Ezrin NH2-terminal domain inhibits the cell extension activity of the COOH-terminal domain.

Abstract

Abstract. Overexpression in insect cells of the full coding sequence of the human membrane cytoskeletal linker ezrin (1-586) was compared with that of a NHEterminal domain (ezrin 1-233) and that of a COOHterminal domain (ezrin 310-586). Ezrin (1-586), as well as ezrin (1-233) enhanced cell adhesion of infected Si x ) cells without inducing gross morphological changes in the cell structure. Ezrin (310-586) enhanced cell adhesion and elicited membrane spreading followed by microspike and lamellipodia extensions by mobilization of Sf9 cell actin. Moreover some microspikes elongated into thin processes, up to 200 #m in length, resembling neurite outgrowths by a mechanism requiring microtubule assembly. Kinetics of videomicroscopic and drug-interference studies demonstrated that mobilization of actin was required for tubulin assembly to proceed. A similar phenotype was observed in CHO cells when a comparable ezrin domain was transiently overexpressed. The shortest domain promoting cell extension was localized between residues 373-586. Removal of residues 566-586, involved in in vitro actin binding (Turunen, O., T. Wahlstr6m, and A. Vaheri. 1994. J. Cell Biol. 126:1445-1453, suppressed the extension activity. Coexpression of ezrin (1-233) with ezrin (310-586) in the same insect cells blocked the constitutive activity of ezrin COOH-terminal domain. The inhibitory activity was mapped within ezrin 115 first NH2-terminal residues. We conclude that ezrin has properties to promote cell adhesion, and that ezrin NHe-terminal domain negatively regulates membrane spreading and elongation properties of ezrin COOH-terminal domain.

Activated Ezrin Promotes Cell Migration through Recruitment of the GEF Dbl to Lipid Rafts and Preferential Downstream Activation of Cdc42

“…A number of studies have utilized the N-ERMAD of ERM proteins as dominant inhibitory constructs and demonstrated that the introduction of this domain alone can alter the function of endogenous ERM proteins, but the molecular mechanism whereby the N-ERMAD achieves dominant inhibition is hitherto poorly understood (Martin et al, 1995;Crepaldi et al, 1997;Amieva et al, 1999). The N-ERMAD can either bind to receptors or scaffolding proteins at the plasma membrane, and thus abrogate binding of endogenous ERM proteins, or interacts directly with the C-ERMAD and thus altering the actin-binding capacities of endogenous ERM proteins.…”

Identification of a phosphatidylinositol‐4,5‐bisphosphate‐binding domain in the N‐terminal region of ezrin

“…The N-terminal domain of ezrin, when expressed in mammalian cells, locates to the plasma membrane, whereas the C-terminal domain is enriched in the actin cytoskeleton [5], possibly via a recently identified actin binding site in that region [6]. Overexpressed intact ezrin accumulates at the plasma membrane of insect cells [7], and overexpression of the C-terminal site in these cells induces formation of cell extensions [8]. Interestingly, the N-terminal domain inhibits the latter effect [8].…”

“…Overexpressed intact ezrin accumulates at the plasma membrane of insect cells [7], and overexpression of the C-terminal site in these cells induces formation of cell extensions [8]. Interestingly, the N-terminal domain inhibits the latter effect [8]. Recently, the presence of two self-associating domains in ezrin, termed N-and C-ERMADs (ezrin-radixinmoesin association domains) has been demonstrated [9].…”

Interdomain binding mediates tumor growth suppression by the NF2 gene product

“…The N-termini of radixin and ezrin can exert a regulatory in¯uence over their C-termini either in cis or in trans Martin et al, 1995) via an intramolecular association (Gary and Bretscher, 1995;Magendantz et al, 1995). We tested whether such interactions are required for the growth inhibiting functions of merlin by stably transfecting RT4-D6P2T cells with both NF2-N and NF2-C17 using di erent selectable markers.…”