Ezrin-Radixin-Moesin-binding Phosphoprotein-50/Na+/H+ Exchanger Regulatory Factor (EBP50/NHERF) Blocks U50,488H-induced Down-regulation of the Human κ Opioid Receptor by Enhancing Its Recycling Rate

Li, Jianguo; Chen, Chongguang; Liu‐Chen, Lee‐Yuan

doi:10.1074/jbc.m200058200

Cited by 111 publications

(123 citation statements)

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“…EBP50 has been shown to stabilize GPCRs, including b2-adrenergic, PTH and k-opioid receptors, at the membrane by tethering these proteins to membrane cytoskeleton via ezrin, and therefore reducing receptor internalization. 8,9,39 This conclusion was based on an EBP50 mutant deleted of its ezrinbinding region, which had also lost membrane-tethering capacity. However, we were unable to identify an EBP50-deletion mutant that had this type of regulation.…”

Section: Discussionmentioning

confidence: 99%

“…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.…”

mentioning

confidence: 99%

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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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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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

Chen

Jou

2012

Cell Death Differ

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“…Although 13 of the tails in the library end in a type 1 PDZ recognition sequence (Table II), none of these tails bound EBP50. It has been suggested that EBP50 could be involved in the recycling of the KOP receptor, which does not contain a PDZ recognition sequence, as demonstrated by co-immunoprecipitation experiments (47). However, neither the KOP receptor tail nor any of the other tails in the library bound EBP50 (Fig.…”

Section: Tm Receptor Tails and Post-endocytic Adaptor Proteinsmentioning

confidence: 99%

A Library of 7TM Receptor C-terminal Tails

Heydorn

Søndergaard

Ersbøll

et al. 2004

Journal of Biological Chemistry

145

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Adaptor and scaffolding proteins determine the cellular targeting, the spatial, and thereby the functional association of G protein-coupled seven-transmembrane receptors with co-receptors, transducers, and downstream effectors and the adaptors determine post-signaling events such as receptor sequestration through interactions, mainly with the C-terminal intracellular tails of the receptors. A library of tails from 59 representative members of the super family of seven-transmembrane receptors was probed as glutathione S-transferase fusion proteins for interactions with four different adaptor proteins previously proposed to be involved in postendocytotic sorting of receptors. Of the two proteins suggested to target receptors for recycling to the cell membrane, which is the route believed to be taken by a majority of receptors, ERM (ezrin-radixin-moesin)-binding phosphoprotein 50 (EBP50) bound only a single receptor tail, i.e. the ␤ 2 -adrenergic receptor, whereas N-ethylmaleimide-sensitive factor bound 11 of the tailfusion proteins. Of the two proteins proposed to target receptors for lysosomal degradation, sorting nexin 1 (SNX1) bound 10 and the C-terminal domain of G proteincoupled receptor-associated sorting protein bound 23 of the 59 tail proteins. Surface plasmon resonance analysis of the binding kinetics of selected hits from the glutathione S-transferase pull-down experiments, i.e. the tails of the virally encoded receptor US28 and the ␦-opioid receptor, confirmed the expected nanomolar affinities for interaction with SNX1. Truncations of the NK 1 receptor revealed that an extended binding epitope is responsible for the interaction with both SNX1 and G protein-coupled receptor-associated sorting protein as well as with Nethylmaleimide-sensitive factor. It is concluded that the tail library provides useful information on the general importance of certain adaptor proteins, for example, in this case, ruling out EBP50 as being a broad spectrumrecycling adaptor.Interaction of receptors with adaptor and scaffolding proteins is important for their biogenesis, their cellular sorting and targeting to the cell membrane, and their function at the membrane in complex with transducer molecules and downstream effector molecules as well as the subsequent internalization and post-endocytotic sorting of the receptors (1, 2). These interactions among receptors, adaptors, and scaffolding proteins are highly regulated processes that can be controlled by phosphorylation events (3), expression of receptor activating, or inactivating variants of adaptor proteins (4), by competition among adaptor proteins, and by competition between adaptor proteins and effector molecules (5). For the large family of G protein-coupled seven-transmembrane segment receptors (7TM 1 receptors), this field is still in its infancy and only a rather sketchy picture has emerged of relative importance of specific adaptor and scaffolding proteins for the biogenesis, function, and desensitization of these receptors. Methods such as yeast two-hybrid screening...

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“…4B). Considering that the complex-glycosylated form stability reflects the overall characteristic of post-Golgi, cell surface, and endosomal CFTR pools, and NHERF may facilitate recycling of CFTR as observed for G-protein-coupled receptors (24,26,35,50), next we evaluated the effect of C-terminal truncation on the apical localization and channel function of CFTR.…”

Section: Cftr-nherf Interaction Is Dispensable For the Stability Of Tmentioning

confidence: 99%

“…The D1 and D2 domains bind with nanomolar affinity to the PDZbinding motifs of CFTR ( 1477 DTRL) and to the C terminus of other transport proteins, signaling molecules, and receptors (18,(22)(23)(24)(25)(26). The ERM-binding domain tethers the complex via ezrin to cytoskeletal elements in a phosphorylation-dependent manner and to the catalytic and regulatory subunit of PKA (19,(27)(28)(29).…”

mentioning

confidence: 99%

The Role of the C Terminus and Na+/H+ Exchanger Regulatory Factor in the Functional Expression of Cystic Fibrosis Transmembrane Conductance Regulator in Nonpolarized Cells and Epithelia

Benharouga

Sharma

et al. 2003

Journal of Biological Chemistry

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The conserved C-terminal peptide motif ( 1476 DTRL) of the cystic fibrosis transmembrane conductance regulator (CFTR) ensures high affinity binding to different PSD-95/Disc-large/zonula occludens-1 (PDZ) domaincontaining molecules, including the Na ؉ /H ؉ exchanger regulatory factor (NHERF)/ezrin-radixin-moesin-binding phosphoprotein of 50 kDa. The physiological relevance of NHERF binding to CFTR is not fully understood. Individuals with mutations resulting in premature termination of CFTR (S1455X or ⌬26 CFTR) have moderately elevated sweat Cl ؊ concentration, without an obvious lung and pancreatic phenotype, implying that the CFTR function is largely preserved. Surprisingly, when expressed heterologously, the ⌬26 mutation was reported to abrogate channel activity by destabilizing the protein at the apical domain and inducing its accumulation at the basolateral membrane

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Ezrin-Radixin-Moesin-binding Phosphoprotein-50/Na+/H+ Exchanger Regulatory Factor (EBP50/NHERF) Blocks U50,488H-induced Down-regulation of the Human κ Opioid Receptor by Enhancing Its Recycling Rate

Cited by 111 publications

References 44 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

A Library of 7TM Receptor C-terminal Tails

The Role of the C Terminus and Na+/H+ Exchanger Regulatory Factor in the Functional Expression of Cystic Fibrosis Transmembrane Conductance Regulator in Nonpolarized Cells and Epithelia

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