Ezrin, a member of a family of proteins involved in the interaction of the microfilament cytoskeleton with the plasma membrane, plays a role in membrane translocation in gastric parietal cells (Hanzel, D., Reggio, H., Bretscher, A., Forte, J. G. and Mangeat, P. (1991). EMBO J. 10, 2363–2373). Human ezrin was expressed in and purified from Escherichia coli. It possesses all the major biophysical, immunological and physiological properties of natural ezrin. Upon microinjection in live gastric HGT-1 cells, ezrin was incorporated into the dorsal microvilli, a site where the endogeneous protein is localized. By coimmunoprecipitation and ezrin-affinity assays, two HGT-1 cell proteins of 77 and 72 kDa behaved as ezrin-binding proteins. In enriched gastric apical membranes, 125I-ezrin labelled proteins of 80, 77 and 72 kDa by overlay assay. The 80 kDa protein was identified as ezrin and the 77 and 72 kDa proteins as gastric forms of proteins structurally related to ezrin, such as radixin and moesin. In insect cells infected with a recombinant baculovirus, one-third of over-expressed ezrin accumulated at the plasma membrane. Ezrin bound a 77 kDa endogenous peripheral membrane protein, behaving as an insect counterpart of the mammalian ezrin family. In addition to the respective role of the amino- and carboxyl-terminal domains of ezrin in linking the membrane and the cytoskeleton (Algrain, M., Turunen, O., Vaheri, A., Louvard, D. and Arpin, M. (1993). J. Cell Biol. 120, 129–139), both domains interacted synergistically in a salt-dependent manner to trigger self-association of ezrin. Ezrin's self-association properties could represent another way of regulating the number of ezrin molecules bound at specific membrane sites.

REFERENCES

Algrain
M.
,
Turunen
O.
,
Vaheri
A.
,
Louvard
D.
,
Arpin
M.
(
1993
).
Ezrin contains cytoskeleton and membrane binding domains accounting for its proposed role as a membrane-cytoskeletal linker.
J. Cell Biol
120
,
129
139
Avnur
Z.
,
Small
J. V.
,
Geigier
B.
(
1983
).
Actin-independent association of vinculin with the cytoplasmic aspect of the plasma membrane in cell-contact areas.
J. Cell Biol
96
,
1622
1630
Berryman
M.
,
Franck
Z.
,
Bretscher
A.
(
1993
).
Ezrin is concentrated in the apical microvilli of a wide variety of epithelial cells whereas moesin is found primarily in endothelial cells.
J. Cell Sci
105
,
1025
1043
Birgbauer
E.
,
Dinsmore
J. H.
,
Winckler
B.
,
Lander
A. D.
,
Solomon
F.
(
1991
).
Association of ezrin isoforms with the neuronal cytoskeleton.
J. Neurosci. Res
30
,
232
241
Bordier
C.
(
1981
).
Phase separation of integral membrane proteins in Triton X-114 solution.
J. Biol. Chem
256
,
1604
1607
Bretscher
A.
(
1983
).
Purification of an 80,000-dalton protein that is a component of the isolated microvillus cytoskeleton, and its localisation in non muscle cells.
J. Cell Biol
97
,
425
432
Bretscher
A.
(
1989
).
Rapid phosphorylation and reorganisation of ezrin and spectrin accompagny morphological changes induced in A-431 cells by epidermal growth factor.
J. Cell Biol
108
,
921
930
Bretscher
A.
(
1993
).
Microfilaments and membranes.
Curr. Opin. Cell Biol
5
,
653
660
Burridge
K.
,
Mangeat
P.
(
1984
).
An interaction between vinculin and talin.
Nature
308
,
744
746
Chew
C. S.
,
Ljungström
M.
,
Smolka
A.
,
Brown
M. R.
(
1989
).
Primary culture of secretagogue-responsive parietal cells from rabbit gastric mucosa.
Am. J. Physiol
253
,
823
–.
Conboy
J.
,
Kan
Y. W.
,
Shodet
S. B.
,
Mohandas
N.
(
1986
).
Molecular cloning of protein 4.1, a major structural element of the human erythrocyte membrane skeleton.
Proc. Nat. Acad. Sci. USA
83
,
9512
9516
Correas
I.
,
Leto
T. L.
,
Speicher
D. W.
,
Marchesi
V. T.
(
1986
).
Identification of the functional site of erythrocyte protein 4.1 involved in spectrin-actin associations.
J. Biol. Chem
261
,
3310
3315
Croizier
G.
,
Croizier
L.
,
Quiot
J. M.
,
Lereclus
L.
(
1988
).
Recombination of Autographa californica and Rachiplusia ou nuclear polyhedrosis viruses in Galleria mellonella L.
J. Gen. Virol
69
,
177
185
Everett
A. W.
,
Nichol
K. A.
(
1990
).
Ezrin immunoreactivity in neuron subpopulation: cellular distribution in relation to cytoskeletal proteins in sensory neurons.
J. Histochem. Cytochem
38
,
1137
1144
Franck
Z.
,
Gary
R.
,
Brestcher
A.
(
1993
).
Moesin, like ezrin, colocalizes with actin in the cortical cytoskeleton in cultured cells, but its expression is more variable.
J. Cell Sci
105
,
219
231
Frosch
P. M.
,
Frosch
M.
,
Pfister
T.
,
Shaad
T. V.
,
Bitter-Suermann
D.
(
1991
).
Cloning and characterisation of an immunodominant major surface antigen of Echinococcus multilocularis.
Mol. Biochem. Parasitol
48
,
121
130
Fujiki
Y.
,
Hubbard
A.
,
Fowler
S.
,
Lazarow
P. B.
(
1982
).
Isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplasmic reticulum.
J. Cell Biol
93
,
97
102
Funayama
N.
,
Nagafuchi
A.
,
Sato
N.
,
Tsukita
Sa.
,
Tsukita
Sh
(
1991
).
Radixin is a novel member of the band 4.1 family.
J. Cell Biol
115
,
1039
1048
Furthmayr
H.
,
Lankes
W.
,
Amieva
M.
(
1992
).
Moesin, a new cytoskeletal protein and constituent of filopodia: its role in cellular functions.
Kidney Int
41
,
665
670
Gary
R.
,
Bretscher
A.
(
1993
).
Heterotypic and homotypic associations between ezrin and moesin, two putative membrane-cytoskeletal linking proteins.
Proc. Nat. Acad. Sci. USA
90
,
10846
10850
Gould
K. L.
,
Cooper
J. A.
,
Bretscher
A.
,
Hunter
T.
(
1986
).
The protein-tyrosine kinase substrat, p81, is homologous to a chicken microvillar core protein.
J. Cell Biol
102
,
660
669
Gould
K. L.
,
Bretscher
A.
,
Esch
F. S.
,
Hunter
T.
(
1989
).
cDNAcloning and sequencing of the protein-tyrosine kinase substrate, ezrin, reveals homology to band 4.1.
EMBO J
8
,
4133
4142
Gu
M.
,
York
J. D.
,
Warshawsky
I.
,
Majerus
P. W.
(
1991
).
Identification, cloning, and expression of a cytosolic megakaryocyte protein-tyrosine phosphatase with sequence homology to cytoskeletal protein 4.1.
Proc. Nat. Acad. Sci. USA
88
,
5867
5871
Hanzel
D. K.
,
Urushidani
T.
,
Usinger
W. R.
,
Smolka
A.
,
Forte
J. G.
(
1989
).
Immunological localization of an 80K phosphoprotein to the apical membrane of gastric parietal cells.
Am. J. Physiol
256
,
1082
–.
Hanzel
D.
,
Reggio
H.
,
Bretscher
A.
,
Forte
J. G.
,
Mangeat
P.
(
1991
).
The secretion-stimulated 80K phospho-protein of parietal cells is ezrin, and has properties of a membrane cytoskeletal linker in the induced apical microvilli.
EMBO J
10
,
2363
2373
Hunter
T.
,
Cooper
J. A.
(
1981
).
Epidermal growth factor induces tyrosine phosphorylation of proteins in A431 human tumor cells.
Cell
24
,
741
752
Hunter
W. M.
,
Greenwood
F.
(
1962
).
Preparation of iodine-131 labeled human growth hormone of high specific activity.
Nature
194
,
495
496
Krieg
J.
,
Hunter
T.
(
1992
).
Identification of the two major epidermal growth factor-induced tyrosine phosphorylation sites in the microvillar core protein ezrin.
J. Biol. Chem
267
,
19258
19265
Lankes
W. T.
,
Furthmayr
H.
(
1991
).
Moesin: a member of the protein 4. 1-talin-ezrin family of proteins.
Proc. Nat. Acad. Sci. USA
88
,
8297
8301
Luna
E. J.
,
Hitt
A. L.
(
1992
).
Cytoskeleton-plasma membrane interactions.
Science
258
,
955
964
Mangeat
P. H.
,
Burridge
K.
(
1984
).
Immunoprecipitation of nonerythrocyte spectrin within live cells following microinjection of specific antibodies: relation with cytoskeletal structures.
J. Cell Biol
98
,
1363
1377
Mangeat
P. H.
(
1988
).
Interaction of biological membranes with the cytoskeletal framework of living cells. Biol.
Cell
64
,
261
281
Mangeat
P. H.
,
Gusdinar
T.
,
Sahuquet
A.
,
Hanzel
D. K.
,
Forte
J. G.
,
Magous
R.
(
1990
).
Acid secretion and membrane reorganization in single gastric parietal cell in primary culture. Biol.
Cell
69
,
223
232
Matsudaira
P.
(
1990
).
Limited N-terminal sequence analysis.
Meth. Enzymol
182
,
602
613
Mercier
F.
,
Reggio
H.
,
Devilliers
G.
,
Bataille
D.
,
Mangeat
P.
(
1989
).
A marker of acid-secreting membrane movement in rat gastric parietal cells. Biol.
Cell
65
,
7
20
Mercier
F.
,
Reggio
H.
,
Devilliers
G.
,
Bataille
D.
,
Mangeat
P.
(
1989
).
Membrane-cytoskeleton dynamics in rat parietal cells: mobilization of actin and spectrin upon stimulation of gastric acid secretion.
J. Cell Biol
108
,
441
453
Pakkanen
R.
(
1988
).
Immunofluorescent and immunochemical evidence for the expression of cytovillin in the microvilli of a wide range of cultured human cells.
J. Cell. Biochem
38
,
65
75
Pakkanen
R.
,
Vaheri
A.
(
1989
).
Cytovillin and other microvillar proteins of human choriocarcinoma cells.
J. Cell. Biochem
41
,
1
12
Reenstra
W. W.
,
Forte
J. G.
(
1990
).
Isolation of H+,K+-ATPase-containing membranes from the gastric oxyntic cell.
Meth. Enzymol
192
,
151
164
Rees
D. J. G.
,
Ades
S. E.
,
Singer
S. J.
,
Hynes
R. O.
(
1990
).
Sequence and domain structure of talin.
Nature
347
,
685
689
Rouleau
G. A.
,
Merel
P.
,
Lutchman
M.
,
Sanson
M.
,
Zucman
J.
,
Marineau
C.
,
Hoang-Xuan
K.
,
Demczuk
S.
,
Desmaze
C.
,
Plougastel
B.
,
Pulst
S. M.
,
Lenoir
G.
,
Bijlsma
E.
,
Fashold
R.
,
Dumanski
J.
,
Jong
P.
,
Parry
D.
,
Eldridge
R.
,
Aurias
A.
,
Delattre
O.
,
Thomas
G.
(
1993
).
Alteration in a new gene encoding a putative membrane-organizing protein causes neuro-fibromatosis type 2.
Nature
363
,
515
521
Royer
M.
,
Hong
S. S.
,
Gay
B.
,
Cerutti
M.
,
Boulanger
P.
(
1992
).
Expression and extracellular release of human immunodeficiency virus type 1 gag precursors by recombinant baculovirus-infected cells.
J. Virol
66
,
3230
3235
Sato
N.
,
Yonemura
S.
,
Obinata
T.
,
Tsukita
Sa.
,
Tsukita
Sh
(
1991
).
Radixin, a barbed-end capping actin-modulating protein, is concentrated at the cleavage furrow during cytokinesis.
J. Cell Biol
113
,
321
330
Sato
N.
,
Funayama
N.
,
Nagafuchi
A.
,
Yonemura
S.
,
Tsukita
Sa.
,
Tsukita
Sh
(
1992
).
A gene family consisting of ezrin, radixin and moesin.
J. Cell Sci
103
,
1
13
Smith
D. B.
,
Johnson
K. S.
(
1988
).
Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase.
Gene
67
,
31
40
Trofatter
J. A.
,
MacCollin
M. M.
,
Rutter
J. L.
,
Murrell
J. R.
,
Duryao
M. P.
,
Parry
D. M.
,
Eldridge
R.
,
Kley
M. N.
,
Menon
A. G.
,
Pulaski
K.
,
Haase
V. H.
,
Ambrose
C. M.
,
Munroe
D.
,
Bove
C.
,
Haines
J. L.
,
Martuza
R. L.
,
Mac Donald
M. E.
,
Seizinger
B. R.
,
Short
M. P.
,
Buckler
A. J.
,
Gusella
J. F.
(
1993
).
A novel moesin-, ezrin-, radixin-like gene is a candidate for the neurofibromatosis tumor suppressor.
Cell
72
,
791
800
Tsukita
Sa.
,
Hieda
Y.
,
Tsukita
Sh
(
1989
).
A new 82 kD barbed end-capping protein (radixin) localized in the cell-to-cell adherens junction: Purification and characterization.
J. Cell Biol
108
,
2369
2382
Tsukita
Sh.
,
Itoh
M.
,
Nagafuchi
A.
,
Yonemura
Sh.
,
Tsukita
Sa
(
1993
).
Submembranous junctional plaque proteins include potential tumor molecules.
J. Cell Biol
123
,
1049
1053
Turunen
O.
,
Winqvist
R.
,
Pakkanen
R.
,
Grzeschik
K. H.
,
Wahlström
T.
,
Vaheri
A.
(
1989
).
Cytovillin, a microvillar Mr75,000 protein. cDNA sequence, prokaryotic expression, and chromosomal localisation.
J. Biol. Chem
264
,
16727
16732
Ullrich
S. J.
,
Robinson
E. A.
,
Appella
E.
(
1986
).
Characterization of a chemically homogeneous tumor antigen from a methylcholanthrene-induced sarcoma, meth A.
Mol. Immunol
23
,
545
555
Urushidani
T.
,
Hanzel
D. K.
,
Forte
J. G.
(
1987
).
Protein phosphorylation associated with stimulation of rabbit gastric glands.
Biochim. Biophys. Acta
930
,
209
219
Urushidani
T.
,
Hanzel
D. K.
,
Forte
J. G.
(
1989
).
Characterization of an 80 kD phosphoprotein involved in parietal cell stimulation.
Am. J. Physiol
256
,
1070
–.
Volkman
L. E.
,
Talhouk
S. N.
,
Oppenheimer
D. I.
,
Charlton
C. A.
(
1992
).
Nuclear F-actin: a functional component of baculovirus-infected lepidopteran cells?.
J. Cell Sci
103
,
15
22
Waterston
R.
,
Martin
C.
,
Craxton
N.
,
Huynh
C.
,
Coulion
A.
,
Hillier
L.
,
Durbin
R. K.
,
Green
P.
,
Shownkeen
R.
,
Halloran
N.
,
Hawkins
T.
,
Wilson
R.
,
Berks
M.
,
Du
Z.
,
Thomas
K.
,
Thierry-Mieg
J.
,
Sulston
J.
(
1992
).
A survey of expressed genes in Caenorhabditis elegans.
Nature Genet
1
,
114
123
Yang
Q.
,
Tonks
N. K.
(
1991
).
Isolation of a cDNA clone encoding a human protein-tyrosine phosphatase with homology to the cytoskeletal-associated proteins band 4.1, ezrin, and talin.
Proc. Nat. Acad. Sci. USA
88
,
5949
5953
This content is only available via PDF.