Туристический кластер Псковской области

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A Disintegrin and Metalloenzyme (ADAM) 17 Activation Is Regulated by α5β1 Integrin in Kidney Mesangial Cells

The disintegrin and metalloenzyme ADAM17 participates in numerous inflammatory and proliferative diseases, and its pathophysiological role was implicated in kidney fibrosis, polycystic kidney disease and other chronic kidney diseases. At present, we have little understanding how the enzyme activity is regulated. In this study we wanted to characterize the role of α5β1 integrin in ADAM17 activity regulation during G protein-coupled receptor (GPCR) stimulation.We showed previously that the profibrotic GPCR agonist serotonin (5-HT) induced kidney mesangial cell proliferation through ADAM17 activation and heparin-binding epidermal growth factor (HB-EGF) shedding. In the present studies we observed that in unstimulated mesangial cell lysates α5β1 integrin co-precipitated with ADAM17 and that 5-HT treatment of the cells induced dissociation of α5β1 integrin from ADAM17. Using fluorescence immunostaining and in situ proximity ligation assay, we identified the perinuclear region as the localization of the ADAM17/α5β1 integrin interaction. In cell-free assays, we showed that purified α5β1 integrin and β1 integrin dose-dependently bound to and inhibited activity of recombinant ADAM17. We provided evidence that the conformation of the integrin determines its ADAM17-binding ability. To study the effect of β1 integrin on ADAM17 sheddase activity, we employed alkaline phosphatase-tagged HB-EGF. Overexpression of β1 integrin lead to complete inhibition of 5-HT-induced HB-EGF shedding and silencing β1 integrin by siRNA significantly increased mesangial cells ADAM17 responsiveness to 5-HT.Our data show for the first time that β1 integrin has an important physiological role in ADAM17 activity regulation. We suggest that regulating α5β1 integrin binding to ADAM17 could be an attractive therapeutic target in chronic kidney diseases

Multiple Mechanisms for E2F Binding Inhibition by Phosphorylation of the Retinoblastoma Protein C-Terminal Domain

The retinoblastoma proteinC-terminal domain (RbC) is necessary for the tumor suppressor protein's activities in growth suppression and E2F transcription factor inhibition. Cyclin-dependent kinase phosphorylation of RbC contributes to Rb inactivation and weakens the Rb-E2F inhibitory complex. Here we demonstratetwo mechanisms for how RbC phosphorylation inhibits E2F binding. We find that phosphorylation of S788 and S795 weakens the direct association between the N-terminal portion of RbC (RbC(N)) and the marked-box domains of E2F and its heterodimerization partner DP. Phosphorylation of these sites and S807/S811 also induces an intramolecular association between RbC and the pocket domain,which overlaps with the site of E2F transactivation domain binding. Areduction in E2F binding affinity occurs with S788/S795 phosphorylation that is additive with the effects of phosphorylation at other sites, and we propose a structural mechanism that explains this additivity. We find that different Rb phosphorylation events have distinct effects on activating E2F family members, which suggests a novel mechanism for how Rb may differentially regulate E2F activities

Utilizing the Luminex Magnetic Bead-Based Suspension Array for Rapid Multiplexed Phosphoprotein Quantification.

The study of protein phosphorylation is critical for the advancement of our understanding of cellular responses to external and internal stimuli. Phosphorylation, the addition of phosphate groups, most often occurs on serine, threonine, or tyrosine residues due to the action of protein kinases. This structural change causes the protein to become activated (or deactivated) and enables it in turn to initiate the phosphorylation of other proteins in a cascade, eventually causing cell-wide changes such as apoptosis, cell differentiation, and growth (among others). Cellular phosphoprotein pathway dysregulation by mutation or chromosomal instability can often give the cell a selective advantage and lead to cancer. Obviously the understanding of these systems is of huge importance to the field of oncology.This chapter aims to provide a "how to" manual for one such technology, the 96-well plate-based xMAP® platform from Luminex. The system utilizes antibody-bound free-floating magnetic spheres which can easily be removed from suspension via magnetization. There are 100 unique bead sets (moving up to 500 bead sets for the most recent system) identified by the ratio of two dyes coating the microsphere. Each bead set is conjugated to a specific antibody which allows targeted protein extraction from low-concentration lysate solution. Biotinylated secondary antibodies/streptavidin-R-phycoerythrin (SAPE) complexes provide the quantification mechanism for the phosphoprotein of interest

Heterodimerization of serotonin receptors 5-HT1A and 5-HT7 differentially regulates receptor signalling and trafficking

Serotonin receptors 5-HT1A and 5-HT7 are highly coexpressed in brain regions implicated in depression. However, their functional interaction has not been established. In the present study we show that 5-HT1A and 5-HT7 receptors form heterodimers both in vitro and in vivo. Foerster resonance energy transfer-based assays revealed that, in addition to heterodimers, homodimers composed either of 5-HT1A or 5-HT7 receptors together with monomers coexist in cells. The highest affinity for complex formation was obtained for the 5-HT7–5-HT7 homodimers, followed by the 5-HT7–5-HT1A heterodimers and 5-HT1A–5-HT1A homodimers. Functionally, heterodimerization decreases 5-HT1A-receptor-mediated activation of Gi protein without affecting 5-HT7-receptor-mediated signalling. Moreover, heterodimerization markedly decreases the ability of the 5-HT1A receptor to activate G-protein-gated inwardly rectifying potassium channels in a heterologous system. The inhibitory effect on such channels was also preserved in hippocampal neurons, demonstrating a physiological relevance of heteromerization in vivo. In addition, heterodimerization is crucially involved in initiation of the serotonin-mediated 5-HT1A receptor internalization and also enhances the ability of the 5-HT1A receptor to activate the mitogen-activated protein kinases. Finally, we found that production of 5-HT7 receptors in the hippocampus continuously decreases during postnatal development, indicating that the relative concentration of 5-HT1A–5-HT7 heterodimers and, consequently, their functional importance undergoes pronounced developmental changes

The bradykinin B2 receptor induces multiple cellular responses leading to the proliferation of human renal carcinoma cell lines

Inga I Kramarenko1, Thomas A Morinelli1,2, Marlene A Bunni1,2, John R Raymond Sr3, Maria N Garnovskaya11Department of Medicine (Nephrology Division), Medical University of South Carolina, Charleston, SC, USA; 2Medical and Research Services of the Ralph H Johnson Veterans Affairs Medical Center, Charleston, SC, USA; 3Medical College of Wisconsin, Milwaukee, WI, USABackground: The vasoactive peptide bradykinin (BK) acts as a potent growth factor for normal kidney cells, but there have been few studies on the role of BK in renal cell carcinomas.Purpose: In this study, we tested the hypothesis that BK also acts as a mitogen in kidney carcinomas, and explored the effects of BK in human renal carcinoma A498 cells.Methods: The presence of mRNAs for BK B1 and BK B2 receptors in A498 cells was demonstrated by reverse transcription–polymerase chain reaction. To study BK signaling pathways, we employed fluorescent measurements of intracellular Ca2+, measured changes in extracellular pH as a reflection of Na+/H+ exchange (NHE) with a Cytosensor microphysiometer, and assessed extracellular signal-regulated kinase (ERK) activation by Western blotting.Results: Exposure to 100 nM of BK resulted in the rapid elevation of intracellular Ca2+, caused a ≥30% increase in NHE activity, and a ≥300% increase in ERK phosphorylation. All BK signals were blocked by HOE140, a BK B2 receptor antagonist, but not by a B1 receptor antagonist. Inhibitor studies suggest that BK-induced ERK activation requires phospholipase C and protein kinase C activities, and is Ca2+/calmodulin-dependent. The amiloride analog 5-(N-methyl-N-isobutyl)-amiloride (MIA) blocked short-term NHE activation and inhibited ERK phosphorylation, suggesting that NHE is critical for ERK activation by BK. BK induced an approximately 40% increase in the proliferation of A498 cells as assessed by bromodeoxyuridine uptake. This effect was blocked by the ERK inhibitor PD98059, and was dependent on NHE activity.Conclusion: We conclude that BK exerts mitogenic effects in A498 cells via the BK B2 receptor activation of growth-associated NHE and ERK.Keywords: A498 cells, G protein-coupled receptors, signal transduction, Na+/H+ exchange, extracellular signal-regulated protein kinas