Identification of two distinct structural motifs that, when added to the C-terminal tail of the rat LH receptor, redirect the internalized hormone-receptor complex from a degradation to a recycling pathway.

We show that most of the internalized rat LH receptor is routed to a lysosomal degradation pathway whereas a substantial portion of the human LH receptor is routed to a recycling pathway. Chimeras of these two receptors identified a linear amino acid sequence (GTALL) present near the C terminus of the human LH receptor that, when grafted onto the rat LH receptor, redirects most of the rat LH receptor to a recycling pathway. Removal of the GTALL sequence from the human LH receptor failed to affect its routing, however. The GTALL sequence shows homology with the C-terminal tetrapeptide (DSLL) of the beta2-adrenergic receptor, a motif that has been reported to mediate the recycling of the internalized beta2-adrenergic receptor by binding to ezrin-radixin-moesin-binding phosphoprotein-50. Addition of the DSLL tetrapeptide to the C terminus of the rat LH receptor also redirects most of the internalized rat LH receptor to a recycling pathway but, like the recycling of the human LH receptor, this rerouting is not mediated by ezrin-radixin-moesin-binding phosphoprotein-50. We conclude that most of the internalized rat LH receptor is degraded because its C-terminal tail lacks motifs that promote recycling and that two distinct, but homologous, motifs (DSLL at the C terminus or GTALL near the C terminus) can reroute the internalized rat LH receptor to a recycling pathway that is independent of ezrin-radixin-moesin-binding phosphoprotein-50.

Identification of EPI64, a TBC/rabGAP domain-containing microvillar protein that binds to the first PDZ domain of EBP50 and E3KARP.

The cortical scaffolding proteins EBP50 (ERM-binding phosphoprotein-50) and E3KARP (NHE3 kinase A regulatory protein) contain two PDZ (PSD-95/DlgA/ZO-1-like) domains followed by a COOH-terminal sequence that binds to active ERM family members. Using affinity chromatography, we identified polypeptides from placental microvilli that bind the PDZ domains of EBP50. Among these are 64- and/or 65-kD differentially phosphorylated polypeptides that bind preferentially to the first PDZ domain of EBP50, as well as to E3KARP, and that we call EPI64 (EBP50-PDZ interactor of 64 kD). The gene for human EPI64 lies on chromosome 22 where nine exons specify a protein of 508 residues that contains a Tre/Bub2/Cdc16 (TBC)/rab GTPase-activating protein (GAP) domain. EPI64 terminates in DTYL, which is necessary for binding to the PDZ domains of EBP50, as a mutant ending in DTYLA no longer interacts. EPI64 colocalizes with EBP50 and ezrin in syncytiotrophoblast and cultured cell microvilli, and this localization in cultured cells is abolished by introduction of the DTYLA mutation. In addition to EPI64, immobilized EBP50 PDZ domains retain several polypeptides from placental microvilli, including an isoform of nadrin, a rhoGAP domain-containing protein implicated in regulating vesicular transport. Nadrin binds EBP50 directly, probably through its COOH-terminal STAL sequence. Thus, EBP50 appears to bind membrane proteins as well as factors potentially involved in regulating membrane traffic.

Hierarchy of merlin and ezrin N- and C-terminal domain interactions in homo- and heterotypic associations and their relationship to binding of scaffolding proteins EBP50 and E3KARP.

The neurofibromatosis 2 tumor suppressor gene product merlin has strong sequence identity to the ezrin-radixin-moesin (ERM) family over its approximately 300-residue N-terminal domain. ERM proteins are membrane cytoskeletal linkers that are negatively regulated by an intramolecular association between domains known as NH(2)- and COOH-ERM association domains (N- and C-ERMADs) that mask sites for binding membrane-associated proteins, such as EBP50 and E3KARP, and F-actin. Here we show that merlin has self-association regions analogous to the N- and C-ERMADs. Moreover, the N-/C-ERMAD interaction in merlin is relatively weak and dynamic, and this property is reflected by the ability of full-length recombinant merlin to form homo-oligomers. Remarkably, the merlin C-ERMAD has a higher affinity for the N-ERMAD of ezrin than the N-ERMAD of merlin. Both the ezrin and merlin N-ERMAD bind EBP50. This interaction with the ezrin N-ERMAD can be inhibited by the presence of the ezrin C-ERMAD, whereas interaction with the merlin N-ERMAD is not inhibited by either C-ERMAD. E3KARP binds tightly to the ezrin N-ERMAD but has little affinity for the merlin N-ERMAD. The implications of these associations and the hierarchies of binding for the function and regulation of merlin and ERM proteins are discussed.

ERM-Merlin and EBP50 protein families in plasma membrane organization and function.

The ezrin-radixin-moesin (ERM) family of proteins have emerged as key regulatory molecules in linking F-actin to specific membrane proteins, especially in cell surface structures. Merlin, the product of the NF2 tumor suppressor gene, has sequence similarity to ERM proteins and binds to some of the same membrane proteins, but lacks a C-terminal F-actin binding site. In this review we discuss how ERM proteins and merlin are negatively regulated by an intramolecular association between their N- and C-terminal domains. Activation of at least ERM proteins can be accomplished by C-terminal phosphorylation in the presence of PIP2. We also discuss membrane proteins to which ERM and merlin bind, including those making an indirect linkage through the PDZ-containing adaptor molecules EBP50 and E3KARP. Finally, the function of these proteins in cortical structure, endocytic traffic, signal transduction, and growth control is discussed.

Structure of the ERM protein moesin reveals the FERM domain fold masked by an extended actin binding tail domain.

The ezrin-radixin-moesin (ERM) protein family link actin filaments of cell surface structures to the plasma membrane, using a C-terminal F-actin binding segment and an N-terminal FERM domain, a common membrane binding module. ERM proteins are regulated by an intramolecular association of the FERM and C-terminal tail domains that masks their binding sites. The crystal structure of a dormant moesin FERM/tail complex reveals that the FERM domain has three compact lobes including an integrated PTB/PH/ EVH1 fold, with the C-terminal segment bound as an extended peptide masking a large surface of the FERM domain. This extended binding mode suggests a novel mechanism for how different signals could produce varying levels of activation. Sequence conservation suggests a similar regulation of the tumor suppressor merlin.

A kinase-regulated PDZ-domain interaction controls endocytic sorting of the beta2-adrenergic receptor.

A fundamental question in cell biology is how membrane proteins are sorted in the endocytic pathway. The sorting of internalized beta2-adrenergic receptors between recycling endosomes and lysosomes is responsible for opposite effects on signal transduction and is regulated by physiological stimuli. Here we describe a mechanism that controls this sorting operation, which is mediated by a family of conserved protein-interaction modules called PDZ domains. The phosphoprotein EBP50 (for ezrinradixin-moesin(ERM)-binding phosphoprotein-50) binds to the cytoplasmic tail of the beta2-adrenergic receptor through a PDZ domain and to the cortical actin cytoskeleton through an ERM-binding domain. Disrupting the interaction of EBP50 with either domain or depolymerization of the actin cytoskeleton itself causes missorting of endocytosed beta2-adrenergic receptors but does not affect the recycling of transferrin receptors. A serine residue at position 411 in the tail of the beta2-adrenergic receptor is a substrate for phosphorylation by GRK-5 (for G-protein-coupled-receptor kinase-5) and is required for interaction with EBP50 and for proper recycling of the receptor. Our results identify a new role for PDZ-domain-mediated protein interactions and for the actin cytoskeleton in endocytic sorting, and suggest a mechanism by which GRK-mediated phosphorylation could regulate membrane trafficking of G-protein-coupled receptors after endocytosis.

The carboxyl-terminal region of EBP50 binds to a site in the amino-terminal domain of ezrin that is masked in the dormant molecule.

EBP50 (ezrin-radixin-moesin-binding phosphoprotein 50) was recently identified by affinity chromatography on the immobilized NH2-terminal domain of ezrin. Here we map and characterize the regions in EBP50 and ezrin necessary for this association. Using blot overlays and in solution binding assays, the COOH-terminal 30 residues of EBP50 were found to be sufficient for an association with residues 1-286 of ezrin. EBP50 did not bind to full-length (1-585) ezrin, indicating that the EBP50 binding site is masked in the full-length molecule. Ezrin contains two complementary self-association domains known as N- and C-ERMADs (ezrin-radixin-moesin-association domains), encompassing residues 1-296 and 479-585, respectively. An ezrin 1-583 construct lacking the two terminal residues necessary for this association was found to have an unmasked EBP50 binding site. Moreover, binding of EBP50 and the C-ERMAD to ezrin residues 1-296 was found to be mutually exclusive, with the C-ERMAD having a higher affinity. These results suggest that in full-length ezrin, the binding site for EBP50 is masked through an intramolecular N/C-ERMAD association. Based on these and additional results, we propose a model whereby dormant ezrin can be activated to bind EBP50 on its NH2-terminal end and F-actin on its COOH-terminal end. Since EBP50 is proposed to bind membrane proteins through its PDZ domains, this provides a molecular description of the regulated linkage of microfilaments to membranes in cell surface microvilli.

An apical PDZ protein anchors the cystic fibrosis transmembrane conductance regulator to the cytoskeleton.

The function of the cystic fibrosis transmembrane conductance regulator (CFTR) as a Cl- channel in the apical membrane of epithelial cells is extensively documented. However, less is known about the molecular determinants of CFTR residence in the apical membrane, basal regulation of its Cl- channel activity, and its reported effects on the function of other transporters. These aspects of CFTR function likely require specific interactions between CFTR and unknown proteins in the apical compartment of epithelial cells. Here we report that CFTR interacts with the recently discovered protein, EBP50 (ERM-binding phosphoprotein 50). EBP50 is concentrated at the apical membrane in human airway epithelial cells, in vivo, and CFTR and EBP50 associate in in vitro binding assays. The CFTR-EBP50 interaction requires the COOH-terminal DTRL sequence of CFTR and utilizes either PDZ1 or PDZ2 of EBP50, although binding to PDZ1 is of greater affinity. Through formation of a complex, the interaction between CFTR and EBP50 may influence the stability and/or regulation of CFTR Cl- channel function in the cell membrane and provides a potential mechanism through which CFTR can affect the activity of other apical membrane proteins.

C-terminal threonine phosphorylation activates ERM proteins to link the cell's cortical lipid bilayer to the cytoskeleton.

The plasma membrane consists of a lipid bilayer with integral membrane proteins stabilized by regulated linkages to the cortical actin cytoskeleton. The regulation is necessary for cells to change shape ormigrate. The ERM (ezrin-radixin-moesin) proteins are believed to provide such links, with the N-terminal halves associating with integral membrane proteins, either directly or indirectly through adapter molecules like EBP50 (ERM binding phosphoprotein, 50 kDa), and their C-terminal halves associating with F-actin. However, isolated ERM proteins largely exist in a dormant state by virtue of an intramolecular interaction between amino- and carboxyl-terminal domains, thereby masking membrane and cytoskeletal association sites. C-terminal threonine phosphorylation of a fragment of radixin has been found to destroy its ability to bind the amino-terminal domain without affecting the C-terminal F-actin binding site. Here we show that C-terminal phosphorylation of full-length, dormant ezrin and moesin by protein kinase C-theta simultaneously unmasks both the F-actin and EBP50 binding sites. Increased phosphorylation of moesin in cells correlated with increased association of moesin with the cortical actin cytoskeleton. These results show that activation of ERM proteins can be accomplished by phosphorylation of a single C-terminal threonine residue.

Identification of EBP50: A PDZ-containing phosphoprotein that associates with members of the ezrin-radixin-moesin family.

Members of the ezrin-radixin-moesin (ERM) family of membrane-cytoskeletal linking proteins have NH2- and COOH-terminal domains that associate with the plasma membrane and the actin cytoskeleton, respectively. To search for ERM binding partners potentially involved in membrane association, tissue lysates were subjected to affinity chromatography on the immobilized NH2-terminal domains of ezrin and moesin, which comprise the ezrin-radixin-moesin-association domain (N-ERMAD). A collection of polypeptides at 50-53 kD from human placenta and at 58-59 kD from bovine brain bound directly to both N-ERMADs. The 50-53-kD placental proteins migrated as a major 50-kD species after phosphatase treatment, indicating that the heterogeneity is due to different phosphorylation states. We refer to these polypeptides as ERM-binding phosphoprotein 50 (EBP50). Sequence analysis of human EBP50 was used to identify an approximately 2-kb human cDNA that encodes a 357-residue polypeptide. Recombinant EBP50 binds tightly to the N-ERMADs of ezrin and moesin. Peptide sequences from the brain candidate indicated that it is closely related to EBP50. EBP50 has two PSD-95/DlgA/ZO-1-like (PDZ) domains and is most likely a homologue of rabbit protein cofactor, which is involved in the protein kinase A regulation of the renal brush border Na+/H+ exchanger. EBP50 is widely distributed in tissues, and is particularly enriched in those containing polarized epithelia. Immunofluorescence microscopy of cultured cells and tissues revealed that EBP50 colocalizes with actin and ezrin in the apical microvilli of epithelial cells, and immunoelectron microscopy demonstrated that it is specifically associated with the microvilli of the placental syncytiotrophoblast. Moreover, EBP50 and ezrin can be coimmunoprecipitated as a complex from isolated human placental microvilli. These findings show that EBP50 is a physiologically relevant ezrin binding protein. Since PDZ domains are known to mediate associations with integral membrane proteins, one mode of membrane attachment of ezrin is likely to be mediated through EBP50.

Ezrin: a protein requiring conformational activation to link microfilaments to the plasma membrane in the assembly of cell surface structures.

The cortical cytoskeleton of eucaryotic cells provides structural support to the plasma membrane and also contributes to dynamic processes such as endocytosis, exocytosis, and transmembrane signaling pathways. The ERM (ezrin-radixin-moesin) family of proteins, of which ezrin is the best studied member, play structural and regulatory roles in the assembly and stabilization of specialized plasma membrane domains. Ezrin and related molecules are concentrated in surface projections such as microvilli and membrane ruffles where they link the microfilaments to the membrane. The present knowledge about ezrin is discussed from an historical perspective. Both biochemical and cell biological studies have revealed that ezrin can exist in a dormant conformation that requires activation to expose otherwise masked association sites. Current results indicate that activated ezrin monomers or head-to-tail oligomers associate directly with F-actin through a domain in its C terminus, and with the membrane through its N-terminal domain. The association of ezrin with transmembrane proteins can be direct, as in the case of CD44, or indirect through EBP50. Other binding partners, including the regulatory subunit of protein kinase A and rho-GDI, suggest that ezrin is an integral component of these signaling pathways. Although the membrane-cytoskeletal linking function is clear, further studies are necessary to reveal how the activation of ezrin and its association with different binding partners is regulated.

Modification of high-heeled shoes to decrease pronation during gait.

One of the reasons that high heels may contribute to the formation of halux valgus is that the wearers pronate during propulsion. This pilot study was performed to determine whether relocation of the heel under the counter of a fashion high-heeled pump could change the degree of pronation of the foot during the gait cycle. The authors report that more foot stability was experienced by the subjects when the center of the heel was offset between 2 and 4 mm medial to the center of the heel counter. This study is designed to promote further research into whether the shoe industry should change the design parameters of high-heeled fashion shoes in order to improve foot function.