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.

Ras regulates the polarity of the yeast actin cytoskeleton through the stress response pathway.

Polarized growth in yeast requires cooperation between the polarized actin cytoskeleton and delivery of post-Golgi secretory vesicles. We have previously reported that loss of the major tropomyosin isoform, Tpm1p, results in cells sensitive to perturbations in cell polarity. To identify components that bridge these processes, we sought mutations with both a conditional defect in secretion and a partial defect in polarity. Thus, we set up a genetic screen for mutations that conferred a conditional growth defect, showed synthetic lethality with tpm1Delta, and simultaneously became denser at the restrictive temperature, a hallmark of secretion-defective cells. Of the 10 complementation groups recovered, the group with the largest number of independent isolates was functionally null alleles of RAS2. Consistent with this, ras2Delta and tpm1Delta are synthetically lethal at 35 degrees C. We show that ras2Delta confers temperature-sensitive growth and temperature-dependent depolarization of the actin cytoskeleton. Furthermore, we show that at elevated temperatures ras2Delta cells are partially defective in endocytosis and show a delocalization of two key polarity markers, Myo2p and Cdc42p. However, the conditional enhanced density phenotype of ras2Delta cells is not a defect in secretion. All the phenotypes of ras2Delta cells can be fully suppressed by expression of yeast RAS1 or RAS2 genes, human Ha-ras, or the double disruption of the stress response genes msn2Deltamsn4Delta. Although the best characterized pathway of Ras function in yeast involves activation of the cAMP-dependent protein kinase A pathway, activation of the protein kinase A pathway does not fully suppress the actin polarity defects, suggesting that there is an additional pathway from Ras2p to Msn2/4p. Thus, Ras2p regulates cytoskeletal polarity in yeast under conditions of mild temperature stress through the stress response pathway.

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.

Nodes of Ranvier form in association with ezrin-radixin-moesin (ERM)-positive Schwann cell processes.

In the adult peripheral nerve, microvillous processes of myelinating Schwann cells project to the nodes of Ranvier; their composition and physiologic function have not been established. As the ezrin-radixin-moesin (ERM) proteins are expressed in the microvilli of many epithelial cells, we have examined the expression and distribution of these proteins in Schwann cells and neurons in vitro and in vivo. Cultured Schwann cells express high levels of all three proteins and the ezrin-binding protein 50, whereas neurons express much lower, although detectable, levels of radixin and moesin. Ezrin is specific for Schwann cells. All three ERM proteins are expressed predominantly at the membrane of cultured Schwann cells, notably in their microvilli. In vivo, the ERM proteins are concentrated strikingly in the nodal processes of myelinating Schwann cells. Because these processes are devoid of myelin proteins, they represent a unique compartment of the myelinating Schwann cell. During development, the ERM proteins become concentrated at the ends of Schwann cells before myelin basic protein expression, demonstrating that Schwann cells are polarized longitudinally at the onset of myelination. ERM-positive Schwann cell processes overlie and are associated closely with nascent nodes of Ranvier, identified by clusters of ankyrin G. Ankyrin accumulation at the node precedes that of Caspr at the paranodes and therefore does not depend on the presence of mature paranodal junctions. These results demonstrate that nodes of Ranvier in the peripheral nervous system form in contact with specialized processes of myelinating Schwann cells that are highly enriched in ERM proteins.

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.

Myosin V orientates the mitotic spindle in yeast.

Coordination of spindle orientation with the axis of cell division is an essential process in all eukaryotes. In addition to ensuring accurate chromosomal segregation, proper spindle orientation also establishes differential cell fates and proper morphogenesis. In both animal and yeast cells, this process is dependent on cytoplasmic microtubules interacting with the cortical actin-based cytoskeleton, although the motive force was unknown. Here we show that yeast Myo2, a myosin V that translocates along polarized actin cables into the bud, orientates the spindle early in the cell cycle by binding and polarizing the microtubule-associated protein Kar9 (refs 7-9). The tail domain of Myo2 that binds Kar9 also interacts with secretory vesicles and vacuolar elements, making it a pivotal component of yeast cell polarization.

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.

Identification of a novel member of the chloride intracellular channel gene family (CLIC5) that associates with the actin cytoskeleton of placental microvilli.

The chloride intracellular channel (CLIC) gene family has been implicated in chloride ion transport within various subcellular compartments. We report here the molecular, biochemical, and cellular characterization of a new member of this gene family termed CLIC5. CLIC5 was isolated from extracts of placental microvilli as a component of a multimeric complex consisting of several known cytoskeletal proteins, including actin, ezrin, alpha-actinin, gelsolin, and IQGAP1. We cloned human cDNAs and generated antibodies specific for CLIC5, CLIC1/NCC27, and CLIC4/huH1/p64H1. CLIC5 shares 52-76% overall identity with human CLIC1, CLIC2, CLIC3, and CLIC4. Northern blot analysis showed that CLIC5 has a distinct pattern of expression compared with CLIC1 and CLIC4. Immunoblot analysis of extracts from placental tissues demonstrated that CLIC4 and CLIC5 are enriched in isolated placental microvilli, whereas CLIC1 is not. Moreover, in contrast to CLIC1 and CLIC4, CLIC5 is associated with the detergent-insoluble cytoskeletal fraction of microvilli. Indirect immunofluorescence microscopy revealed that CLIC4 and CLIC5 are concentrated within the apical region of the trophoblast, whereas CLIC1 is distributed throughout the cytoplasm. These studies suggest that CLIC1, CLIC4, and CLIC5 play distinct roles in chloride transport and that CLIC5 interacts with the cortical actin cytoskeleton in polarized epithelial cells.

Polarization of cell growth in yeast.

The actin cytoskeleton provides the structural basis for cell polarity in Saccharomyces cerevisiae as well as most other eukaryotes. In Part I of this two-part commentary, presented in the previous issue of Journal of Cell Science, we discussed the basis by which yeast establishes and maintains different states of polarity through &Rgr; GTPases and cyclin-dependent protein kinase signaling. Here we discuss how, in response to those signals, the actin cytoskeleton guides growth of the yeast cell. A polarized array of actin cables at the cell cortex is the primary structural determinant of polarity. Motors such as class V myosins use this array to transport secretory vesicles, mRNA and organelles towards growth sites, where they are anchored by a cap of cytoskeletal and regulatory proteins. Cortical actin patches enhance and maintain this polarity, probably through endocytic recycling, which allows reuse of materials and prevents continued growth at old sites. The dynamic arrangement of targeting and recycling provides flexibility for the precise control of morphogenesis.

Polarization of cell growth in yeast. I. Establishment and maintenance of polarity states.

The ability to polarize is a fundamental property of cells. The yeast Saccharomyces cerevisiae has proven to be a fertile ground for dissecting the molecular mechanisms that regulate cell polarity during growth. Here we discuss the signaling pathways that regulate polarity. In the second installment of this two-part commentary, which appears in the next issue of Journal of Cell Science, we discuss how the actin cytoskeleton responds to these signals and guides the polarity of essentially all events in the yeast cell cycle. During the cell cycle, yeast cells assume alternative states of polarized growth, which range from tightly focused apical growth to non-focused isotropic growth. RhoGTPases, and in particular Cdc42p, are essential to guiding this polarity. The distribution of Cdc42p at the cell cortex establishes cell polarity. Cyclin-dependent protein kinase, Ras, and heterotrimeric G proteins all modulate yeast cell polarity in part by altering the distribution of Cdc42p. In turn, Cdc42p generates feedback signals to these molecules in order to establish stable polarity states and coordinate cytoskeletal organization with the cell cycle. Given that many of these signaling pathways are present in both fungi and animals, they are probably ancient and conserved mechanisms for regulating polarity.

The COOH-terminal domain of Myo2p, a yeast myosin V, has a direct role in secretory vesicle targeting.

MYO2 encodes a type V myosin heavy chain needed for the targeting of vacuoles and secretory vesicles to the growing bud of yeast. Here we describe new myo2 alleles containing conditional lethal mutations in the COOH-terminal tail domain. Within 5 min of shifting to the restrictive temperature, the polarized distribution of secretory vesicles is abolished without affecting the distribution of actin or the mutant Myo2p, showing that the tail has a direct role in vesicle targeting. We also show that the actin cable-dependent translocation of Myo2p to growth sites does not require secretory vesicle cargo. Although a fusion protein containing the Myo2p tail also concentrates at growth sites, this accumulation depends on the polarized delivery of secretory vesicles, implying that the Myo2p tail binds to secretory vesicles. Most of the new mutations alter a region of the Myo2p tail conserved with vertebrate myosin Vs but divergent from Myo4p, the myosin V involved in mRNA transport, and genetic data suggest that the tail interacts with Smy1p, a kinesin homologue, and Sec4p, a vesicle-associated Rab protein. The data support a model in which the Myo2p tail tethers secretory vesicles, and the motor transports them down polarized actin cables to the site of exocytosis.

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 cytoskeletal linker protein moesin: decreased levels in Wiskott-Aldrich syndrome platelets and identification of a cleavage pathway in normal platelets.

The Wiskott-Aldrich syndrome (WAS) is a severe disease of platelets (small size, thrombocytopenia) and lymphocytes (immunodeficiency) arising from mutations of the X-chromosome gene WASP. Because of the prominent role of cytoskeletal abnormalities, particularly the paucity of surface microvilli, in the cellular pathology of this disease, blood cells from WAS patients were examined for moesin, a cytoskeletal linker protein that stabilizes cell surface microvilli, filopodia and lamellipodia. Comparison of patient and normal lymphocytes by immunofluorescence microscopy and immunoblotting showed normal levels and distribution of moesin in lymphocytes of WAS patients. In contrast, platelets from WAS patients stained only dimly for moesin relative to normal platelets. Quantitation by immunoblot revealed significantly decreased moesin levels in WAS patient platelets relative to normal platelets (63.5 +/- 4.9% of normal levels, n = 8, P < 0.0001). A novel reaction of normal platelets was discovered that may play a role in the depletion of moesin in patient platelets, namely the cleavage of moesin as a late event in platelet activation in response to certain platelet agonists.

Moesin, the major ERM protein of lymphocytes and platelets, differs from ezrin in its insensitivity to calpain.

The ERM proteins, ezrin, radixin and moesin, provide regulated linkage of the cytoskeleton with the plasma membrane, particularly in cell surface projections. Ezrin and moesin were found co-expressed, and radixin was not detected, in human blood lymphocytes, monocytes and neutrophils. Moesin is the quantitatively dominant ERM protein in these cells and the only one in platelets. Because Ca signaling pathways involving calpain cleavages are important in blood cells, we examined ERM protein sensitivity to this protease. A striking difference was discovered: sensitivity of ezrin and resistance of moesin (and radixin) to calpain. In intact stimulated lymphocytes, ezrin was cleaved, while moesin was not, strongly suggesting that differential sensitivity to calpain contributes to specialized functions of these proteins.

Regulation of cortical structure by the ezrin-radixin-moesin protein family.

Molecules involved in ERM (ezrin-radixin-moesin) based attachment of membrane proteins to the cortical cytoskeleton in cell surface structures have been identified. In lymphocytes, a direct interaction is seen with extracellular matrix receptors and intercellular adhesion molecules. In polarized epithelial cells, an adaptor molecule named EBP50 provides a bridge between the amino-terminal domain of ezrin and the cytoplasmic regions of plasma membrane proteins, including the cystic fibrosis transmembrane conductance regulator (CFTR) and the beta2 adrenergic receptor. ERM proteins are conformationally regulated - binding sites for EBP50 and F actin are masked in the dormant molecules and activation leads to exposure of these sites. The mechanism of activation, however, remains to be fully elucidated. ERM proteins also play a role in the Rho and Rac signaling pathways: activated ERM proteins can dissociate Rho-GDI (GDP dissociation inhibitor) from Rho and thereby activate Rho-dependent pathways.

Tropomyosin-containing actin cables direct the Myo2p-dependent polarized delivery of secretory vesicles in budding yeast.

The actin cytoskeleton in budding yeast consists of cortical patches and cables, both of which polarize toward regions of cell growth. Tropomyosin localizes specifically to actin cables and not cortical patches. Upon shifting cells with conditionally defective tropomyosin to restrictive temperatures, actin cables disappear within 1 min and both the unconventional class V myosin Myo2p and the secretory vesicle-associated Rab GTPase Sec4p depolarize rapidly. Bud growth ceases and the mother cell grows isotropically. When returned to permissive temperatures, tropomyosin-containing cables reform within 1 min in polarized arrays. Cable reassembly permits rapid enrichment of Myo2p at the focus of nascent cables as well as the Myo2p- dependent recruitment of Sec4p and the exocyst protein Sec8p, and the initiation of bud emergence. With the loss of actin cables, cortical patches slowly assume an isotropic distribution within the cell and will repolarize only after restoration of cables. Therefore, actin cables respond to polarity cues independently of the overall distribution of cortical patches and are able to directly target the Myo2p-dependent delivery of secretory vesicles and polarization of growth.