Ezrin-radixin-moesin Family Research Articles

Inflammatory stimuli upregulate Rho-kinase in human coronary vascular smooth muscle cells

Recent studies have demonstrated that upregulated Rho-kinase plays an important role in the pathogenesis of arteriosclerosis and vasospasm in both animals and humans. However, little is known about the molecular mechanism(s) involved in the Rho-kinase upregulation. Since inflammatory mechanisms have been implicated in the pathogenesis of arteriosclerosis and vasospasm, we examined whether inflammatory stimuli upregulate Rho-kinase in vitro and in vivo. In cultured human coronary vascular smooth muscle cells (hcVSMC), inflammatory stimuli, such as angiotensin II and interleukin-1β, increased Rho-kinase expression (at both mRNA and protein levels) and function (as evaluated by the extent of the phosphorylation of the ERM (the ezrin/radixin/moesin) family, substrates of Rho-kinase) in a time- and concentration-dependent manner. The expression of Rho-kinase was inhibited by blockades of protein kinase C (PKC) (by either GF109253 or prolonged treatment with phorbol myristate acetate for 24 h) and an adenovirus-mediated gene transfer of dominant-active Iκ-B, suggesting an involvement of PKC and NF-κB in the intracellular signal transduction pathway for the Rho-kinase expression. Furthermore, coronary vascular lesion formation (characterized by medial thickening and perivascular fibrosis) induced by a long-term administration of angiotensin II was markedly suppressed in NF-κB –/– mice with reduced expression and activity of Rho-kinase in vivo. These results indicate that the expression and function of Rho-kinase are upregulated by inflammatory stimuli (e.g. angiotensin II and IL-1β) in hcVSMC with an involvement of PKC and NF-κB both in vitro and in vivo.

Phosphorylation of ezrin on threonine T567 plays a crucial role during compaction in the mouse early embryo

The preimplantation development of the mouse embryo leads to the divergence of the first two cell lineages, the inner cell mass and the trophectoderm. The formation of a microvillus pole during compaction at the eight-cell stage and its asymmetric inheritance during mitosis are key events in the emergence of these two cell populations. Ezrin, a member of the ERM protein family, seems to be involved in the formation and stabilization of this apical microvillus pole. To further characterize its function in early development, we mutated the key residue T567, which was reported to be essential for regulation of ezrin function through phosphorylation. Here, we show that expression of ezrin mutants in which the COOH-terminal threonine T567 was replaced by an aspartate (to mimic a phosphorylated residue; T567D) or by an alanine (to avoid phosphorylation; T567A) interferes with E-cadherin function and disrupts the first morphogenetic events of development: compaction and cavitation. The active mutant ezrin-T567D induces the formation of numerous and abnormally long microvilli at the surface of blastomeres. Moreover, it localizes all around the cell cortex and inhibits cell–cell adhesion and cell polarization at the eight-cell stage. During the following stages, only half of the embryos are able to compact and finally to cavitate. In those embryos, the amount of ezrin-T567D decreases in the basolateral areas, while the proportion of adherens junctions increases. The reverse inactive mutant ezrin-T567A is mainly cytoplasmic and does not perturb compaction at the eight-cell stage. However, at the 16-cell stage, it relocalizes at the basolateral cortex, leading to a strong decrease in the surface of adherens junctions, and finally, embryos abort development. Our results show that ezrin is directly involved in the formation of microvilli in the early mouse embryo. Moreover, they indicate that maintenance of ezrin in basolateral areas prevents microvilli breakdown and inhibits the formation of normal cell–cell contacts mediated by E-cadherin, thereby impairing blastomeres polarization and morphogenesis of the blastocyst.

Ezrin gene, coding for a membrane-cytoskeleton linker protein, is regionally expressed in the developing mouse neuroepithelium

Ezrin is a member of the Ezrin, Radixin, Moesin (ERM) proteins family that are proposed to act as linkers between the cytoskeleton and plasma membrane. Ezrin regulates cell–cell and cell–matrix interactions playing a role in the regulation of cellular adhesion, movement and morphology in epithelia. Alterations in the expression of Ezrin and other members of ERM family have also been observed in brain tumours. Here we report the expression pattern of Ezrin during mouse neural development, from early stages to postnatal stages. In young and middle gestation embryos, Ezrin is expressed in the roof plate of the neural tube, in the presumptive domain of the choroidal plexus, and in some precise domains of ventricular epithelium. These domains are distributed in basal and alar neuroepithelial regions, some of them in relation to the expression of cadherins. At later gestation and postnatal stages, Ezrin expression is maintained on the mature choroidal plexus and is weakly detected in the proliferative regions of the mature brain.

Ezrin, a key component in tumor metastasis

Identification of the key regulatory molecules in metastasis is crucial for understanding tumor dissemination and for the development of novel interventions. The recent identification of ezrin as a necessary component in the metastasis of osteosarcoma and rhabdomyosarcoma is, therefore, an important advance. Ezrin has been implicated in many roles, for example, as a conduit for signals between metastasis-associated cell-surface molecules and signal transduction components. This suggests that ezrin and, potentially, other members of the ERM (ezrin–radixin–moesin) family have key roles in the coordination of signals and cellular complexes that are required for the successful metastasis of these and other malignancies.

Cyclic AMP-dependent Protein Kinase Phosphorylates Merlin at Serine 518 Independently of p21-activated Kinase and Promotes Merlin-Ezrin Heterodimerization

Mutations in the NF2 tumor suppressor gene encoding merlin induce the development of tumors of the nervous system. Merlin is highly homologous to the ERM (ezrin-radixin-moesin) family of membrane/cytoskeleton linker proteins. However, the mechanism for the tumor suppressing activity of merlin is not well understood. Previously, we characterized a novel role for merlin as a protein kinase A (PKA)-anchoring protein, which links merlin to the cAMP/PKA signaling pathway. In this study we show that merlin is also a target for PKA-induced phosphorylation. In vitro [gamma-(33)P]ATP labeling revealed that both the merlin N and C termini are phosphorylated by PKA. Furthermore, both in vitro and in vivo phosphorylation studies of the wild-type and mutated C termini demonstrated that PKA can phosphorylate merlin at serine 518, a site that is phosphorylated also by p21-activated kinases (PAKs). Merlin was phosphorylated by PKA in cells in which PAK activity was suppressed, indicating that the two kinases function independently. Both in vitro and in vivo interaction studies indicated that phosphorylation of serine 518 promotes heterodimerization between merlin and ezrin, an event suggested to convert merlin from a growth-suppressive to a growth-permissive state. This study provides further evidence on the connection between merlin and cAMP/PKA signaling and suggests a role for merlin in the cAMP/PKA transduction pathway.

Nuclear ERM (ezrin, radixin, moesin) proteins: regulation by cell density and nuclear import

The highly conserved ERM (ezrin-radixin-moesin) family of proteins function as molecular linkers between the actin cytoskeleton and transmembrane receptors. We now provide unequivocal evidence that full-length endogenous ezrin and moesin also localise to the nucleus in two independent mammalian cell lines. All three ERM family members can localise to the nucleus upon exogenous expression of their GFP-tagged counterparts, suggesting a common family trend. Furthermore, Dmoesin, the Drosophila ERM homologue, is present in the nucleus of an insect cell line and can localise to the nucleus when exogenously expressed in MDCK cells. The nuclear localisation of endogenous ezrin and moesin is regulated by cell density and is resistant to detergent extraction, suggesting tight association with nuclear structures. Furthermore, phosphorylation in the actin-binding domain is not a prerequisite for nuclear localisation. We have identified a specific nuclear localisation sequence, which is conserved and functional in all ERM family members, implying specific regulated nuclear import. Although the precise nuclear function of the ERM proteins is unknown, these data provide further evidence that an increasing number of cytoskeletal components directly link the plasma membrane with nuclear events.

Cellular retinaldehyde-binding protein interacts with ERM-binding phosphoprotein 50 in retinal pigment epithelium.

To characterize mechanisms of apical localization of visual cycle components in retinal pigment epithelium (RPE) by the identification of cellular retinaldehyde-binding protein (CRALBP) interaction partners. An overlay assay was used to detect interactions of CRALBP with components of RPE microsomes. Interacting proteins were identified with two-dimensional (2D)-PAGE and liquid chromatography tandem mass spectrometry (LC MS/MS). Protein interactions were characterized by affinity chromatography, peptide competition, and expression of protein domains. Protein colocalization in mouse retina was examined using double-label immunocytochemistry and confocal microscopy. CRALBP bound to a 54-kDa protein in RPE microsomes, which was identified as ERM (ezrin, radixin, moesin)-binding phosphoprotein 50 (EBP50), a PDZ domain protein, also known as sodium/hydrogen exchanger regulatory factory type 1 (NHERF-1). EBP50 and ezrin in solubilized microsomes bound to CRALBP-agarose but not to a control agarose column. CRALBP bound to both recombinant PDZ domains of EBP50 but not to the C-terminal ezrin-binding domain. In outer retina, EBP50 and ezrin were localized to RPE and Müller apical processes. CRALBP was distributed throughout both RPE and Müller cells, including their apical processes. RM proteins are multivalent linkers that connect plasma membrane proteins with the cortical actin cytoskeleton. EBP50 interacts with ERM family members through a C-terminal domain and binds targets such as CRALBP through its PDZ domains, thus contributing to an apical localization of target proteins. Our results provide a structural basis for apical localization of a retinoid-processing complex in RPE cells and offer insight into the cell biology of retinoid processing and trafficking in RPE.

Rho/Rho-Kinase Pathway in the Brainstem Contributes to Hypertension Caused by Chronic Nitric Oxide Synthase Inhibition

Central nervous system mechanisms are involved in hypertension caused by chronic inhibition of nitric oxide (NO) synthesis. Chronic inhibition of NO synthesis might also activate the Rho/Rho-kinase pathway in the vasculature. We recently demonstrated that activation of the Rho/Rho-kinase pathway in the nucleus tractus solitarii (NTS) contributes to hypertensive mechanisms in spontaneously hypertensive rats. The aim of the present study was to determine whether activation of this pathway also contributes to neurogenic hypertensive mechanisms caused by chronic NO synthesis inhibition. The NO synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) was administered to Wistar-Kyoto rats in their drinking water (1 mg/mL) for 2 weeks. Bilateral microinjection of Y-27632, a specific Rho-kinase inhibitor, into the NTS elicited decreases in arterial pressure, heart rate, and renal sympathetic nerve activity in control rats and L-NAME-treated rats. The magnitude of the decrease, however, was significantly greater in L-NAME-treated than in control rats. In another group of rats, the specific Rho-kinase inhibitor, Y-27632, was administered intracisternally for 2 weeks with a mini-osmotic pump from the beginning of treatment with L-NAME. Y-27632 co-treatment significantly attenuated the increase in arterial pressure. Furthermore, the expression level of membranous RhoA and phosphorylation of the target proteins of Rho-kinase, the ERM (ezrin, radixin, moesin) family members, was significantly greater in L-NAME-treated rats than in control rats. These results indicate that activation of the Rho/Rho-kinase pathway in the NTS contributes to neurogenic hypertension caused by chronic NO synthase inhibition.

Foxj1 is required for apical localization of ezrin in airway epithelial cells.

Establishment and maintenance of epithelial cell polarity depend on cytoskeletal organization and protein trafficking to polarized cortical membranes. ERM (ezrin, radixin, moesin) family members link polarized proteins with cytoskeletal actin. Although ERMs are often considered to be functionally similar, we found that, in airway epithelial cells, apical localization of ERMs depend on cell differentiation and is independently regulated. Moesin was present in the apical membrane of all undifferentiated epithelial cells. However, in differentiated cells, ezrin and moesin were selectively localized to apical membranes of ciliated airway cells and were absent from secretory cells. To identify regulatory proteins required for selective ERM trafficking, we evaluated airway epithelial cells lacking Foxj1, an F-box factor that directs programs required for cilia formation at the apical membrane. Interestingly, Foxj1 expression was also required for localization of apical ezrin, but not moesin. Additionally, membrane-cytoskeletal and threonine-phosphorylated ezrin were decreased in Foxj1-null cells, consistent with absent apical ezrin. Although apical moesin expression was present in null cells, it could not compensate for ezrin because ERM-associated EBP50 and the beta2 adrenergic receptor failed to localize apically in the absence of Foxj1. These findings indicate that Foxj1 regulates ERM proteins differentially to selectively direct the apical localization of ezrin for the organization of multi-protein complexes in apical membranes of airway epithelial cells.

Shifts in cellular localization of moesin in normal oral epithelium, oral epithelial dysplasia, verrucous carcinoma and oral squamous cell carcinoma.

Moesin, a member of ERM (ezrin/radixin/moesin) family, links actin filaments of cell surface structure to the cell membrane. The purpose of the study is to assess the shifts in cellular distribution of moesin in normal oral epithelium, oral epithelial dysplasia (OED), verrucous carcinoma (VC), and oral squamous cell carcinoma (OSCC). The expression of moesin was evaluated immunohistochemically in paraffin-embedded tissues of 59 specimens of OSCC, 35 specimens of OED, 17 specimens of VC, and five specimens of normal oral epithelium. In the normal oral epithelia, all specimens showed a pattern of membranous expression against the anti-moesin antibody in the basal layer cells. In the OED specimens, moesin was dominantly expressed in the cell membrane except for the cornified layer. In VC and OSCC specimens, almost the whole of the carcinoma cells were stained with anti-moesin antibody. However, in OSCC samples, moesin was markedly expressed increasingly in the cytoplasm and decreasingly in the cell membrane, as compared with OED and VC. In addition, there was a significant correlation between the pattern of moesin expression and tumor differentiation in OSCC. Our results suggest that it is useful to detect the moesin expression as adjunct to screening mucosal lesions in the oral cavity.

Structure of the Active N-terminal Domain of Ezrin: CONFORMATIONAL AND MOBILITY CHANGES IDENTIFY KEYSTONE INTERACTIONS

Ezrin is a member of the ERM (ezrin, radixin, moesin) family of proteins that cross-link the actin cytoskeleton to the plasma membrane and also may function in signaling cascades that regulate the assembly of actin stress fibers. Here, we report a crystal structure for the free (activated) FERM domain (residues 2-297) of recombinant human ezrin at 2.3 A resolution. Structural comparison among the dormant moesin FERM domain structure and the three known active FERM domain structures (radixin, moesin, and now ezrin) allows the clear definition of regions that undergo structural changes during activation. The key regions affected are residues 135-150 and 155-180 in lobe F2 and residues 210-214 and 235-267 in lobe F3. Furthermore, we show that a large increase in the mobilities of lobes F2 and F3 accompanies activation, suggesting that their integrity is compromised. This leads us to propose a new concept that we refer to as keystone interactions. Keystone interactions occur when one protein (or protein part) contributes residues that allow another protein to complete folding, meaning that it becomes an integral part of the structure and would rarely dissociate. Such interactions are well suited for long-lived cytoskeletal protein interactions. The keystone interactions concept leads us to predict two specific docking sites within lobes F2 and F3 that are likely to bind target proteins.

Crystallization and preliminary crystallographic analysis of the ezrin FERM domain.

Ezrin is a member of the ERM (ezrin/radixin/moesin) family of proteins that cross-link the actin cytoskeleton to the plasma membrane and that also function, both upstream and downstream of the small G-protein Rho, in signaling cascades that regulate the assembly of actin stress fibers. In this study, crystals were obtained of the amino-terminal 297 residues (referred to as FERM) of ezrin. The crystals of the FERM domain of ezrin belong to the monoclinic space group P2(1), with unit-cell parameters a = 48.5, b = 112.8, c = 66.3 A, beta = 102.3 degrees, and contain two molecules in the asymmetric unit. A 2.3 A data set was collected using synchrotron radiation at CHESS A1.

Microvilli-like structures are associated with the internalization of virulent capsulated Neisseria meningitidis into vascular endothelial cells.

Bacterial pathogens are internalized into non-phagocytic cells either by a zipper mechanism involving a direct contact between a bacterial ligand and a cellular receptor or a trigger mechanism secondary to the formation of membrane ruffles. Here we show that internalization of capsulated Neisseria meningitidis within endothelial cells following type IV pilus-mediated adhesion is associated with the formation of cellular protrusions at the site of bacterial attachment. These protrusions, like microvilli, are highly enriched in ezrin and moesin, two members of the ERM (ezrin/radixin/moesin) family, whereas vinculin and paxillin are absent. ERM-binding transmembrane proteins, such as CD44, and cortical actin polymerization colocalized within these membrane protrusions. Expression of dominant-negative ezrin largely prevented cortical actin polymerization, thus confirming the role of this molecule in bacteria-induced cytoskeletal modifications. Moreover, using selective inhibitors and dominant-negative mutants of the Rho family GTPases, we show that bacteria-induced actin polymerization required the activation of both Rho and Cdc42 but not of Rac1. Whereas GTPase inhibition dramatically reduced actin polymerization at the site of bacterial attachment, ezrin recruitment was not affected, indicating that bacterial adhesion promotes ezrin recruitment independently of the activity of the Rho-GTPases. Furthermore, GTPase inhibition largely reduced N. meningitidis entry into endothelial cells without affecting adhesion. We thus propose that following pilus-mediated adhesion, capsulated N. meningitidis recruit ERM-binding transmembrane proteins, as well as ezrin and moesin, and that both Rho and Cdc42 are critical for the subsequent cytoskeletal modifications responsible for the formation of microvilli-like cellular protrusions and bacterial internalization.

The NADPH Oxidase Components p47phox and p40phox Bind to Moesin through Their PX Domain

The NADPH oxidase of phagocytes is a membrane-bound heterodimeric flavocytochrome which catalyses the transfer of electrons from NADPH in the cytoplasm to oxygen in the phagosome. A number of cytosolic proteins are involved in its activation/deactivation: p47phox, p67phox, p40phox and the small GTP-binding protein, rac. The cytosolic phox proteins interact with the cytoskeleton in human neutrophils and, in particular, an interaction with coronin has been reported (Grogan A., Reeves, E., Keep, N. H., Wientjes, F., Totty, N., Burlingame, N. L., Hsuan, J., and Segal, A. W. (1997) J. Cell Sci. 110, 3071–3081). Here, we report on the interaction of another cytoskeletal protein, moesin, with the phox proteins. Moesin belongs to the ezrin–radixin–moesin family of F-actin-binding proteins and we show that it binds to p47phox and p40phox in a phosphoinositide-dependent manner. Furthermore, we show that its N-terminal part binds to the PX domain of p47phox and p40phox.

Expanding the role of NHERF, a PDZ-domain containing protein adapter, to growth regulation.

NHERF (Na+/H+ exchanger regulatory factor or NHERF-1) and E3KARP (NHE3 kinase A regulatory protein or NHERF-2) are structurally related protein adapters that are highly expressed in epithelial tissues. NHERF proteins contain two tandem PDZ domains and a C-terminal sequence that binds several members of the ERM (ezrin-radixin-moesin) family of membrane-cytoskeletal adapters. Although identified as a regulator of NHE3, recent evidence points to a broadening role for NHERF in the function, localization and/or turnover of G-protein coupled receptors, platelet-derived growth factor receptor and ion transporters such as CFTR, Na/Pi cotransporter, Na/HCO3 cotransporter and Trp (calcium) channels. NHERF also recruits non-membrane proteins such as the c-Yes/YAP-65 complex, members of the phospholipase Cbeta family and the GRK6A protein kinase to apical surface of polarized epithelial cells where they regulate or respond to membrane signals. While two distinct models have been proposed for NHERF's role in signal transduction, the common theme is NHERF's ability to bring together membrane and non-membrane proteins to regulate cell metabolism and growth. NHERF overexpression in human breast cancers and mutations in NHERF targets, such as CFTR and merlin, the product of Neurofibromatosis NF2 tumor suppressor gene, that impair NHERF binding suggest that aberrant NHERF function contributes to human disease.

Ezrin-Dependent Promotion of Glioma Cell Clonogenicity, Motility, and Invasion Mediated by BCL-2 and Transforming Growth Factor-β2

Ezrin belongs to the ezrin-radixin-moesin family proteins, which cross-link actin cytoskeleton and plasma membrane. Malignant glioma cells are paradigmatic for their strong migratory and invasive properties. Here, we report that the expression of dominant-negative ezrins inhibits clonogenicity, migration, and invasiveness of human malignant glioma cells. Furthermore, dominant-negative ezrins block hepatocyte growth factor (HGF)-mediated stimulation of clonogenicity and migration, without altering HGF-induced protein kinase B/Akt and focal adhesion kinase phosphorylation. Glioma cells expressing dominant-negative ezrins exhibit a shift of the BCL-2/BAX rheostat toward apoptosis, reduced alpha(V)beta(3) integrin expression and reduced matrix metalloproteinase (MMP) expression and activity. These changes are associated with a dramatic loss of transforming growth factor beta(2) (TGF-beta(2)) release. Exogenous supplementation of TGF-beta(2) overcomes the inhibitory effects of dominant-negative ezrins on migration and clonogenicity. A neutralizing TGF-beta(2) antibody mimics the effects of dominant-negative ezrins on clonogenicity and migration. Exogenous HGF markedly induces TGF-beta(2) protein levels, and a neutralizing TGF-beta(2) antibody abolishes the HGF-mediated increase in glioma cell motility. Finally, TGF-beta(2) does not modulate BCL-2 or BAX expression, but BCL-2 gene transfer increases the levels of latent and active TGF-beta(2). Intracranial xenografts of U87MG glioma cells transfected with the dominant-negative ezrins in athymic mice grow to significantly smaller volumes, and the median survival of these mice is 50 d compared with 28 d in the control group. These data define a novel pathway for HGF-induced glioma cell migration and invasion, which requires ezrin, changes in the BCL-2/BAX rheostat, and the induction of TGF-beta(2) expression in vitro, and underscore the important role of HGF signaling in vivo.

Ezrin-radixin-moesin–binding phosphoprotein 50 is expressed at the apical membrane of rat liver epithelia

Ezrin-radixin-moesin (ERM)-binding phosphoprotein 50 (EBP50) and NHE3 Kinase A regulatory protein (E3KARP) are membrane-cytoskeleton linking proteins that utilize 2 PSD-95/DIg/ZO-1 (PDZ) domains and an ERM binding site to coordinate cyclic adenosine monophosphate (cAMP)-regulated ion transport in a number of distinct epithelia. ERM family members serve to anchor EBP50 and E3KARP to the actin cytoskeleton and sequester protein kinase A (PKA) to these protein complexes. In hepatocytes and cholangiocytes, the epithelial cells of the bile secretory unit, cAMP-activated PKA stimulates secretion and bile formation, but the molecular mechanisms, including the potential contribution of EBP50 and E3KARP, remain undetermined. The present studies evaluated the comparative expression and localization of EBP50 and E3KARP in rat hepatocytes and cholangiocytes. Complementary DNAs encoding rat EBP50 and E3KARP were identified by reverse transcription-polymerase chain reaction in both epithelial cell types and subsequently sequenced. Northern and Western analysis showed the presence of EBP50 messenger RNA and protein in both hepatocytes and cholangiocytes. Confocal immunofluorescence revealed EBP50 was concentrated at the apical domain of both cell types. E3KARP was also expressed in cholangiocytes but had a distinct cytoplasmic/nuclear distribution. In dominant-negative transfection studies, patch clamp analysis of Mz-ChA1 cholangiocarcinoma cells showed that expression of the PDZ1 domain of EBP50 selectively decreased the endogenous cAMP-mediated Cl secretory response. The apical expression of EBP50, presence of specific ERM proteins, and functional effects of PDZ1 expression on cholangiocyte secretion suggest EBP50 is positioned to contribute to the organization and regulation of bile secretory proteins in both hepatocytes and cholangiocytes. (H EPATOLOGY 2001;33:166-176.)

Protein Kinase A Associates with Cystic Fibrosis Transmembrane Conductance Regulator via an Interaction with Ezrin

The cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial Cl(-) channel whose activity is controlled by cAMP-dependent protein kinase (PKA)-mediated phosphorylation. We found that CFTR immunoprecipitates from Calu-3 airway cells contain endogenous PKA, which is capable of phosphorylating CFTR. This phosphorylation is stimulated by cAMP and inhibited by the PKA inhibitory peptide. The endogenous PKA that co-precipitates with CFTR could also phosphorylate the PKA substrate peptide, Leu-Arg-Arg-Ala-Ser-Leu-Gly (kemptide). Both the catalytic and type II regulatory subunits of PKA are identified by immunoblotting CFTR immunoprecipitates, demonstrating that the endogenous kinase associated with CFTR is PKA, type II (PKA II). Phosphorylation reactions mediated by CFTR-associated PKA II are inhibited by Ht31 peptide but not by the control peptide Ht31P, indicating that a protein kinase A anchoring protein (AKAP) is responsible for the association between PKA and CFTR. Ezrin may function as this AKAP, since it is expressed in Calu-3 and T84 epithelia, ezrin binds RII in overlay assays, and RII is immunoprecipitated with ezrin from Calu-3 cells. Whole-cell patch clamp of Calu-3 cells shows that Ht31 peptide reduces cAMP-stimulated CFTR Cl(-) current, but Ht31P does not. Taken together, these data demonstrate that PKA II is linked physically and functionally to CFTR by an AKAP interaction, and they suggest that ezrin serves as an AKAP for PKA-mediated phosphorylation of CFTR.

Cloning and expression profile of chicken radixin

Radixin is a member of the ERM (ezrin/radixin/moesin) family of cytoskeletal linkers. We have cloned chicken radixin as a 4.3 kb cDNA, which encodes an 80 kDa protein that is more than 98% identical to radixin from evolutionarily diverse species. High sequence homology (70–80%) also extends into the 3′-untranslated region (UTR) of the radixin gene. The 3′-UTR of moesin, but not ezrin, was also conserved, suggesting an essential, and possibly specific, regulatory function. A distinct pattern of radixin expression is seen in chicken tissues, suggesting a cell-type-specific function.

Ezrin promotes morphogenesis of apical microvilli and basal infoldings in retinal pigment epithelium.

Ezrin, a member of the ezrin/radixin/moesin (ERM) family, localizes to microvilli of epithelia in vivo, where it bridges actin filaments and plasma membrane proteins. Here, we demonstrate two specific morphogenetic roles of ezrin in the retinal pigment epithelium (RPE), i.e., the formation of very long apical microvilli and of elaborate basal infoldings typical of these cells, and characterize the role of ezrin in these processes using antisense and transfection approaches. In the adult rat RPE, only ezrin (no moesin or radixin) was detected at high levels by immunofluorescence and immunoelectron microscopy at microvilli and basal infoldings. At the time when these morphological differentiations develop, in the first two weeks after birth, ezrin levels increased fourfold to adult levels. Addition of ezrin antisense oligonucleotides to primary cultures of rat RPE drastically decreased both apical microvilli and basal infoldings. Transfection of ezrin cDNA into the RPE-J cell line, which has only trace amounts of ezrin and moesin, sparse and stubby apical microvilli, and no basal infoldings, induced maturation of microvilli and the formation of basal infoldings without changing moesin expression levels. Taken together, the results indicate that ezrin is a major determinant in the maturation of surface differentiations of RPE independently of other ERM family members.