The β2-adrenergic receptor interacts with the Na+/H+-exchanger regulatory factor to control Na+/H+ exchange

Hall, Randy A.; Premont, Richard T.; Chow, Chung‐Wai; Blitzer, Jeremy T.; Pitcher, Julie A.; Claing, Audrey; Stoffel, Robert H.; Barak, Larry S.; Shenolikar, Shirish; Weinman, Edward J.; Grinstein, Sergio; Lefkowitz, Robert J.

doi:10.1038/33458

Cited by 544 publications

(448 citation statements)

References 25 publications

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“…2,3 EBP50 binds proteins that contain a PDZ-binding motif including ion exchangers, 4,5 ion channels, 6 G protein-coupled receptors, [7][8][9] growth factor tyrosine kinase receptors 10,11 and PTEN. 10 EBP50 participates in the modulation of ion transport, organization of apical microvilli, cancer development, and the trafficking and stabilization of membrane proteins.…”

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confidence: 99%

Phosphorylation of EBP50 negatively regulates β-PIX-dependent Rac1 activity in anoikis

Chen

Jou

2012

Cell Death Differ

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We demonstrated a protein kinase C (PKC)-dependent phosphorylation of canine ezrin/radixin/moesin (ERM)-binding phosphoprotein 50 (EBP50) at serine 347/348 by site-directed mutagenesis and a phospho-specific antibody. Cell fractionation and confocal imaging revealed the relocation of EBP50 from the plasma membrane to cytosol that accompanied this phosphorylation event. Increased phosphorylation at these serine residues led to the dissociation of EBP50 from ezrin and b-PIX, which are two upstream regulators of Rac1 activation. Cells overexpressing an EBP50 mutant, mimicking serine 347/348 phosphorylation, became refractory to hepatocyte growth factor-induced cell spreading and scattering, which is normally mediated by Rac1 activation. Detachment of cells from the substratum also elicited an increase in EBP50 phosphorylation, apparently due to counteracting activities of PKC and protein phosphastase 2A, which resulted in decreased Rac1 activation and induction of anoikis. Cells overexpressing an EBP50 mutant defective in serine 347/348 phosphorylation did not undergo apoptosis in suspension culture. These studies reveal a signaling cascade in which different phosphorylation states and subcellular localization of EBP50 regulate Rac1 function. Scaffold proteins have many modular domains that mediate protein-protein interactions involved in orchestrating different signaling cascades. As a result, scaffold proteins are thought to act as stable structural proteins that facilitate many cell signaling pathways. However, this traditional role of scaffold proteins has been challenged, 1 and the dynamic role of scaffold as both an activator and suppressor of cellular signaling remains poorly characterized. Furthermore, the factors that modulate such a bifunctional role of scaffold proteins remain largely unknown.Ezrin/radixin/moesin (ERM)-binding phosphoprotein 50 (EBP50) is a scaffold protein with two tandem PDZ domains and a C-terminal ERM-binding domain. 2,3 EBP50 binds proteins that contain a PDZ-binding motif including ion exchangers, 4,5 ion channels, 6 G protein-coupled receptors, 7-9 growth factor tyrosine kinase receptors 10,11 and PTEN. 10 EBP50 participates in the modulation of ion transport, organization of apical microvilli, cancer development, and the trafficking and stabilization of membrane proteins. 9,12-14 Understanding the mechanisms that regulate the function of EBP50 is, therefore, a critical problem.Ras family proteins are locked in a GDP-bound, inactivated state by binding GDI in the GTP-rich cellular environment. 15,16 Dissociation of small GTPases from GDI is required, but is not sufficient for their activation. GEFs drive the exchange of GTP for GDP, leading to the activation of small GTPases. 17 EBP50 can activate Rho-family small GTPases by recruiting ezrin, which is thought to cause the dissociation of GDI from the small GTPase, and b-PIX, a Rac1 GEF that contains a PDZ-binding motif. 18,19 EBP50 is a serine/threonine-phosphorylated protein. 3 The phosphorylation state of EBP50 regulates the acc...

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confidence: 99%

Phosphorylation of EBP50 negatively regulates β-PIX-dependent Rac1 activity in anoikis

Chen

Jou

2012

Cell Death Differ

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“…In Vitro Binding Assay. Far Western in vitro binding assays were used to assess binding of Kir2.1 C-terminal tails to PSD95 PDZ1,2 (24). Briefly, GST-fused proteins (1 µg) were separated by SDS-PAGE, transferred to nitrocellulose, and then stained with Ponceau-S (Sigma) to visualize transferred proteins.…”

Section: Methodsmentioning

confidence: 99%

NMR Studies of Interactions between C-Terminal Tail of Kir2.1 Channel and PDZ1,2 Domains of PSD95

et al. 2007

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Control of surface expression of inwardly rectifying potassium (Kir) channels is important for regulating membrane excitability. Kir2 channels have been shown to interact directly with PDZ-containing proteins in the postsynaptic density (PSD). These scaffold proteins, such as PSD95, bind to Kir2.1 channels via a PDZ-binding motif (T/S-x-Φ) in the C-terminal tail (SEI428). By utilizing a multidimensional solution NMR approach, we show that the previously unresolved structure of Kir2.1 tail (residues 372−428) is highly flexible. Using in vitro binding assays, we determined that shortening the flexible tail of Kir2.1 preceding the C-terminal region (residues 414−428) does not significantly disrupt PDZ binding. We also investigated which amino acids in the Kir2.1 tail associated with PSD95 PDZ1,2 by NMR spectroscopy, revealing that a stretch of 12 C-terminal amino acids is involved in interaction with both PDZ domains (residues 417−428). Deletion of the 11 amino acids preceding the C-terminal tail, Δ414−424, completely disrupts binding to PSD95 PDZ1,2. Therefore, the molecular interfaces formed between PDZ domains and Kir2.1 tail involve regions outside the previously identified binding motif (SEI428) and may be important for additional channel-specific interactions with associating PDZ-containing proteins.

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“…NHERF contains two tandem PDZ domains (PDZDs) at its NH 2 -terminus that have been shown to interact with a variety of proteins (Hall et al, 1998;Wang et al, 1998;Mohler et al, 1999;Weinman et al, 2000), and a MERLIN-binding motif at its COOHterminus. Wild-type PDZDs of NHERF were shown to bind to GST-IDB but not GST itself, confirming the physical binding of the two molecules ( Figure 1c).…”

Section: Confirmation Of Nherf and Syk Interactionmentioning

confidence: 99%

NHERF (Na+/H+ Exchanger Regulatory Factor) gene mutations in human breast cancer

et al. 2004

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Yeast two-hybrid screening was used to explore novel proteins that interact with a breast tumor or metastasis suppressor, SYK (spleen tyrosine kinase). The screening yielded NHERF (Na + /H + exchanger regulatory factor, also known as NHERF1 or EBP-50) that binds to the interdomain B of SYK. NHERF is an estrogen-responsive gene that encodes an inhibitory factor for epithelial Na + /H + exchanger isoform 3 (NHE3). We found intragenic mutation of the NHERF gene accompanied by loss of heterzygosity (LOH) in B3% (3/85) of breast cancer cell lines and primary breast tumors. Mutations occurred at the conserved PDZ domains at NHERF NH 2 -terminus that bound to SYK, or at its COOH-terminus motif that binds to MERLIN, the product of Neurofibromatosis 2 (NF2) tumor suppressor gene. NHERF tumorigenic mutations decreased or abolished its interaction with SYK or MERLIN, suggesting a pathway link among these three molecules that may play a critical role in mammary neoplastic progression. Primary breast tumors with LOH at the NHERF locus had clinical presentations of higher aggressiveness, indicating that deregulated NHERF signaling may be associated with disease progression. Moreover, the LOH was inversely correlated with SYK promoter methylation, suggesting that NHERF and SYK may transduce a common suppressive signal. Taken together, the results indicated NHERF to be a candidate tumor suppressor gene in human breast carcinoma that may be interconnected to the SYK and MERLIN suppressors.

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The β2-adrenergic receptor interacts with the Na+/H+-exchanger regulatory factor to control Na+/H+ exchange

Cited by 544 publications

References 25 publications

Phosphorylation of EBP50 negatively regulates β-PIX-dependent Rac1 activity in anoikis

Phosphorylation of EBP50 negatively regulates β-PIX-dependent Rac1 activity in anoikis

NMR Studies of Interactions between C-Terminal Tail of Kir2.1 Channel and PDZ1,2 Domains of PSD95

NHERF (Na+/H+ Exchanger Regulatory Factor) gene mutations in human breast cancer

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