Abstract 052: Alpha1a-Adrenoceptor Genetic Variant-Triggered Hyperproliferation in Cardiovascular Cells is Mediated by Novel Interacting Protein Spinophilin

Hypertension ◽  
2014 ◽  
Vol 64 (suppl_1) ◽  
Author(s):  
Ekaterina Babaeva ◽  
Irina Gradinaru ◽  
Debra A Schwinn ◽  
Anush Oganesian
Keyword(s):  
Intracellular Signaling ◽  
Genetic Variant ◽  
Cell Counting ◽  
Intracellular Loop ◽  
Interacting Protein ◽  
Naturally Occurring ◽  
Specific Proteins ◽  
Cardiovascular Cells ◽  
Novel Target ◽  
G Protein Coupled

Objectives: Alpha1-Adrenergic Receptors (α1ARs), members of the G protein-coupled receptor (GPCR) superfamily, play a major role in regulating cardiovascular (CV) function. Recently, we discovered that naturally occurring human α1aAR-G247R (247R) genetic variant, identified in the 3rd intracellular loop (3iL) of the receptor in highly hypertensive patient, triggers constitutive hyperproliferation in fibroblasts, cardiomyoblasts and smooth muscle cells (SMC). Specific proteins mediating this signaling remain unknown. Spinophilin (SPL) is a ubiquitously expressed protein controlling GPCR signaling by binding its 3rd intracellular loop (3iL). We hypothesize that SPL mediates α1aAR signaling and examined whether SPL directly interacts with α1aAR-WT (WT) and 247R. Methods: Cells were co-transfected with HA-α1ARs and full length Myc-SPL or its fragments to determine SPL domains responsible for binding to α1ARs. Cell lysates were co-immunoprecipitated with HA-tag antibodies. SPL levels were analyzed by Western blotting. SPL knockdown experiments were performed by transiently transfecting cells with SPL or scrambled siRNA, cell proliferation was determined by cell counting. Results: We demonstrate a distinct interaction of SPL with WT and 247R, WT interaction being the strongest. Different domains of SPL differentially interact with WT or 247R. SPL 1-480aa fragment interacts stronger with WT indicating interaction with 3iL, while SPL 480-817 fragment interacts stronger with 247R. Endogenous SPL levels are increased in 247R cells compared to WT or control cells. Inhibition of SPL expression in SMC with siRNA reduces 247R-triggered hyperproliferation to near normal levels and has no effect in WT cells. Conclusions: We identified SPL as a novel interacting protein involved in mediating intracellular signaling of α1aAR and its genetic variant in CV cells. Different domains of SPL differentially bind to WT or 247R indicating that SPL has a distinct role in regulation of their signaling pathways. Our findings also reveal that SPL is critical for 247R-triggered EGFR transactivation pathways. Thus SPL could be considered as a potentially novel target in α1aAR-mediated cardiovascular disorders.

Abstract 200: Novel Interaction of Spinophilin with Alpha1a-Adrenergic Receptor and its Genetic Variant in Cardiovascular Cells

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Ekaterina Babaeva ◽  
Irina Gradinaru ◽  
Debra A Schwinn ◽  
Anush Oganesian
Keyword(s):  
Genetic Variant ◽  
Direct Interaction ◽  
Cell Types ◽  
Intracellular Loop ◽  
Preliminary Results ◽  
Protein Levels ◽  
Cardiovascular Cells ◽  
Novel Target ◽  
G Protein Coupled

Activation of α 1 -Adrenergic Receptors (α 1 ARs), members of the G protein-coupled receptor (GPCR) superfamily, in response to stimulation of the sympathetic nervous system by catecholamines plays a major role in regulating cardiovascular (CV) function. Among three α 1 AR subtypes (α 1a ,α 1b ,α 1d ), α 1a ARs predominate in human resistant vessels and in heart. Recently, we discovered that naturally occurring human α 1a AR-G247R (247R) genetic variant, identified in the 3 rd intracellular loop (3iL) of the receptor in highly hypertensive patient, triggers constitutive hyperproliferation in CV cells (cardiomyoblasts, smooth muscle cells (SMC) and fibroblasts), which may lead to myocardial fibrosis and remodeling. In fibroblasts and cardiomyoblasts 247R triggered hyperproliferation is due to constitutive active coupling to Gq-independent βarrestin1/MMP/EGFR/ERK dependent pathway, while in SMC it is Gq- and MMP/EGFR/ERK-dependent. Here we report that α 1a AR-WT (WT) and 247R differentially interact with ubiquitous multi-domain scaffold protein spinophilin (SPL) that binds to 3iL of several GPCRs competing with arrestin thereby prolonging their signaling. The role of SPL in CV regulation is poorly studied. We hypothesized that SPL mediates constitutive signaling of 247R and examined whether SPL directly interacts with α 1a AR-WT or 247R. Our preliminary results reveal a direct interaction of SPL with WT and 247R: the SPL-WT interaction appears to be stronger as determined by co-immunoprecipitation. Different domains of SPL differentially interact with WT or 247R. SPL 1-480aa fragment interacts stronger with WT indicating interaction with 3iL, while SPL 480-817 fragment interacts stronger with 247R. Our preliminary results also demonstrate that 247R expression in all three cell types elevates endogenous SPL protein levels. Importantly, inhibition of SPL expression with specific siRNA reduces 247R-triggered hyperproliferation in SMC and cardiomyoblasts to near normal levels, while SPL knockdown has no effect in WT cells. Thus, we identified SPL as a novel protein involved in interacting and signaling of α 1a AR and its genetic variant in CV cells and that SPL could be considered as a potentially novel target in α 1a AR-mediated cardiovascular disorders.

Abstract P194: A Novel Signalosome in the Alpha1a-Adrenergic Receptor and its Genetic Variant Signaling Pathway

Hypertension ◽  
2015 ◽  
Vol 66 (suppl_1) ◽  
Author(s):  
Ekaterina Babaeva ◽  
Irina Gradinaru ◽  
Debra A Schwinn ◽  
Anush Oganesian
Keyword(s):  
Protein Interactions ◽  
Genetic Variant ◽  
Inhibitory Effect ◽  
Hek293 Cells ◽  
Scaffolding Protein ◽  
Receptor Protein ◽  
Intracellular Loop ◽  
Protein Levels ◽  
Cardiovascular Cells ◽  
Novel Target

Objectives : Human α 1 adrenergic receptors (AR), members of G protein-coupled receptor superfamily (GPCR) regulate blood pressure via smooth muscle cell (SMC) proliferation and vasoconstriction. We showed that α 1a AR-G247R (247R) genetic variant, identified in a hypertensive patient, constitutively couples to β-arrestin1/MMP/EGFR transactivation pathway leading to hyperproliferation in fibroblasts, cardiomyoblasts and SMCs. A scaffolding protein spinophilin (SPL) serves as a docking site for many regulatory proteins including RGS2 (negative R egulators of G -protein S ignaling). SPL also binds 3 rd intracellular loop of some GPCRs. Hypothesis: differential interaction of 247R or α 1a AR-WT (WT) with SPL/RGS2/β-arrestin signalosome mediates unique signaling of 247R and hyperproliferation in cardiovascular cells. Methods: Receptor-protein interactions were determined by co-immunoprecipitation from HEK293 cells expressing HA-α 1 ARs and full length Myc-SPL, its fragments or Flag-RGS2. Protein levels were analyzed by Western. SPL knockdown or RGS2 overexpression was performed using SPL-specific or scrambled siRNA or Flag-RGS2. Results: Our results reveal that in SMCs or cardiomyoblasts 247R but not WT upregulates endogenous SPL. SPL exhibits stronger (~2-fold) interaction with WT compared to 247R or α 1b , recruits RGS2 and inhibits receptor signaling. In contrast, weak SPL-247R interaction diminishes RGS2 inhibitory effect permitting hyperproliferation of 247R cells. SPL knockdown inhibits 247R-induced proliferation by allowing RGS2 directly bind to 247R as observed with RGS2 overexpression. Overexpression of SPL with RGS2 restores hyperproliferation suggesting that in 247R cells SPL binds RGS2 preventing RGS2-247R interaction. Conclusions: We present SPL/RGS2/β-arrestin as a novel signalosome responsible for α 1a AR-247R genetic variant triggered hyperproliferation in different cardiovascular cells. We reveal that SPL regulates α 1a AR signaling by differentially binding WT or 247R receptors and recruiting RGS2 protein to the receptor. These novel findings unravel critical roles of SPL and RGS2 in α 1 AR signaling, as well as identify SPL as a potential novel target for treatment of α 1 AR-mediated cardiovascular disorders.

Abstract 348: α1a-Adrenoceptor Genetic Variant-Triggered Hyperproliferation Is Mediated Through Aberrant Interaction With Spinophilin in Smooth Muscle Cells

2014 ◽  
Vol 34 (suppl_1) ◽  
Author(s):  
Ekaterina Babaeva ◽  
Irina Gradinaru ◽  
Debra A Schwinn ◽  
Anush Oganesian
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Genetic Variant ◽  
Muscle Cells ◽  
Intracellular Loop ◽  
Naturally Occurring ◽  
Pathway Expression ◽  
G Protein Coupled ◽  
Dependent Pathway ◽  
Unique Mechanism

Activation of α1-Adrenergic Receptors (α1ARs), members of the G protein-coupled receptor (GPCR) superfamily, in response to stimulation of the sympathetic nervous system by catecholamines plays a major role in regulating cardiovascular (CV) function and blood pressure (BP). Among three α1AR subtypes (α1a,α1b,α1d) expressed in vasculature α1aARs predominate in human vascular smooth muscle cells (SMC), particularly in resistant vessels most involved in BP control. Polymorphisms in the α1aAR gene are also related to hypertension (HTN), one of the major CV risk factors. Recently, we discovered a novel, unique mechanism of hypertension triggered by naturally occurring human α1aAR-G247R (247R) genetic variant, identified in the 3rd intracellular loop (3iL) of the receptor in hypertensive patient. In fibroblasts, 247R signals via constitutive active coupling to the βarrestin1/MMP7/EGFR pathway. Here we report that 247R expression in SMC triggers constitutive, Gq- and MMP/EGFR/ERK-dependent hyperproliferation. Agonist (phenylephrine) treatment of cells inhibits hyperproliferation and induces hypertrophy and α1aAR inverse agonist prazosin inhibits hyperproliferation and hypertrophy indicating Gq-dependent pathway. Expression of 247R in SMC also triggers upregulation of spinophilin (SPL), a ubiquitous multi-domain scaffold protein that binds to 3iL of several GPCRs and competes with βarrestins for 3iL binding. We hypothesized that SPL mediates constitutive signaling of 247R and examined whether SPL directly interacts with α1aAR-WT or 247R. Our preliminary results demonstrate a distinct interaction of α1aAR-WT and 247R with SPL, although the SPL-α1AR-WT interaction appears to be strongest. SPL 1-480aa fragment demonstrates stronger interaction with α1aAR-WT indicating that this domain is responsible for interaction with 3iL. Interestingly the SPL 480-817 fragment has stronger interaction with 247R. Thus, expression of the naturally occurring human α1aAR genetic variant in vascular cells activates distinct signaling pathways leading to aberrant hyperproliferation and hypertrophy, and may eventually result in hypertension and other CV diseases suggesting a possible novel mechanism underlying some forms of human hypertension.

scholarly journals The short third intracellular loop and cytoplasmic tail of bitter taste receptors provide functionally relevant GRK phosphorylation sites in TAS2R14

2020 ◽  
pp. jbc.RA120.016056
Author(s):  
Donghwa Kim ◽  
Maria Castaño ◽  
Lauren K Lujan ◽  
Jung A. Woo ◽  
Stephen B. Liggett
Keyword(s):  
Bitter Taste ◽  
Taste Receptors ◽  
Intracellular Loop ◽  
Early Endosomes ◽  
Bitter Taste Receptors ◽  
Novel Target ◽  
Intracellular Loops ◽  
G Protein Coupled ◽  
Gst Fusion Proteins

For most GPCRs, the third intracellular loops (IL3) and C-terminal tails (CT) are sites for GRK-mediated phosphorylation, leading to b-arrestin binding and agonist-specific desensitization. These regions of the G protein-coupled bitter taste receptors (TAS2Rs) are short compared to the superfamily, and their functional role is unclear. TAS2R14 expressed on human airway smooth muscle (HASM) cells relax the cell, suggesting a novel target for bronchodilators. To assess IL3 and CT in agonist-promoted TAS2R14 desensitization (tachyphylaxis), we generated GST-fusion proteins of both the WT sequence and Ala substituted for Ser/Thr in the IL3 and CT sequences. In vitro, activated GRK2 phosphorylated both WT IL3 and WT CT proteins but not Ala-substituted forms. Next, TAS2R14s with mutations in IL3 (IL-5A), CT (CT-5A) and in both regions (IL/CT-10A) were expressed in HEK-293T cells. IL/CT-10A and CT-5A failed to undergo desensitization of the [Ca2+]i response compared to WT, indicating functional desensitization by GRK-phosphorylation is at residues in the CT. Short-term desensitization of TAS2R14 was blocked by GRK2 knockdown in HASM cells. Receptor:b-arrestin binding was absent with IL/CT-10A and CT-5A, but was also reduced in IL-5A, indicating a role for IL3 phosphorylation in the b-arrestin interaction for this function. Agonist-promoted internalization of IL-5A and CT-5A receptors was impaired and these receptors failed to colocalize with early endosomes. These results show that agonist-promoted functional desensitization of TAS2R14 occurs by GRK phosphorylation of CT residues and b-arrestin binding. However, b-arrestin function in the internalization and trafficking of the receptor requires cooperative GRK phosphorylation of IL3 and CT residues.

A Dinucleotide Deletion in the CD24 Gene Is a Potential Risk Factor for Colorectal Cancer

2020 ◽  
Vol 86 (5) ◽  
pp. 480-485
Author(s):  
Lior Segev ◽  
Ilana Naboishchikov ◽  
Diana Kazanov ◽  
Ezra Bernstein ◽  
Meital Shaked ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Peripheral Blood ◽  
Intracellular Signaling ◽  
Genetic Variant ◽  
Colorectal Carcinogenesis ◽  
Polymorphism Analysis ◽  
Peripheral Blood Leukocytes ◽  
Clinical Value ◽  
Blood Leukocytes ◽  
Multistep Process

Background CD24 is a sialoglycoprotein anchored to the cell surface via glycosylphosphatidylinositol and is involved in intracellular signaling processes. It plays an important role in the early stages of the multistep process of colorectal carcinogenesis. Several single nucleotide polymorphisms in the CD24 gene are reported to exert a diverse effect on cancer risk. We aimed to elucidate whether CD24 TG/del genetic variants are associated with susceptibility to colorectal cancer (CRC). Methods The study included 179 subjects, 36 with CRC (prior to surgery) and 143 healthy control subjects. Deoxyribonucleic acid was purified from peripheral blood leukocytes, and by using restriction fragment length polymorphism analysis, the CD24 gene was genotyped for the specific genetic variant, TG deletion. Additionally, CD24 protein expression levels were determined by Western blotting analysis. Results The incidence of the TG/del was higher among the CRC patients compared with healthy controls, 14% and 10%, respectively ( P = .54). CD24 protein levels were significantly higher among CRC patients. There were no significant differences in CD24 expression between CRC patients at different stages of the disease or between patients who carry the mutation and those who did not. Conclusions CD24 genetic variant might be of clinical value for risk assessment as part of cancer prevention programs. Further study on larger populations is needed to validate the importance of this dinucleotide deletion in CRC development. Overexpression of CD24 protein occurs early along the multistep process of CRC carcinogenesis, and a simple blood sample based on CD24 expression on peripheral blood leukocytes can contribute to early diagnosis.

scholarly journals G Protein-Coupled Estrogen Receptor 1 (GPER) as a Novel Target for Schizophrenia Drug Treatment

2020 ◽  
Vol 1 (1) ◽  
Author(s):  
Danielle S Macêdo ◽  
Lia Lira Olivier Sanders ◽  
Raimunda das Candeias ◽  
Cyntia de Freitas Montenegro ◽  
David Freitas de Lucena ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
Estrogen Receptors ◽  
G Protein ◽  
Brain Inflammation ◽  
Antipsychotic Treatment ◽  
New Class ◽  
Estrogen Receptor Modulators ◽  
Novel Target ◽  
G Protein Coupled ◽  
Estrogen Receptor 1

Abstract The observation that a person’s sex influences the onset age of schizophrenia, the course of the disease, and antipsychotic treatment response suggests a possible role for estrogen receptors in the pathophysiology of schizophrenia. Indeed, treatment with adjunctive estrogen or selective estrogen receptor modulators (SERMs) are known to reduce schizophrenia symptoms. While estrogen receptors (ER)α and ERβ have been studied, a third and more recently discovered estrogen receptor, the G protein-coupled estrogen receptor 1 (GPER), has been largely neglected. GPER is a membrane receptor that regulates non-genomic estrogen functions, such as the modulation of emotion and inflammatory response. This review discusses the possible role of GPER in brain impairments seen in schizophrenia and in its potential as a therapeutic target. We conducted a comprehensive literature search in the PubMed/MEDLINE database, using the following search terms: “Schizophrenia,” “Psychosis,” “GPER1 protein,” “Estrogen receptors,” “SERMS,” “GPER1 agonism, “Behavioral symptoms,” “Brain Inflammation.” Studies involving GPER in schizophrenia, whether preclinical or human studies, have been scarce, but the results are encouraging. Agonism of the GPER receptor could prove to be an essential mechanism of action for a new class of “anti-schizophrenia” drugs.

scholarly journals Circ_0003266 sponges miR-503-5p to suppress colorectal cancer progression via regulating PDCD4 expression

BMC Cancer ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Caihong Wen ◽  
Xiaoqing Feng ◽  
Honggang Yuan ◽  
Yong Gong ◽  
Guangsheng Wang
Keyword(s):  
Colorectal Cancer ◽  
Cell Proliferation ◽  
Cell Lines ◽  
Cancer Progression ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Circular Rnas ◽  
Pdcd4 Expression ◽  
Novel Target

Abstract Background Circular RNAs (circRNAs) feature prominently in tumor progression. However, the biological function and molecular mechanism of circ_0003266 in colorectal cancer (CRC) require further investigation. Methods Circ_0003266 expression in 46 pairs CRC tissues / adjacent tissues, and CRC cell lines was detected by quantitative real-time polymerase chain reaction (qRT-PCR); after circ_0003266 was overexpressed or knocked down in CRC cells, cell proliferation, apoptosis, migration, and invasion were evaluated by the cell counting kit-8 (CCK-8), flow cytometry, and Transwell assays, respectively; the interaction among circ_0003266, miR-503-5p, and programmed cell death 4 (PDCD4) was confirmed using bioinformatics analysis and dual-luciferase reporter assay; PDCD4 protein expression in CRC cells was quantified using Western blot. Results Circ_0003266 was significantly lowly expressed in CRC tissues and cell lines. Circ_0003266 overexpression markedly repressed CRC cell proliferation, migration, and invasion, and accelerated the cell apoptosis, but its overexpression promoted the malignant phenotypes of CRC cells. PDCD4 was a direct target of miR-503-5p and circ_0003266 promoted PDCD4 expression by competitively sponging miR-503-5p. Conclusion Circ_0003266 suppresses the CRC progression via sponging miR-503-5p and regulating PDCD4 expressions, which suggests that circ_0003266 may serve as a novel target for the treatment of CRC.

LncRNA OIP5-AS1 accelerates ox-LDL-treated HUVECs injury by NF-κB pathway via miR-30c-5p

2021 ◽  
pp. 1-12
Author(s):  
Lei Zhang ◽  
Qiulai Li ◽  
Yanxia Chen ◽  
Qiao Zhu
Keyword(s):  
Oxidative Stress ◽  
Vascular Endothelial Cells ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Vital Role ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Sod Activity ◽  
Interacting Protein ◽  
Rna Immunoprecipitation

BACKGROUND: Oxidized low-density lipoprotein (ox-LDL) could induce endothelial injury and played a vital role in the progression and development of atherosclerosis. This study aimed to investigate the role of Opa-interacting protein 5 antisense RNA 1 (OIP5-AS1) in ox-LDL-induced human umbilical vascular endothelial cells (HUVECs) injury and the potential mechanisms. METHODS: Cell proliferation and apoptosis were evaluated by Cell Counting Kit-8 (CCK-8) assay and flow cytometry assay, respectively. The levels of lactate dehydrogenase (LDH), reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD) and nitric oxide (NO) were detected by corresponding detection kits, respectively. Quantitative real-time PCR (qRT-PCR) was conducted to measure the expression of OIP5-AS1 or microRNA-30c-5p (miR-30c-5p) in HUVECs. Binding between OIP5-AS1 and miR-30c-5p was predicted through bioinformatics analysis and confirmed by dual-luciferase reporter assay and RNA immunoprecipitation (RIP). Western blot was used to analyze p-IκB, IκB, p-p65 and p65 levels. RESULTS: In HUVECs, exposure to ox-LDL led to a decrease in cell viability and an increase in LDH release and apoptosis with concomitant enhancement of oxidative stress, as evidenced by increased ROS and MDA generation, as well as decreased SOD activity and NO levels, while OIP5-AS1 knockdown or miR-30c-5p upregulation could rescue these effects above. Mechanically, OIP5-AS1 functioned as a sponge of miR-30c-5p. OIP5-AS1-induced injury and apoptosis, oxidative stress and activation of NF-κB pathway were reversed by miR-30c-5p in ox-LDL-treated HUVECs. CONCLUSION: OIP5-AS1 contributed to ox-LDL-treated HUVECs injury by activation of NF-κB pathway via miR-30c-5p.

scholarly journals Peroxiredoxins as Potential Targets for Cardiovascular Disease

Antioxidants ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1244
Author(s):  
Se-Jin Jeong ◽  
Jong-Gil Park ◽  
Goo Taeg Oh
Keyword(s):  
Cardiovascular Disease ◽  
Intracellular Signaling ◽  
Toxic Substance ◽  
Regulatory Proteins ◽  
The Past ◽  
Glutathione Peroxidases ◽  
Intracellular Signals ◽  
Common Etiology ◽  
Cardiovascular Cells

Increased oxidative stress (OS) is considered a common etiology in the pathogenesis of cardiovascular disease (CVD). Therefore, the precise regulation of reactive oxygen species (ROS) in cardiovascular cells is essential to maintain normal physiological functions. Numerous regulators of cellular homeostasis are reportedly influenced by ROS. Hydrogen peroxide (H2O2), as an endogenous ROS in aerobic cells, is a toxic substance that can induce OS. However, many studies conducted over the past two decades have provided substantial evidence that H2O2 acts as a diffusible intracellular signaling messenger. Antioxidant enzymes, including superoxide dismutases, catalase, glutathione peroxidases, and peroxiredoxins (Prdxs), maintain the balance of ROS levels against augmentation of ROS production during the pathogenesis of CVD. Especially, Prdxs are regulatory sensors of transduced intracellular signals. The intracellular abundance of Prdxs that specifically react with H2O2 act as regulatory proteins. In this review, we focus on the role of Prdxs in the regulation of ROS-induced pathological changes in the development of CVD.

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