Browsing by Subject "Angiotensin II"

Now showing 1 - 5 of 5
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    Activation and targeting of extracellular signal-regulated kinases by beta-arrestin scaffolds.
    (Proc Natl Acad Sci U S A, 2001-02-27) Luttrell, LM; Roudabush, FL; Choy, EW; Miller, WE; Field, ME; Pierce, KL; Lefkowitz, RJ
    Using both confocal immunofluorescence microscopy and biochemical approaches, we have examined the role of beta-arrestins in the activation and targeting of extracellular signal-regulated kinase 2 (ERK2) following stimulation of angiotensin II type 1a receptors (AT1aR). In HEK-293 cells expressing hemagglutinin-tagged AT1aR, angiotensin stimulation triggered beta-arrestin-2 binding to the receptor and internalization of AT1aR-beta-arrestin complexes. Using red fluorescent protein-tagged ERK2 to track the subcellular distribution of ERK2, we found that angiotensin treatment caused the redistribution of activated ERK2 into endosomal vesicles that also contained AT1aR-beta-arrestin complexes. This targeting of ERK2 reflects the formation of multiprotein complexes containing AT1aR, beta-arrestin-2, and the component kinases of the ERK cascade, cRaf-1, MEK1, and ERK2. Myc-tagged cRaf-1, MEK1, and green fluorescent protein-tagged ERK2 coprecipitated with Flag-tagged beta-arrestin-2 from transfected COS-7 cells. Coprecipitation of cRaf-1 with beta-arrestin-2 was independent of MEK1 and ERK2, whereas the coprecipitation of MEK1 and ERK2 with beta-arrestin-2 was significantly enhanced in the presence of overexpressed cRaf-1, suggesting that binding of cRaf-1 to beta-arrestin facilitates the assembly of a cRaf-1, MEK1, ERK2 complex. The phosphorylation of ERK2 in beta-arrestin complexes was markedly enhanced by coexpression of cRaf-1, and this effect is blocked by expression of a catalytically inactive dominant inhibitory mutant of MEK1. Stimulation with angiotensin increased the binding of both cRaf-1 and ERK2 to beta-arrestin-2, and the association of beta-arrestin-2, cRaf-1, and ERK2 with AT1aR. These data suggest that beta-arrestins function both as scaffolds to enhance cRaf-1 and MEK-dependent activation of ERK2, and as targeting proteins that direct activated ERK to specific subcellular locations.
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    Effects of Proximal Tubule Angiotensin II Signaling on Energy Metabolism in the Kidney
    (2017-12-12) Jimenez Contreras, Fabian
    Chronic kidney disease (CKD) affects over 26 million adults in the United States, thus it is imperative that we deduce more about the pathogenesis of the disease. CKD is generally multi-factorial, and loss of renal function can result from a number of diseases and pathologic processes. For example, propagation of kidney injury and renal fibrosis can result from abnormal regulation of energy metabolism in kidney cells. In renal proximal tubule epithelial cells, a key segment of the nephron, fatty acids are a major fuel source. As the proximal tubule is responsible for the bulk of sodium reabsorption by the kidney, maintaining adequate energy balance is crucial to this function; therefore, alterations in fatty acid oxidation in the renal proximal tubule may lead to renal dysfunction. Our hypothesis is that angiotensin II (Ang II) signaling, a major effector of the powerful renin-angiotensin system (RAS), alters fatty acid oxidation and this becomes exaggerated in states of renal injury such as hypertension and diabetes where the RAS can be dysregulated. Therefore, we sought to explore the metabolic changes linked to Ang II signaling in the renal proximal tubule. Increased levels of Ang II have previously been shown to induce renal fibrosis and hypertension. For our studies, we used a novel mouse line, one lacking AT1a receptors in renal proximal tubule cells (PTKO mice) and expected that the lack of AT1a receptors helps to maintain normal fatty acid oxidation in disease states. To model pathology which might stress the renal proximal tubule cells, we induced two diseases: hypertension, by infusing Ang II via osmotic mini pumps and diabetes, by employing a genetic model of type 1 diabetes, the Akita model. Our major outcome was the assessment of gene expression of several key metabolic pathways, using a quantitative PCR analysis of samples from mouse renal cortex, which is rich in proximal tubules. We aimed to measure genetic biomarkers in the fatty acid oxidation pathway, glucose oxidation pathway, markers of renal injury and fibrosis. These studies demonstrate how two clinically-relevant diseases influence metabolism in the kidney and how leveraging the RAS may lead to solutions against this disruption, and potentially alter CKD progression.
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    Independent beta-arrestin 2 and G protein-mediated pathways for angiotensin II activation of extracellular signal-regulated kinases 1 and 2.
    (Proc Natl Acad Sci U S A, 2003-09-16) Wei, Huijun; Ahn, Seungkirl; Shenoy, Sudha K; Karnik, Sadashiva S; Hunyady, László; Luttrell, Louis M; Lefkowitz, Robert J
    Stimulation of a mutant angiotensin type 1A receptor (DRY/AAY) with angiotensin II (Ang II) or of a wild-type receptor with an Ang II analog ([sarcosine1,Ile4,Ile8]Ang II) fails to activate classical heterotrimeric G protein signaling but does lead to recruitment of beta-arrestin 2-GFP and activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) (maximum stimulation approximately 50% of wild type). This G protein-independent activation of mitogen-activated protein kinase is abolished by depletion of cellular beta-arrestin 2 but is unaffected by the PKC inhibitor Ro-31-8425. In parallel, stimulation of the wild-type angiotensin type 1A receptor with Ang II robustly stimulates ERK1/2 activation with approximately 60% of the response blocked by the PKC inhibitor (G protein dependent) and the rest of the response blocked by depletion of cellular beta-arrestin 2 by small interfering RNA (beta-arrestin dependent). These findings imply the existence of independent G protein- and beta-arrestin 2-mediated pathways leading to ERK1/2 activation and the existence of distinct "active" conformations of a seven-membrane-spanning receptor coupled to each.
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    Monoclonal antibodies reveal receptor specificity among G-protein-coupled receptor kinases.
    (Proc Natl Acad Sci U S A, 1996-07-23) Oppermann, M; Diversé-Pierluissi, M; Drazner, MH; Dyer, SL; Freedman, NJ; Peppel, KC; Lefkowitz, RJ
    Guanine nucleotide-binding regulatory protein (G protein)-coupled receptor kinases (GRKs) constitute a family of serine/threonine kinases that play a major role in the agonist-induced phosphorylation and desensitization of G-protein-coupled receptors. Herein we describe the generation of monoclonal antibodies (mAbs) that specifically react with GRK2 and GRK3 or with GRK4, GRK5, and GRK6. They are used in several different receptor systems to identify the kinases that are responsible for receptor phosphorylation and desensitization. The ability of these reagents to inhibit GRK- mediated receptor phosphorylation is demonstrated in permeabilized 293 cells that overexpress individual GRKs and the type 1A angiotensin II receptor. We also use this approach to identify the endogenous GRKs that are responsible for the agonist-induced phosphorylation of epitope-tagged beta2- adrenergic receptors (beta2ARs) overexpressed in rabbit ventricular myocytes that are infected with a recombinant adenovirus. In these myocytes, anti-GRK2/3 mAbs inhibit isoproterenol-induced receptor phosphorylation by 77%, while GRK4-6-specific mAbs have no effect. Consistent with the operation of a betaAR kinase-mediated mechanism, GRK2 is identified by immunoblot analysis as well as in a functional assay as the predominant GRK expressed in these cells. Microinjection of GRK2/3-specific mAbs into chicken sensory neurons, which have been shown to express a GRK3-like protein, abolishes desensitization of the alpha2AR-mediated calcium current inhibition. The intracellular inhibition of endogenous GRKs by mAbs represents a novel approach to the study of receptor specificities among GRKs that should be widely applicable to many G-protein-coupled receptors.
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    Podoplanin neutralization improves cardiac remodeling and function after acute myocardial infarction.
    (JCI insight, 2019-07) Cimini, Maria; Garikipati, Venkata Naga Srikanth; de Lucia, Claudio; Cheng, Zhongjian; Wang, Chunlin; Truongcao, May M; Lucchese, Anna Maria; Roy, Rajika; Benedict, Cindy; Goukassian, David A; Koch, Walter J; Kishore, Raj
    Podoplanin, a small mucine-type transmembrane glycoprotein, has been recently shown to be expressed by lymphangiogenic, fibrogenic and mesenchymal progenitor cells in the acutely and chronically infarcted myocardium. Podoplanin binds to CLEC-2, a C-type lectin-like receptor 2 highly expressed by CD11bhigh cells following inflammatory stimuli. Why podoplanin expression appears only after organ injury is currently unknown. Here, we characterize the role of podoplanin in different stages of myocardial repair after infarction and propose a podoplanin-mediated mechanism in the resolution of post-MI inflammatory response and cardiac repair. Neutralization of podoplanin led to significant improvements in the left ventricular functions and scar composition in animals treated with podoplanin neutralizing antibody. The inhibition of the interaction between podoplanin and CLEC-2 expressing immune cells in the heart enhances the cardiac performance, regeneration and angiogenesis post MI. Our data indicates that modulating the interaction between podoplanin positive cells with the immune cells after myocardial infarction positively affects immune cell recruitment and may represent a novel therapeutic target to augment post-MI cardiac repair, regeneration and function.