Relevance of Dopamine Signals Anchoring Dynamin-2 to the Plasma Membrane during Na+,K+-ATPase Endocytosis

Efendiev, Riad; Yudowski, Guillermo A.; Zwiller, Jean; Leibiger, Barbara; Katz, Adrian I.; Berggren, Per Olof; Pedemonte, Carlos H.; Leibiger, Ingo B.; Bertorello, Alejandro M.

doi:10.1074/jbc.m205173200

Cited by 40 publications

(38 citation statements)

References 49 publications

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“…Dynamin is a crucial factor in endocytosis that participates in membrane fission (Roux et al, 2006). Our results showing that dynamin K44A inhibits Na,K-ATPase endocytosis are concordant with a previous study reporting that in renal cells clathrin-dependent Na,K-ATPase endocytosis requires dynamin (Efendiev et al, 2002). Collectively, these results provide evidence that, during hypoxia, changes in Na,K-ATPase protein levels at the plasma membrane are mediated by a defined intracellular signaling mechanism and not as a result of generalized cell damage.…”

supporting

confidence: 82%

Role of the small GTPase RhoA in the hypoxia-induced decrease of plasma membrane Na,K-ATPase in A549 cells

Dada

Novoa

Lecuona

et al. 2007

Journal of Cell Science

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Hypoxia impairs alveolar fluid reabsorption by promoting Na,K-ATPase endocytosis, from the plasma membrane of alveolar epithelial cells. The present study was designed to determine whether hypoxia induces Na,K-ATPase endocytosis via reactive oxygen species (ROS)-mediated RhoA activation. In A549 cells, RhoA activation occurred within 15 minutes of cells exposure to hypoxia. This activation was inhibited in cells infected with adenovirus coding for gluthatione peroxidase (an H 2 O 2 scavenger), in mitochondria depleted ( 0 ) cells or cells expressing decreased levels of the Rieske iron-sulfur protein (inhibitor of mitochondrial complex III), which suggests a role for mitochondrial ROS. Moreover, exogenous H 2 O 2 treatment during normoxia mimicked the effects of hypoxia on RhoA, further supporting a role for ROS. Cells expressing dominant negative RhoA failed to endocytose the Na,KATPase during hypoxia or after H 2 O 2 treatment. Na,KATPase endocytosis was also prevented in cells treated with Y-27632, a Rho-associated kinase (ROCK) inhibitor, and in cells expressing dominant negative ROCK. In summary, we provide evidence that in human alveolar epithelial cells exposed to hypoxia, RhoA/ROCK activation is necessary for Na,K-ATPase endocytosis via a mechanism that requires mitochondrial ROS. Journal of Cell Science 2215 RhoA and Na,K-ATPase endocytosis al., 2006;Dada et al., 2003). In the present study, we report that under hypoxic conditions mitochondrial ROS activate RhoA, and that the RhoA/ROCK regulate Na,K-ATPase endocytosis from the plasma membrane into intracellular compartments. ResultsEffect of hypoxia on alveolar epithelial Na,K-ATPase We have previously described that exposing A549 cells to 1.5% O 2 for 60 minutes resulted in a decrease in plasma membrane Na,K-ATPase ␣ 1 expression by ~50%, while milder hypoxic conditions (3% O 2 ) induced similar results by 120 minutes (Dada et al., 2003). To characterize the specificity of the endocytic process we assessed the levels of plasma membrane transferrin receptor during hypoxia. The decrease in plasma membrane Na,K-ATPase protein levels appears to be specific since the levels of the transferrin receptor were unchanged as assessed by a biotinylation assay (Fig. 1A). It has been previously demonstrated that dynamin is a mediator of plasma membrane protein internalization (De Camilli et al., 1995). Here, we examined the ability of hypoxia to regulate Na,KATPase protein levels in cells transiently transfected with a plasmid coding for a GFP-tagged dominant negative dynamin-2 (DN-GFP-Dyn K44A). As depicted in Fig. 1B, we found hypoxia failed to induce a significant decrease in Na,K-ATPase protein expression at the plasma membrane in cells expressing the DN-dynamin, which suggests a role for dynamin in promoting Na,K-ATPase endocytosis.Actin disrupting agents prevent hypoxia-induced Na,KATPase endocytosis To determine whether actin organization was required for hypoxia-induced Na,K-ATPase endocytosis, A549 cells were pre-incubated with the actin-filament-disrupting ...

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supporting

confidence: 82%

Role of the small GTPase RhoA in the hypoxia-induced decrease of plasma membrane Na,K-ATPase in A549 cells

Dada

Novoa

Lecuona

et al. 2007

Journal of Cell Science

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“…For this, we used the technique of plasma membrane protein biotinylation, which has been validated in several of our previous publications (16,21,24). After treatment of the cells with either PMA or Ang II, the cells were rapidly transferred to a 4°C bath, the temperature at which the plasma membrane proteins were labeled with N-hydroxysulfosuccinimide-biotin.…”

Section: Resultsmentioning

confidence: 99%

“…After separation, the agarose beads were washed four times with IP buffer containing 0.2% Triton X-100 and 0.2% SDS and once with 50 mM Tris-HCl, pH 7.4, and finally resuspended in Laemmli sample buffer. Electrophoresis, Western blot analysis with ExtrAvidin peroxidase conjugate, and densitometric analysis were performed as described previously (16,21,24).…”

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confidence: 99%

G-protein-coupled Receptor-mediated Traffic of Na,K-ATPase to the Plasma Membrane Requires the Binding of Adaptor Protein 1 to a Tyr-255-based Sequence in the α-Subunit

Efendiev

Budu

Bertorello

et al. 2008

Journal of Biological Chemistry

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Motion of integral membrane proteins to the plasma membrane in response to G-protein-coupled receptor signals requires selective cargo recognition motifs that bind adaptor protein 1 and clathrin. Angiotensin II, through the activation of AT1 receptors, promotes the recruitment to the plasma membrane of Na,K-ATPase molecules from intracellular compartments. We present evidence to demonstrate that a tyrosinebased sequence (IVVY-255) present within the Na,K-ATPase ␣1-subunit is involved in the binding of adaptor protein 1. Mutation of Tyr-255 to a phenylalanine residue in the Na,KATPase ␣1-subunit greatly reduces the angiotensin II-dependent activation of Na,K-ATPase, recruitment of Na,K-ATPase molecules to the plasma membrane, and association of adaptor protein 1 with Na,K-ATPase ␣1-subunit molecules. To determine protein-protein interaction, we used fluorescence resonance energy transfer between fluorophores attached to the Na,K-ATPase ␣1-subunit and adaptor protein 1. Although angiotensin II activation of AT1 receptors induces a significant increase in the level of fluorescence resonance energy transfer between the two molecules, this effect was blunted in cells expressing the Tyr-255 mutant. Thus, results from different methods and techniques suggest that the Tyr-255-based sequence within the NKA ␣1-subunit is the site of adaptor protein 1 binding in response to the G-protein-coupled receptor signals produced by angiotensin II binding to AT1 receptors.

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“…Among those signals, binding (to a proline-rich motif within the Na ϩ ,K ϩ -ATPase ␣ 1 -subunit) and activation of PI 3-kinase activity are of utmost relevance for recruiting recognition molecules (adaptins) and clathrin formation (15,17), as well as recruiting dynamin for clathrin vesicle fission (33). Phosphorylation of the Na ϩ ,K ϩ -ATPase ␣ 1 -subunit at Ser-18 is critical for PI 3-kinase binding, but the molecular mechanisms of this interaction remain unknown.…”

Section: Resultsmentioning

confidence: 99%

The 14-3-3 Protein Translates the NA+,K+-ATPase α1-Subunit Phosphorylation Signal into Binding and Activation of Phosphoinositide 3-Kinase during Endocytosis

Efendiev

Chen²,

Krmar³

et al. 2005

Journal of Biological Chemistry

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Relevance of Dopamine Signals Anchoring Dynamin-2 to the Plasma Membrane during Na+,K+-ATPase Endocytosis

Cited by 40 publications

References 49 publications

Role of the small GTPase RhoA in the hypoxia-induced decrease of plasma membrane Na,K-ATPase in A549 cells

Role of the small GTPase RhoA in the hypoxia-induced decrease of plasma membrane Na,K-ATPase in A549 cells

G-protein-coupled Receptor-mediated Traffic of Na,K-ATPase to the Plasma Membrane Requires the Binding of Adaptor Protein 1 to a Tyr-255-based Sequence in the α-Subunit

The 14-3-3 Protein Translates the NA+,K+-ATPase α1-Subunit Phosphorylation Signal into Binding and Activation of Phosphoinositide 3-Kinase during Endocytosis

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