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