literature regarding the role of ERK (extracellular signal-regulated kinase) activity in cardiac hypertrophy. ERK signaling produces beneficial effects during the early phase of chronic pressure overload in response to G protein-coupled receptors (GPCRs) and integrin stimulation. These functions comprise (i) adaptive concentric hypertrophy and (ii) cell death prevention. On the other hand, ERK participates in maladaptive hypertrophy during hypertension and chemotherapy-mediated cardiac side effects. Specific ERK-associated scaffold proteins are implicated in either cardioprotective or detrimental hypertrophic functions. Interestingly, ERK phosphorylated at threonine 188 and activated ERK5 (the big MAPK 1) are associated with pathological forms of hypertrophy. Finally, we examine the connection between ERK activation and hypertrophy in (i) transgenic mice overexpressing constitutively activated RTKs (receptor tyrosine kinases), (ii) animal models with mutated sarcomeric proteins characteristic of inherited hypertrophic cardiomyopathies (HCMs), and (iii) mice reproducing syndromic genetic RASopathies. Overall, the scientific literature suggests that during cardiac hypertrophy, ERK could be a “good” player to be stimulated or a “bad” actor to be mitigated, depending on the pathophysiological context.

scholarly journals Diverse Effects of Lysophosphatidic Acid Receptors on Ovarian Cancer Signaling Pathways

2019 ◽  
Vol 2019 ◽  
pp. 1-10
Author(s):  
Hadil Onallah ◽  
Ben Davidson ◽  
Reuven Reich
Keyword(s):  
Ovarian Cancer ◽  
Growth Factor ◽  
Signaling Pathways ◽  
Cancer Progression ◽  
Lysophosphatidic Acid ◽  
Migration And Invasion ◽  
Erk Activation ◽  
Molecular Machinery ◽  
Opposing Effects ◽  
G Protein Coupled

Lysophosphatidic acid (LPA) is a bioactive phospholipid with mitogenic and growth factor-like activities affecting cell invasion, cancer progression, and resistance. It is produced mainly by autotaxin and acts on six G-protein-coupled receptors, LPAR1-6. LPA has recently been implicated as a growth factor present in ascites of ovarian cancer patients. However, mitogenic pathways stimulated by LPA via its receptors may involve any novel, thus far uncharacterized, signaling pathway(s). Here we show that three LPA receptors are involved in tumor progression by activation of both the AKT and ERK signaling pathways. CRISPR-edited LPAR2 and LPAR3 knockouts have opposing effects on ERK activation, whereas LPAR6 is involved in the activation of AKT, affecting cell migration and invasion. Our study identifies specific molecular machinery triggered by LPA and its receptors that modulates tumor cells and can serve as therapeutic target in this malignancy.

scholarly journals Lysophosphatidylcholine-induced Surface Redistribution Regulates Signaling of the Murine G Protein-coupled Receptor G2A

2005 ◽  
Vol 16 (5) ◽  
pp. 2234-2247 ◽  
Author(s):  
Li Wang ◽  
Caius G. Radu ◽  
Li V. Yang ◽  
Laurent A. Bentolila ◽  
Mireille Riedinger ◽  
...  
Keyword(s):  
G Protein ◽  
Fluorescent Protein ◽  
Spatial Dynamics ◽  
Surface Expression ◽  
Subcellular Fractionation ◽  
Membrane Receptors ◽  
G Protein Coupled Receptor ◽  
Erk Activation ◽  
Fractionation Analysis ◽  
G Protein Coupled

Intracellular trafficking and spatial dynamics of membrane receptors critically regulate receptor function. Using microscopic and subcellular fractionation analysis, we studied the localization of the murine G protein-coupled receptor G2A (muG2A). Evaluating green fluorescent protein-tagged, exogenously expressed as well as the endogenous muG2A, we observed that this receptor was spontaneously internalized and accumulated in endosomal compartments, whereas its surface expression was enhanced and stabilized by lysophosphatidylcholine (LPC) treatment. Monensin, a general inhibitor of recycling pathways, blocked LPC-regulated surface localization of muG2A as well as muG2A-dependent extracellular signal-regulated kinase (ERK) activation and cell migration induced by LPC treatment. Mutation of the conserved DRY motif (R→ A) enhanced the surface expression of muG2A, resulting in its resistance to monensin inhibition of ERK activation. Our data suggest that intracellular sequestration and surface expression regulated by LPC, rather than direct agonistic activity control the signaling responses of murine G2A toward LPC.

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