Four-point-one, Ezrin, Radixin, Moesin Research Articles

The Tumor Suppressors Merlin and Expanded Function Cooperatively to Modulate Receptor Endocytosis and Signaling

The precise coordination of signals that control proliferation is a key feature of growth regulation in developing tissues . While much has been learned about the basic components of signal transduction pathways, less is known about how receptor localization, compartmentalization, and trafficking affect signaling in developing tissues. Here we examine the mechanism by which the Drosophila Neurofibromatosis 2 (NF2) tumor suppressor ortholog Merlin (Mer) and the related tumor suppressor expanded (ex) regulate proliferation and differentiation in imaginal epithelia. Merlin and Expanded are members of the FERM (Four-point one, Ezrin, Radixin, Moesin) domain superfamily, which consists of membrane-associated cytoplasmic proteins that interact with transmembrane proteins and may function as adapters that link to protein complexes and/or the cytoskeleton . We demonstrate that Merlin and Expanded function to regulate the steady-state levels of signaling and adhesion receptors and that loss of these proteins can cause hyperactivation of associated signaling pathways. In addition, pulse-chase labeling of Notch in living tissues indicates that receptor levels are upregulated at the plasma membrane in Mer; ex double mutant cells due to a defect in receptor clearance from the cell surface. We propose that these proteins control proliferation by regulating the abundance, localization, and turnover of cell-surface receptors and that misregulation of these processes may be a key component of tumorigenesis.

The C. elegans ezrin-radixin-moesin protein ERM-1 is necessary for apical junction remodelling and tubulogenesis in the intestine

Members of the ezrin-radixin-moesin (ERM) family of proteins have been found to serve as linkers between membrane proteins and the F-actin cytoskeleton in many organisms. We used RNA interference (RNAi) approach to assay ERM proteins of the Caenorhabditis elegans genome for a possible involvement in apical junction (AJ) assembly or positioning. We identify erm-1 as the only ERM protein required for development and show, by multiple RNA interference, that additional four-point one, ezrin-radixin-moesin (FERM) domain-containing proteins cannot compensate for the depletion of ERM-1. ERM-1 is expressed in most if not all cells of the embryo at low levels but is upregulated in epithelia, like the intestine. ERM-1 protein co-localizes with F-actin and the intermediate filament protein IFB-2 at the apical cell cortex. ERM-1 depletion results in intestine-specific phenotypes like lumenal constrictions or even obstructions. This phenotype arises after epithelial polarization of intestinal cells and can be monitored using markers of the apical junction. We show that the initial steps of epithelial polarization in the intestine are not affected in erm-1(RNAi) embryos but the positioning of apical junction proteins to an apico-lateral position arrests prematurely or fails, resulting in multiple obstructions of the intestinal flow after hatching. Mechanistically, this phenotype might be due to an altered apical cytoskeleton because the apical enrichment of F-actin filaments is lost specifically in the intestine. ERM-1 is the first protein of the apical membrane domain affecting junction remodelling in C. elegans. ERM-1 interacts genetically with the catenin–cadherin system but not with the DLG-1 (Discs large)-dependent establishment of the apical junction.

Talin controls integrin activation

Tight, dynamic control of the affinity of integrin adhesion receptors for their extracellular ligands (integrin activation) is essential for the development and functioning of multicellular organisms. Integrin activation is controlled by intracellular signals that, through their action on integrin cytoplasmic domains, induce conformational changes in integrin extracellular domains, resulting in increased affinity for the ligand. Recent results indicate that the binding of talin, a major actin-binding protein, to integrin beta tails represents a final common step in integrin activation pathways. The major integrin-binding site lies within the talin FERM (four-point-one, ezrin, radixin, moesin) domain, and binding occurs via a variant of the classical PTB domain (phosphotyrosine-binding domain)-NPxY interaction. Formation of this talin-integrin complex plays a critical role in integrin activation, since mutations, in either talin or integrin beta tails, which disrupt complex formation, inhibit integrin activation. Furthermore, use of RNA interference to knockdown talin expression selectively reveals that talin is essential for integrin activation in response to physiological agonists. Thus the association of the cytoskeletal protein talin with integrin beta cytoplasmic domains is a critical step during integrin activation, and regulation of this step may be a final common element in the signalling pathways that control integrin activation.

The Kindler Syndrome Protein Is Regulated by Transforming Growth Factor-β and Involved in Integrin-mediated Adhesion

Transforming growth factor-beta1 (TGF-beta1) contributes to tumor invasion and cancer progression by increasing the motility of tumor cells. To identify genes involved in TGF-beta-mediated cell migration, the transcriptional profiles of human mammary epithelial cells (HMEC) treated with TGF-beta were compared with untreated cells by cDNA microarray analysis. One gene up-regulated by TGF-beta was recently named kindlerin (Jobard, F., Bouadjar, B., Caux, F., Hadj-Rabia, S., Has, C., Matsuda, F., Weissenbach, J., Lathrop, M., Prud'homme, J. F., and Fischer, J. (2003) Hum. Mol. Genet. 12, 925-935). This gene is significantly overexpressed in some cancers (Weinstein, E. J., Bourner, M., Head, R., Zakeri, H., Bauer, C., and Mazzarella, R. (2003) Biochim. Biophys. Acta 1637, 207-216), and mutations in this gene lead to Kindler syndrome, an autosomal-recessive genodermatosis. TGF-beta stimulation of HMEC resulted in a marked induction of kindlerin RNA, and Western blotting demonstrated a corresponding increase in protein abundance. Kindlerin displays a putative FERM (four point one ezrin radixin moesin) domain that is closely related to the sequences in talin that interact with integrin beta subunit cytoplasmic domains. The critical residues in the talin FERM domain that mediate integrin binding show a high degree of conservation in kindlerin. Furthermore, kindlerin is recruited into a molecular complex with the beta1A and beta3 integrin cytoplasmic domains. Consistent with these biochemical findings, kindlerin is present at focal adhesions, sites of integrin-rich, membrane-substratum adhesion. Additionally, kindlerin is required for normal cell spreading. Taken together, these data suggest a role for kindlerin in mediating cell processes that depend on integrins.

Localization of an Integrin Binding Site to the C Terminus of Talin

Talin, consisting of a 47-kDa N-terminal head domain (residues 1-433) and a 190-kDa C-terminal rod domain (residues 434-2541), links integrins to the actin cytoskeleton. We previously reported that the binding stoichiometry of integrin alpha(IIb)beta(3):talin is approximately 2:1. More recently, an integrin binding site has been localized to the talin head domain. In the present study, we identified another integrin binding site at the C-terminal region of the talin rod domain. In a solid phase binding assay, RGD affinity-purified alpha(IIb)beta(3) bound in a dose-dependent manner to microtiter wells coated with the isolated 190-kDa proteolytic fragment of the talin rod domain. Additionally, alpha(IIb)beta(3) also bound to the talin rod domain captured by 8d4, an anti-talin monoclonal antibody. Polyclonal antibodies raised against a recombinant protein fragment corresponding to the entire talin rod domain (anti-talin-R) inhibited alpha(IIb)beta(3) binding to intact talin by approximately 50% but completely blocked alpha(IIb)beta(3) binding to the talin rod domain. To localize the integrin binding site, we examined alpha(IIb)beta(3) binding to recombinant polypeptide fragments corresponding to partial sequences of the talin rod domain. Whereas alpha(IIb)beta(3) bound effectively to talin-(1075-2541) and talin-(1984-2541), it failed to bind to talin-(434-1076) and talin-(434-1975). Furthermore, the binding of alpha(IIb)beta(3) to talin-(1984-2541) was inhibited by anti-talin-R. These results indicate that an integrin binding site is located within residues 1984-2541 of the talin rod domain. Thus, talin contains two integrin binding sites, one in the homologous FERM (band four-point-one, ezrin, radixin, moesin) domain and another near its C terminus. Because talin exists as an anti-parallel homodimer in focal adhesions, the two integrin binding sites in the adjacent talin molecules would be in close proximity with each other.

Calpain Cleavage Promotes Talin Binding to the β3Integrin Cytoplasmic Domain

Talin links integrin beta cytoplasmic domains to the actin cytoskeleton and is involved in the clustering and activation of these receptors. To understand how talin recognizes integrin beta cytoplasmic domains, we configured surface plasmon resonance methodology to measure the interaction of talin with the beta3 integrin cytoplasmic domain. Here we report that the N-terminal approximately 47-kDa talin head domain (talin-H) has a 6-fold higher binding affinity than intact talin for the beta3 tail. The affinity difference is mainly due to a difference in k(on). Calpain cleavage of intact talin released talin-H and resulted in a 16-fold increase in apparent K(a) and a 100-fold increase in apparent k(on). The increase in talin binding after cleavage was greater than predicted for stoichiometric liberation of free talin-H. This additional increase in binding was due to cooperative binding of talin-H and talin rod domain to the beta3 tail. Talin resembles ERM (ezrin, radixin, moesin) proteins in possessing an N-terminal FERM (band four-point-one, ezrin, radixin, moesin) domain. These data show that the talin FERM domain, like that in the ERM proteins, is masked in the intact molecule. Furthermore, they suggest that talin cleavage by calpain may contribute to the effects of the protease on the clustering and activation of integrins.