Regulation of P2X1 receptors by modulators of the cAMP effectors PKA and EPAC

P2X1 receptors are adenosine triphosphate (ATP)-gated cation channels that are functionally important for male fertility, bladder contraction, and platelet aggregation. The activity of P2X1 receptors is modulated by lipids and intracellular messengers such as cAMP, which can stimulate protein kinase A (PKA). Exchange protein activated by cAMP (EPAC) is another cAMP effector; however, its effect on P2X1 receptors has not yet been determined. Here, we demonstrate that P2X1 currents, recorded from human embryonic kidney (HEK) cells transiently transfected with P2X1 cDNA, were inhibited by the highly selective EPAC activator 007-AM. In contrast, EPAC activation enhanced P2X2 current amplitude. The PKA activator 6-MB-cAMP did not affect P2X1 currents, but inhibited P2X2 currents. The inhibitory effects of EPAC on P2X1 were prevented by triple mutation of residues 21 to 23 on the amino terminus of P2X1 subunits to the equivalent amino acids on P2X2 receptors. Double mutation of residues 21 and 22 and single mutation of residue 23 also protected P2X1 receptors from inhibition by EPAC activation. Finally, the inhibitory effects of EPAC on P2X1 were also prevented by NSC23766, an inhibitor of Rac1, a member of the Rho family of small GTPases. These data suggest that EPAC is an important regulator of P2X1 and P2X2 receptors.

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Regulators and Effectors of Small GTPases: Rho Family

10.1016/s0076-6879(06)x1068-6 ◽

2006 ◽

Keyword(s):

Small Gtpases ◽

Rho Family

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PseudoGTPase domains in p190RhoGAP proteins: a mini-review

Biochemical Society Transactions ◽

10.1042/bst20180481 ◽

2018 ◽

Vol 46 (6) ◽

pp. 1713-1720 ◽

Cited By ~ 6

Author(s):

Amy L. Stiegler ◽

Titus J. Boggon

Keyword(s):

Family Members ◽

Small Gtpases ◽

Small Gtpase ◽

Signaling Proteins ◽

New Family ◽

Gtpase Activating Proteins ◽

Rho Family ◽

Biochemical Analyses ◽

Catalytic Cleft

Pseudoenzymes generally lack detectable catalytic activity despite adopting the overall protein fold of their catalytically competent counterparts, indeed ‘pseudo’ family members seem to be incorporated in all enzyme classes. The small GTPase enzymes are important signaling proteins, and recent studies have identified many new family members with noncanonical residues within the catalytic cleft, termed pseudoGTPases. To illustrate recent discoveries in the field, we use the p190RhoGAP proteins as an example. p190RhoGAP proteins (ARHGAP5 and ARHGAP35) are the most abundant GTPase activating proteins for the Rho family of small GTPases. These are key regulators of Rho signaling in processes such as cell migration, adhesion and cytokinesis. Structural biology has complemented and guided biochemical analyses for these proteins and has allowed discovery of two cryptic pseudoGTPase domains, and the re-classification of a third, previously identified, GTPase-fold domain as a pseudoGTPase. The three domains within p190RhoGAP proteins illustrate the diversity of this rapidly expanding pseudoGTPase group.

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Morin Exerts Anti-Diabetic Effects in Human HepG2 Cells Via Down-Regulation of miR-29a

Experimental and Clinical Endocrinology & Diabetes ◽

10.1055/a-0650-4082 ◽

2018 ◽

Vol 127 (09) ◽

pp. 615-622 ◽

Cited By ~ 2

Author(s):

Toktam Razavi ◽

Shideh Montasser Kouhsari ◽

Khalil Abnous

Keyword(s):

Insulin Signaling ◽

High Glucose ◽

Hepg2 Cells ◽

Target Genes ◽

Expression Level ◽

Inhibitory Effects ◽

Down Regulation ◽

Insulin Mimetic ◽

Important Regulator ◽

First Time

Abstract Diabetes mellitus is a complex metabolic disease around the world that is characterized by hyperglycemia resulting from impaired insulin secretion, insulin action, or both. MicroRNA-29a is an important regulator of insulin signaling and gluconeogenesis pathways through IRS2, PI3K and PEPCK expressions which up regulates in Diabetes. Morin is a substantial bioflavonoid which has insulin mimetic effect, and interacting with nucleic acids and proteins. In this study HepG2 cells, were exposed to high glucose to induce diabetic condition. We have determined whether high glucose stimulation might promotes miR-29a expression level in HepG2 cells and subsequently evaluated the Morin treatment effects on this state. In HepG2 cells, high glucose increases miR-29a expression level and decreases its target genes, IRS2 and PI3K expression, and increases associated downstream gene in gluconeogenic pathway, PEPCK. Morin treatment down regulates miR-29a expression level and improves insulin signaling and glucose metabolism. To confirm the inhibitory effects of Morin on miR-29a, we have transfected cells with mimic and inhibitor-miR-29a. This study for the first time identifies that Morin improves diabetic condition through down regulation of the miR-29a level, and suggest that this new inhibitor of miR-29a may be a useful biomedicine to treat diabetes.

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Identification and characterization of a new isoform of small GTPase RhoE

Communications Biology ◽

10.1038/s42003-020-01295-4 ◽

2020 ◽

Vol 3 (1) ◽

Author(s):

Yuan Dai ◽

Weijia Luo ◽

Xiaojing Yue ◽

Wencai Ma ◽

Jing Wang ◽

...

Keyword(s):

Rho Gtpase ◽

Small Gtpases ◽

Small Gtpase ◽

Biological Functions ◽

Coding Region ◽

Alternative Translation ◽

Rho Family ◽

Binding Capability ◽

Identification And Characterization

Abstract The Rho family of GTPases consists of 20 members including RhoE. Here, we discover the existence of a short isoform of RhoE designated as RhoEα, the first Rho GTPase isoform generated from alternative translation. Translation of this new isoform is initiated from an alternative start site downstream of and in-frame with the coding region of the canonical RhoE. RhoEα exhibits a similar subcellular distribution while its protein stability is higher than RhoE. RhoEα contains binding capability to RhoE effectors ROCK1, p190RhoGAP and Syx. The distinct transcriptomes of cells with the expression of RhoE and RhoEα, respectively, are demonstrated. The data propose distinctive and overlapping biological functions of RhoEα compared to RhoE. In conclusion, this study reveals a new Rho GTPase isoform generated from alternative translation. The discovery provides a new scope of understanding the versatile functions of small GTPases and underlines the complexity and diverse roles of small GTPases.

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Double Mutations in Succinate Dehydrogenase Are Involved in SDHI Resistance in Corynespora cassiicola

Microorganisms ◽

10.3390/microorganisms10010132 ◽

2022 ◽

Vol 10 (1) ◽

pp. 132

Author(s):

Bingxue Sun ◽

Guangxue Zhu ◽

Xuewen Xie ◽

Ali Chai ◽

Lei Li ◽

...

Keyword(s):

Succinate Dehydrogenase ◽

Point Mutation ◽

Single Point ◽

Resistance Management ◽

Site Directed Mutagenesis ◽

Single Point Mutation ◽

Single Mutation ◽

Corynespora Cassiicola ◽

Double Mutation ◽

Mutation Analyses

With the further application of succinate dehydrogenase inhibitors (SDHI), the resistance caused by double mutations in target gene is gradually becoming a serious problem, leading to a decrease of control efficacy. It is important to assess the sensitivity and fitness of double mutations to SDHI in Corynespora cassiicola and analysis the evolution of double mutations. We confirmed, by site-directed mutagenesis, that all double mutations (B-I280V+D-D95E/D-G109V/D-H105R, B-H278R+D-D95E/D-G109V, B-H278Y+D-D95E/D-G109V) conferred resistance to all SDHI and exhibited the increased resistance to at least one fungicide than single point mutation. Analyses of fitness showed that all double mutations had lower fitness than the wild type; most of double mutations suffered more fitness penalties than the corresponding single mutants. We also further found that double mutations (B-I280V+D-D95E/D-G109V/D-H105R) containing low SDHI-resistant single point mutation (B-I280V) exhibited higher resistance to SDHI and low fitness penalty than double mutations (B-H278Y+D-D95E/D-G109V) containing high SDHI-resistant single mutations (B-H278Y). Therefore, we may infer that a single mutation conferring low resistance is more likely to evolve into a double mutation conferring higher resistance under the selective pressure of SDHI. Taken together, our results provide some important reference for resistance management.

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SmgGDS: An Emerging Master Regulator of Prenylation and Trafficking by Small GTPases in the Ras and Rho Families

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.685135 ◽

2021 ◽

Vol 8 ◽

Author(s):

Anthony C. Brandt ◽

Olivia J. Koehn ◽

Carol L. Williams

Keyword(s):

Splice Variants ◽

Therapeutic Strategies ◽

Small Gtpases ◽

Peptide Inhibitors ◽

Signaling Cascades ◽

Therapeutic Targeting ◽

Growing Body ◽

History Of ◽

Rho Family ◽

Mutant Forms

Newly synthesized small GTPases in the Ras and Rho families are prenylated by cytosolic prenyltransferases and then escorted by chaperones to membranes, the nucleus, and other sites where the GTPases participate in a variety of signaling cascades. Understanding how prenylation and trafficking are regulated will help define new therapeutic strategies for cancer and other disorders involving abnormal signaling by these small GTPases. A growing body of evidence indicates that splice variants of SmgGDS (gene name RAP1GDS1) are major regulators of the prenylation, post-prenylation processing, and trafficking of Ras and Rho family members. SmgGDS-607 binds pre-prenylated small GTPases, while SmgGDS-558 binds prenylated small GTPases. This review discusses the history of SmgGDS research and explains our current understanding of how SmgGDS splice variants regulate the prenylation and trafficking of small GTPases. We discuss recent evidence that mutant forms of RabL3 and Rab22a control the release of small GTPases from SmgGDS, and review the inhibitory actions of DiRas1, which competitively blocks the binding of other small GTPases to SmgGDS. We conclude with a discussion of current strategies for therapeutic targeting of SmgGDS in cancer involving splice-switching oligonucleotides and peptide inhibitors.

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Co-ordinated Ras and Rac activity shapes macropinocytic cups and enables phagocytosis of geometrically diverse bacteria

10.1101/763748 ◽

2019 ◽

Author(s):

Catherine M. Buckley ◽

Henderikus Pots ◽

Aurelie Gueho ◽

Ben A. Phillips ◽

Bernd Gilsbach ◽

...

Keyword(s):

Dictyostelium Discoideum ◽

Small Gtpases ◽

Small Gtpase ◽

Extracellular Material ◽

Multidomain Protein ◽

Complex Shapes ◽

Rho Family ◽

Phagocytic Cups ◽

Interior Domain ◽

Professional Phagocyte

AbstractEngulfment of extracellular material by phagocytosis or macropinocytosis depends on the ability of cells to generate specialised cup shaped protrusions. To effectively capture and internalise their targets, these cups are organised into a ring or ruffle of actin-driven protrusion encircling a non-protrusive interior domain. These functional domains depend on the combined activities of multiple Ras and Rho family small GTPases, but how their activities are integrated and differentially regulated over space and time is unknown. Here, we show that the amoeba Dictyostelium discoideum coordinates Ras and Rac activity using the multidomain protein RGBARG (RCC1, RhoGEF, BAR and RasGAP-containing protein). We find RGBARG uses a tripartite mechanism of Ras, Rac and phospholipid interactions to localise at the protruding edge and interface with the interior of both macropinocytic and phagocytic cups. There, RGBARG shapes the protrusion by driving Rac activation at the rim whilst suppressing expansion of the active Ras interior domain. Consequently, cells lacking RGBARG form enlarged, flat interior domains unable to generate large macropinosomes. During phagocytosis, we find that disruption of RGBARG causes a geometry-specific defect in engulfing rod-shaped bacteria and ellipsoidal beads. This demonstrates the importance of co-ordinating small GTPase activities during engulfment of more complex shapes and thus the full physiological range of microbes, and how this is achieved in a model professional phagocyte.

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