Regulation mechanism of ERM (ezrin/radixin/moesin) protein/plasma membrane association: possible involvement of phosphatidylinositol turnover and Rho-dependent signaling pathway.

Abstract

Abstract. The ERM proteins, ezrin, radixin, and moesin, are involved in the actin filament/plasma membrane interaction as cross-linkers. CD44 has been identified as one of the major membrane binding partners for ERM proteins. To examine the CD44/ERM protein interaction in vitro, we produced mouse ezrin, radixin, moesin, and the glutathione-S-transferase (GST)/CD44 cytoplasmic domain fusion protein (GST-CD44cyt) by means of recombinant baculovirus infection, and constructed an in vitro assay for the binding between ERM proteins and the cytoplasmic domain of CD44. In this system, ERM proteins bound to GST-CD44cyt with high affinity (K s of moesin was 9.3 _+ 1.6nM) at a low ionic strength, but with low affinity at a physiological ionic strength. However, in the presence of phosphoinositides (phosphatidylinositol [PI], phosphatidylinositol 4-monophosphate [4-PIP], and phosphatidylinositol 4,5-bisphosphate [4,5-PIP2]), ERM proteins bound with a relatively high affinity to GST-CD44cyt even at a physiological ionic strength : 4,5-PIP 2 showed a marked effect (Kd of moesin in the presence of 4,5-PIPE was 9.3 _+ 4.8 nM). Next, to examine the regulation mechanism of CD44/ERM interaction in vivo, we reexamined the immunoprecipitated CD44/ERM complex from BHK cells and found that it contains Rho-GDP dissociation inhibitor (GDI), a regulator of Rho GTPase. We then evaluated the involvement of Rho in the regulation of the CD44/ERM complex formation. When recombinant ERM proteins were added and incubated with lysates of cultured BHK cells followed by centrifugation, a portion of the recombinant ERM proteins was recovered in the insoluble fraction. This binding was enhanced by GTP~S and markedly suppressed by C3 toxin, a specific inhibitor of Rho, indicating that the GTP form of Rho in the lysate is required for this binding. A mAb specific for the cytoplasmic domain of CD44 also markedly suppressed this binding, identifying most of the binding partners for exogenous ERM proteins in the insoluble fraction as CD44. Consistent with this binding analysis, in living BHK cells treated with C3 toxin, most insoluble ERM proteins moved to soluble compartments in the cytoplasm, leaving CD44 free from ERM. These findings indicate that Rho regulates the CD44/ERM complex formation in vivo and that the phosphatidylinositol turnover may be involved in this regulation mechanism. ! r'~IRM (Ezrin/Radixin/Moesin) family proteins are F'~I thought to function as general cross-linkers between plasma membranes and actin fdaments

Inhibition of the hyaluronan-CD44 interaction by merlin contributes to the tumor-suppressor activity of merlin

“…The best-characterized intracellular binding partners of CD44 are the ERM proteins and merlin (Tsukita et al, 1994;Hirao et al, 1996;Sainio et al, 1997). CD44 and ezrin play important roles in promoting tumor metastasis (Yu et al, 1997;Stamenkovic 1999, 2000;Yu et al, 2004;Khanna et al, 2001Khanna et al, , 2004, whereas merlin acts as a tumor suppressor (Rouleau et al, 1993;Trofatter et al, 1993;Lutchman and Rouleau, 1995;Sherman et al, 1997;McClatchey et al, 1998;Giovannini et al, 1999Giovannini et al, , 2000.…”

“…As a receptor for a major ECM component, CD44 plays an important role in regulating cell adhesion and migration (Lesley et al, 1993;Sherman et al, 1994;Stamenkovic 2000). CD44 interacts with both merlin and the ERM proteins (Tsukita et al, 1994;Hirao et al, 1996;Sainio et al, 1997;Tsukita and Yonemura, 1997). ERM proteins, especially ezrin, are thought to serve as key linkers between CD44 and the actin cytoskeleton.…”

“…CD44, the principal cell-surface receptor for hyaluronan (HA; Stamenkovic et al, 1989;Aruffo et al, 1990), is one of the transmembrane proteins that interact with ERM proteins and merlin (Tsukita et al, 1994;Hirao et al, 1996;Sainio et al, 1997;Tsukita and Yonemura, 1997;Morrison et al, 2001). There are numerous isoforms of CD44, the most commonly expressed isoform is the standard form of CD44 (CD44s) that lacks variant exon products.…”

“…Only merlin isoform I has been shown to exhibit tumor-suppressing activity (Sherman et al, 1997). The NH 2 -terminus of merlin shares high sequence homology with the NH 2 -terminal halves of ERM proteins that mediate the binding of these proteins to a variety of molecules in the plasma membrane (Tsukita et al, 1994;Hirao et al, 1996;Yonemura et al, 1998). Accordingly, merlin mutants that lack part of the NH 2 -terminal ERM-homology domains lose their ability to associate with the plasma membrane (Deguen et al, 1998;Koga et al, 1998).…”

Recognition of CD146 as an ERM-binding protein offers novel mechanisms for melanoma cell migration

“…Therefore, GDIs modulate the cycling of Rho GTPases between cytosol and membranes. Importantly, RhoG-DI1 was found to associate with a protein complex containing ERM proteins and CD44, which might be important for cell cytoskeleton remodeling and cell motility (Hirao et al, 1996). CD146, also known as MCAM, MUC18, A32 antigen and S-Endo-1, was initially identified as a melanoma marker .…”