REGULATION MECHANISM OF ERM (EZRIN RADIXIN/MOESIN) PROTEIN/PLASMA MEMBRANE ASSOCIATION - POSSIBLE INVOLVEMENT OF PHOSPHATIDYLINOSITOL TURNOVER AND RHO-DEPENDENT SIGNALING PATHWAY/

Citation
M. Hirao et al., REGULATION MECHANISM OF ERM (EZRIN RADIXIN/MOESIN) PROTEIN/PLASMA MEMBRANE ASSOCIATION - POSSIBLE INVOLVEMENT OF PHOSPHATIDYLINOSITOL TURNOVER AND RHO-DEPENDENT SIGNALING PATHWAY/, The Journal of cell biology, 135(1), 1996, pp. 37-51
Citations number
90
Categorie Soggetti
Cell Biology
Journal title
ISSN journal
00219525
Volume
135
Issue
1
Year of publication
1996
Pages
37 - 51
Database
ISI
SICI code
0021-9525(1996)135:1<37:RMOE(R>2.0.ZU;2-C
Abstract
The ERM proteins, ezrin, radixin, and moesin, are involved in the acti n filament/plasma membrane interaction as cross-linkers. CD44 has been identified as one of the major membrane binding partners for ERM prot eins. To examine the CD44/ERM protein interaction in vitro, we produce d mouse ezrin, radixin, moesin, and the glutathione-S-transferase (GST )/CD44 cytoplasmic domain fusion protein (GST-CD44cyt) by means of rec ombinant baculovirus infection, and constructed an in vitro assay for the binding between ERM proteins and the cytoplasmic domain of CD44. I n this system, ERM proteins bound to GST-CD44cyt with high affinity (K -d 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 o f phosphoinositides (phosphatidylinositol [PI], phosphatidylinositol 4 -monophosphate [4-PIP], and phosphatidylinosilol 4,5-bisphosphate [4,5 -PIP2]), ERM proteins bound with a relatively high affinity to GST-CD4 4cyt even at a physiological ionic strength:4,5-PIP2 showed a marked e ffect (K-d of moesin in the presence of 4,5-PIP2 was 9.3 +/- 4.8 nM). Next, to examine the regulation mechanism of CD44/ERM interaction in v ivo, we reexamined the immunoprecipitated CD44/ERM complex from BHK ce lls 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 gamma 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 al so markedly suppressed this binding, identifying most of the binding p artners for exogenous ERM proteins in the insoluble fraction as CD44. Consistent with this binding analysis, in living BHK cells treated wit h 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 mech anism.