Discriminatory aptamer reveals serum response element transcription regulated by cytohesin-2

Theis, Mirko; Knorre, Alexander; Kellersch, Bettina; Moelleken, Jörg; Wieland, Felix; Kolanus, Waldemar; Famulok, Michael

doi:10.1073/pnas.0402901101

Cited by 60 publications

(44 citation statements)

References 33 publications

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“…The aptamer possesses high specificity toward GRK2 as compared to other kinases and thus represents a promising starting point for the functional interference and analysis of endogenous GRK2 function inside cells and living organisms. For example the aptamer can be either directly transfected into cells or expressed from plasmids that encode the aptamer sequence under certain promoter elements (Mayer et al 2001;Theis et al 2004;Famulok and Mayer 2005). Recent studies demonstrated that aptamers can be converted into small molecule inhibitors using so-called aptamer-displacement assays (Hafner et al 2006).…”

Section: Discussionmentioning

confidence: 99%

An RNA molecule that specifically inhibits G-protein-coupled receptor kinase 2 in vitro

Mayer

Wulffen²,

Brockmann³

et al. 2008

RNA

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G-protein-coupled receptors are desensitized by a two-step process. In a first step, G-protein-coupled receptor kinases (GRKs) phosphorylate agonist-activated receptors that subsequently bind to a second class of proteins, the arrestins. GRKs can be classified into three subfamilies, which have been implicated in various diseases. The physiological role(s) of GRKs have been difficult to study as selective inhibitors are not available. We have used SELEX (systematic evolution of ligands by exponential enrichment) to develop RNA aptamers that potently and selectively inhibit GRK2. This process has yielded an aptamer, C13, which bound to GRK2 with a high affinity and inhibited GRK2-catalyzed rhodopsin phosphorylation with an IC 50 of 4.1 nM. Phosphorylation of rhodopsin catalyzed by GRK5 was also inhibited, albeit with 20-fold lower potency (IC 50 of 79 nM). Furthermore, C13 reveals significant specificity, since almost no inhibitory activity was detectable testing it against a panel of 14 other kinases. The aptamer is two orders of magnitude more potent than the best GRK2 inhibitors described previously and shows high selectivity for the GRK family of protein kinases.

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Section: Discussionmentioning

confidence: 99%

An RNA molecule that specifically inhibits G-protein-coupled receptor kinase 2 in vitro

Mayer

Wulffen²,

Brockmann³

et al. 2008

RNA

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“…It is at present unknown how the interaction of ARNO/cytohesin-2 with the A 2A receptor supports sustained activation of MAP kinase. However, it is not without precedent; inhibition of ARNO/cytohesin-2 was recently found to down-regulate mitogen-activated protein kinase activation in HeLa cells (28).…”

Section: Discussionmentioning

confidence: 99%

Heterotrimeric G Protein-independent Signaling of a G Protein-coupled Receptor

Gsandtner

Charalambous

Stefan

et al. 2005

Journal of Biological Chemistry

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The A 2A adenosine receptor is a prototypical G s -coupled receptor, but it also signals, e.g. to mitogen-activated protein (MAP) kinase, via a pathway that is independent of heterotrimeric G proteins. Truncation of the carboxyl terminus affects the strength of the signal through these alternative pathways. In a yeast two-hybrid interaction hunt, we screened a human brain library for proteins that bound to the juxtamembrane portion of the carboxyl terminus of the A 2A receptor. This approach identified ARNO/cytohesin-2, a nucleotide exchange factor for the small (monomeric) G proteins of the Arf (ADP-ribosylation factor) family, as a potential interaction partner. We confirmed a direct interaction by mutual pull down (of fusion proteins expressed in bacteria) and by immunoprecipitation of the proteins expressed in mammalian cells. To circumvent the long term toxicity associated with overexpression of ARNO/cytohesin-2, we created stable cell lines that stably expressed the A 2A receptor and where ARNO/cytohesin-2 or the dominant negative version E156K-ARNO/ cytohesin-2 was inducible by mifepristone. Cyclic AMP accumulation induced by an A 2A -specific agonist was neither altered by ARNO/cytohesin-2 nor by the dominant negative version. This was also true for agonistinduced desensitization. In contrast, expression of dominant negative E156K-ARNO/cytohesin-2 and of dominant negative T27N-Arf6 abrogated the sustained phase of MAP kinase stimulation induced by the A 2A receptor. We therefore conclude that ARNO/cytohesin-2 is required to support the alternative, heterotrimeric G protein-independent, signaling pathway of A 2A receptor, which is stimulation of MAP kinase.Over the last decade, it has been increasingly accepted that G protein-coupled receptors also bind regulatory proteins other than G proteins, arrestins, and G protein-coupled kinases, which are involved in effector regulation and desensitization, respectively (1). These accessory proteins include components of signaling cascades and bind to the carboxyl termini of various G protein-coupled receptors (2). The A 2A adenosine receptor has an unusually long intracellular carboxyl-terminal tail, 122 amino acids in man, when compared, for instance, to 34 residues in the carboxyl terminus of the A 1 adenosine receptor. Circumstantial evidence suggests that accessory proteins bind to the carboxyl terminus of the A 2A receptor (3). A 2A receptors can activate mitogen-activated protein (MAP) 1 kinase by a G␣ s -independent signaling pathway; this can be seen both in endothelial cells where the receptor is endogenously expressed (4) and upon heterologous expression in HEK293 cells (5). Truncation of the carboxyl terminus does not impair the ability of the A 2A adenosine receptor to stimulate MAP kinase but blunts stimulation of cAMP accumulation. In addition, fulllength and truncated receptors differ in their constitutive (agonist-independent) activity; this difference is only seen in intact cells and is lost upon membrane preparation, suggesting the loss of one o...

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“…For example, an in vitro selected RNA aptamer was used to discriminate the roles of two highly homologous protein, cytohesin 1 and cytohesin 2, in regulating gene expression in response to serum stimulation (30). This aptamer binds to the N-terminal segment of cytohesin-2 specifically and can down-regulate expression of genes mediated though its serum response element and reduce mitogen-activated protein kinase activation in HeLa cells, suggesting a specific role of cytohesin-2 but not cytohesin-1 in serum-mediated transcriptional activation in nonimmune cells.…”

Section: Discussionmentioning

confidence: 99%

RNA Aptamers Targeting the Cell Death Inhibitor CED-9 Induce Cell Killing in Caenorhabditis elegans

Yang

Yan

Parish

et al. 2006

Journal of Biological Chemistry

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Bcl-2 family proteins include anti-and proapoptotic factors that play important roles in regulating apoptosis in diverse species. Identification of compounds that can modulate the activities of Bcl-2 family proteins will facilitate development of drugs for treatment of apoptosis-related human diseases. We used an in vitro selection method named systematic evolution of ligands by exponential enrichment (SELEX) to isolate RNA aptamers that bind the Caenorhabditis elegans Bcl-2 homolog CED-9 with high affinity and specificity and tested whether these aptamers modulate programmed cell death in C. elegans. Five CED-9 aptamers were isolated and classified into three groups based on their predicted secondary structures. Biochemical analyses indicated that two of these aptamers, R9-2 and R9-7, and EGL-1, an endogenous CED-9-binding proapoptotic protein, bound to distinct regions of CED-9. However, these two aptamers shared overlapping CED-9 binding sites with CED-4, another CED-9-binding proapoptotic factor. Importantly ectopic expression of these two aptamers in touch receptor neurons induced efficient killing of these neurons largely in a CED-3 caspase-dependent manner. These findings suggest that RNA aptamers can be used to modulate programmed cell death in vivo and can potentially be used to develop drugs to treat human diseases caused by abnormal apoptosis.Apoptosis is an essential cellular process that is critical for tissue homeostasis and animal development in metazoans. Abnormal inactivation of apoptosis can result in uncontrolled cell growth, leading to development of cancer and autoimmune disorders. By contrast, inappropriate activation of apoptosis can cause too much cell death, leading to neurodegenerative diseases and immunodeficiency (1, 2). Development of effective therapeutic methods that can correct or reverse inappropriate apoptosis is thus a critical issue in clinical medicine.Apoptosis is controlled and executed by an evolutionarily conserved cell death pathway (3, 4). At the center of this pathway is a family of conserved cell death regulators first defined by the human proto-oncogene bcl-2, which promotes cell survival and was identified by virtue of its overexpression in a number of B-cell lymphomas (5-8). Subsequently a family of Bcl-2-related proteins, characterized by the presence of at least one of four conserved Bcl-2 homology (BH) 3 domains, has been discovered and found in organisms as distantly related as Caenorhabditis elegans and humans (8). Members of this family can be either antiapoptotic or proapoptotic and can form heterodimers with selected family members to affect apoptosis. The mechanisms by which Bcl-2 family proteins regulate cell death appear to be quite complicated but likely involve modulation of the mitochondrial permeability and the release of crucial apoptogenic factors such as cytochrome c, apoptosisinducing factor, and endonuclease G, which promote activation of caspases, the cell death executors, and other cell death events such as chromosome fragmentation (8, 9). In ...

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Discriminatory aptamer reveals serum response element transcription regulated by cytohesin-2

Cited by 60 publications

References 33 publications

An RNA molecule that specifically inhibits G-protein-coupled receptor kinase 2 in vitro

An RNA molecule that specifically inhibits G-protein-coupled receptor kinase 2 in vitro

Heterotrimeric G Protein-independent Signaling of a G Protein-coupled Receptor

RNA Aptamers Targeting the Cell Death Inhibitor CED-9 Induce Cell Killing in Caenorhabditis elegans

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