Proc. Nati. Acad. Sci. USA Vol. 88, pp. 8297-8301, October 1991 Cell Biology

Moesin: A member of the protein 4.1-talin-ezrin family of proteins (cell-matrix interaction/heparan sulfate/membrane-cytoskeleton)

WOLFGANG T. LANKES

AND

HEINZ FURTHMAYR

Laboratory of Experimental Oncology, Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305

Communicated by Vincent T. Marchesi, June 7, 1991

ABSTRACT Moesin (membrane-organizing extension spike protein, pronounced md' ez in) has previously been isolated from bovine uterus and characterized as a possible receptor protein for heparan sulfate. We now have cloned and sequenced its complete cDNA, which represents a single 4.2kilobase mRNA encoding a protein of 577 amino acids. It contains no apparent signal peptide or transmembrane domain. In addition, the protein shows significant sequence identity (72%) to ezrin (cytovillin, p81), as well as similarity to protein 4.1 and talin. All of the latter proteins have been postulated to serve as structural links between the plasma membrane and the cytoskeleton. A similar role for moesin is implied by structure and domain predictions derived from the cDNA-deduced peptide sequence. Furthermore, our data indicate that moesin is identical to the 77-kDa band that copurifies with ezrin in its isolation from human placenta [Bretscher, A. (1989) J. Cel Biol. 108, 921-930].

Because of our interest in the molecular mechanisms of the interaction of basement membrane heparan sulfate and cells, we have previously isolated a protein named moesin (membrane-organizing extension spike protein, pronounced mW' ez in). By means of heparin affinity chromatography and gel filtration we purified it from the microsomal fraction of bovine uteri as a double band with an apparent molecular mass of 78 kDa on SDS/PAGE (1). Available data suggested that moesin is a candidate receptor for heparin or heparan sulfate (1). To further investigate this possibility, we have determined the primary structure of moesin,* which reveals a significant homology to the sequences for ezrin (2, 3), protein 4.1 (4), and talin (5). It has recently become clear that these proteins constitute a family with structural and, probably, functional relationships. Ezrin, originally isolated and described as an 80-kDa protein from chicken intestinal microvillar cores (6) and subsequently from human placenta (7), is now known to be identical to the tyrosine kinase substrate p81 (8), to a protein found in chicken erythrocyte marginal bands (9), to cytovillin (10), and to the gastric 80-kDa phosphoprotein (11). It is 37% identical to the N-terminal 300 amino acids of human erythrocyte protein 4.1, an 80-kDa protein shown to be relevant in linking and modifying the interaction of cytoskeletal and integral membrane proteins (12). A similar function, namely linking vinculin to the integrins, and thus the cytoskeleton to extracellular matrix receptors, is assumed for talin (13), which has a mass of 270 kDa but shares 23% N-terminal identity with ezrin (5). As all of these proteins are known to be localized to the submembraneous cytoskeleton, one has to consider a similar localization and function for moesin. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

MATERIALS AND METHODS Protein Purification and Analysis. Moesin was isolated from bovine uteri by a method described earlier (1). The protein was separated on SDS gels into two bands, and twodimensional peptide mapping was performed as described (14), using in-gel 125I labeling with chloramine T, chymotrypsin digestion, and autoradiography. For isolation ofmoesin from HL-60 cells (ATCC CCL 240), 1 x 109 cells were grown in roller bottles and collected by centrifugation at 600 x g for 15 min, washed twice with phosphate-buffered saline; homogenized in 5 ml of buffer containing 100 mM Tris HCI, 100 mM NaCl, 1 mM EDTA, 1 mM phenylmethanesulfonyl fluoride, 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) at pH 7.85; and centrifuged for 10 min at 100,000 rpm in a table-top ultracentrifuge (Beckman, 100.3 rotor). The supernatant was loaded onto a 5-ml heparin-Sepharose column, which was then extensively washed, and bound proteins were eluted with a 50-ml gradient from 0.1 M to 0.8 M NaCI in the same buffer. Further purification was done as described (1). A 10-,ul sample of the fractionated eluate was taken for analysis by SDS/PAGE (15). Moesin was also isolated from human placenta as described (7) with further separation by heparin affinity chromatography. Cell and tissue lysates for Western blots were prepared from the 15,000 x g supernatant of a 1% Nonidet P-40 (NP-40) extract. Equal amounts of protein were subjected to SDS/PAGE and then transferred to reinforced nitrocellulose membranes (Schleicher & Schuell) by using a semidryblotting apparatus (Biometra) (16). After blocking with 3% bovine serum albumin and incubation with primary antibody appropriately diluted in 10 mM Tris-HCl/150 mM NaCl/ 0.05% Tween-20, pH 8.0, the detection was performed with alkaline-phosphatase- or horseradish-peroxidase-conjugated species-specific secondary antibodies (Promega). For N-terminal sequencing, the proteins were separated on an SDS/7% polyacrylamide gel, transferred to polyvinylidene difluoride (PVDF) membrane (Millipore), cut out, and subjected to automated Edman degradation on an Applied Biosystems 470A gas-phase sequencer. Internal peptides were generated by digestion of the PVDF-bound protein with endoproteinase Lys-C (Boehringer), peptide separation by reversephase HPLC, and sequencing as above (J. Fernandez, M. DeMott, and S. M. Mische, personal communication). Antibodies. Mouse monoclonal antibody (mAb) 38/87 (kindly provided by R. Schwartz-Albiez, Heidelberg) was developed by immunization with bovine moesin and has been described earlier (1). Rabbit antisera 90-7 and 90-3 against purified human placenta moesin and ezrin, respectively, were elicited in rabbits by a standard procedure (14). Screening of Libraries, Cloning, and Sequencing. Three positive clones were isolated from 2 x 106 plaques of an *The sequence reported in this paper has been deposited in the GenBank data base (accession no. M69066).

8297

8298

Cell Biology: Lankes and Furthmayr

A 1,m --Kua

E3-is

Proc. Natl. Acad. Sci. USA 88 (1991)

quenced by the dideoxy chain-termination method (21), using Sequenase 2.0 T7 DNA polymerase (United States Biochemical) and deoxyadenosine 5'-a-[35S]thiotriphosphate (Amersham). Sequences were analyzed by using the University of Wisconsin Genetics Computer Group software package (22) on a Digital Equipment VAX computer. Northern Blot Analysis. Total cytoplasmic RNA from cultured cells was isolated by the guanidinium isothiocyanate method (23). Tissue cytoplasmic RNA was purified by a modification of the same method. Poly(A)+ RNA was enriched for with oligo(dT)-cellulose (24). For Northern hybridizations, 10 ,ug of RNA was separated on a 1.2% agarose/ formaldehyde gel with an RNA standard loaded for size assessment (BRL) and then transferred to Hybond N (Amersham) with a vacuum transfer apparatus (Pharmacia LKB). Hybridizations were carried out with a random-primed probe of the insert of clone M3 for 15 hr at 420C in 50%o (vol/vol) formamide/l M NaCl71% SDS/1% dextran sulfate containing salmon sperm DNA at 500 ,.g/ml. Membranes were then washed twice in 2x SSC/0.1% SDS at ambient temperature and twice in 0.1 x SSC/0.1% SDS for 60 min at 600C (1x SSC = 0.15 M NaCl/0.015 M sodium citrate, pH 7.0). In Vitro Translation. We generated a G(5')ppp(5')G (Pharmacia) capped transcript of clone UIII with T3 RNA polymerase (Boehringer) for in vitro translation with rabbit reticulocyte lysate (Promega) in the presence of an amino acid mixture containing [355]methionine. Immunoprecipitation. Immunoprecipitation was carried out in the presence of 1% NP-40 as described earlier (1).

4--78-kDa¢

_

Ezrin

Moesin

B Lower 78-kDa band

Upper 78-kDa band

FIG. 1. Separation and distinction of moesin and ezrin polypeptides. (A) Western blot (Left) of HL-60 total cell lysate fractions eluted from a heparin column. The five fractions correspond, from left to right, to eluates at approximately 0.2, 0.3, 0.4, 0.5, and 0.6 M NaCl of a hinear NaCl gradient. mAb 38/87 (anti-moe~in) and antiserum 90-3 (anti-ezrin) were used simultaneously and visualized with an anti-mhouse lgG conjugate with alkaline phosphatase and with an anti-rabbit IgG conjugate with perxidase. Moesin can be further resolved into two bands (Right) by SDS/PAGE and staining with Coomassie blue. (B) Autoradiography of two-dimensional peptide maps of the upper (Left) and lower (Right) band of moesin obtained by chymotryptic cleavage.

RESULTS Purification of Moesin from HL-60 Cells. Moesin and ezrin both bind to heparin-Sepharose and can be eluted with a gradient of NaCl or increasing concentrations of heparin. The proteins are discriminated by specific antibody mAb 38/87 (anti-moesin) and antiserum 90-3 (anti-ezrin) (Fig. 1A). Both proteins copurify when the protocol for the isolation of ezrin from human placenta is employed, and moesin very likely is identical to the 77-kDa band described by Bretscher (7). After SDS/PAGE and in Western blots, moesin appears as a closely spaced doublet, with the intensity of the fainter upper band varying between tissues (cf. Fig. 6) and different preparations for currently unknown reasons. This is not apparent in Fig. 1A, but when 5% polyacrylamide PAGE is used, these two bands can be separated (Fig. 1B and Fig. 6). When the proteins are subjected to "2I-labeling and chymotrypsin digestion, the two-dimensional peptide maps shown in Fig. 1B are obtained. Only minor differences are observed, indicating near identity of the two bands. Ezrin; on the other hand, can be easily distinguished from moesin by onedimensional peptide mapping with V8 protease (data not shown). In contrast to Western blotting, immunoprecipitation of HL-60 cell lysate with antibodies to ezrin or moesin

HL-60 cDNA library in A Zap (Stratagene) (kindly provided by T. Leto, National Institutes of Health) (18) that were immunoscreened with mAb 38/87 and anti-mouse IgG conjugated to alkaline phosphatase (19). Subsequently, another 14 clones were identified by hybridizing the same library with a [12P~dCTP random-pri'med probe generated from -the insert of an antibody-screen-positive clone. To obtain the 5' untranslated re~gion, we had to additionally employ a sizeselected [3- to 4.5-kilobase (kb)] HL-60 cDNA library, constructed in A Uni-Zap (Stratagene) (kindly provided by P. Murphy, National Institutes -of Health).- Single plaques were used to generate pBluescript SK+ plasmids (Stratagene) by superinfection and excision with the R408 helper phage. For complete sequencing, exoniuclease 111/51 nuclease nested deletions of the insert from both T3 and T7 ends (20), as well as three oligonucleotide sequencing primers, were generated. Denatured double-stranded plasmid templates were seSr-_x =

cr

rz

13

c

(O

mz

(

:c

to (i

>

()

O u

E~-~ --

--

5,

--

--

3,

M3 M2 M7 Fragment used as probe to rescreen

M13z

M3z M4z M2z M7z M8z

UIII

kb l 0.5

l 1

l

l

l

15

2

2.5

l 3

3.5

FIG. 2. Restriction map of full-length cDNA and fragments obtained from library screening. The open reading frame is boxed. Three clones obtained from HL-60 cDNA library screening with mAb 38/87 (M2, M3, and M7) are shown, along with 6 of the other 14 clones (Mlz-M14z) isolated by probe hybridization and clone UIII from a size-selected library.

Cell Biology: Lankes and Furthmayr

Proc. Natl. Acad. Sci. USA 88 (1991)

8299

A M

21

P

K

T

S

V

47 87

14 1

GCAGTACCAGGACACTKAGGTTTCTCCACCTGGCTGACTCAATAAGAAGGTGACTGCCCAGGATGTGCGGAAGGQAGCCCCCTGCTCTTTAGTTCCGTGCCAGTTCTACCCPGA Q Y Q D T K G F S T W r -IX L N K K V T A Q D V R K E S P L L F K F R A K F Y P E

261

GGATGTGTCCGAGGAATTGATTCAGGACATCACTCAGCGCCTGTTCTTTCTGCAAGTGAAAGAGGGCATTCTCAATGATGATATTTACTGCCCGCCTGAGACCGCTGTGCTGCTGGCCTC

381

501

GTATGCTGTCCAGTCTAAGTATGGCGACTTCAATAAGGAAGTGCATAAGTCTGGCTACCTGGCCGGAGACAAGTTGCTCCCGCAGAGAGTCCTGGACAGCACAAACTCAACAGGACCA Y A V Q S K Y G D F N K E V H K S G Y L A G D K L L P Q R V L E O H K L N K D Q GTGGGAGGAGCGGATCCAGGTGTGGCATGAGGAACACCGTGGCATGCTCAGGGAGGATGCTGTCCTGGAATATCTGAGATTGCTCAAGATCTGGAGATGTATGGTGTGAACTACTTCAG

621

CATCAAGAACAAGAAAGGCTCAGAGCTGTGGCTGGGGGTGGATGCCCTGGGTCTCAACATCTATGAGCAGAATGACAGACTAACTCCCAAGATAGGCTTCCCCTGGAGTGAAATCAGGAA

D

W

V

E

S

E

E

R

E

L

I

QD

Q V N H

I

I

E

T Q R

E

H

R

L

G

F

M

F

L

L Q V

R

E

D

K

A

G

E

V

L

I

E

L

N

Y

L

D

K

D

I

I

Y

C

P

A Q D

P

L

E

E

M

T

Y

A

G

V

V

L

L

N

Y

A

F

S

S

207 247

741

861

CCATGAACTATACATGCGCCGTCGCAAGCCTGATACCATTGAGGTGCAGCAGATGAAGGCACAGGCCCGGGAGGAGAAGCACCAGAAGCAGATGGAGCGTGCTATGCTGGAAAATGAGAA H

E

L

Y

R

M

R

E

L

N L

G

V

D

A

L

L

G

N

I

E Q N

Y

D

R

L

T

P

K

I

G

F

P

W

S

E

I

R

ATTTGTCATCAAGCCCATTGACAAAAAAGCCCCGGACTTCGTCTTCTATGCTCCCCGGCTGCGGATTAACAAGCGGATCTTGGCCTTGTGCATGGGGAA F

V

R K

I

P

K

P

D

T

I

I

D

E

K K A

D

P

F

F

V

V Q Q H K A Q A

Y R

P

A

R

E K

E

L

R

HQ

I

N

KQ

K M

R E

I R

L A

A

L

C

L

E

Q Q

E

M

M

G

N

E

K

E

E

N

981

GAAGAAGCGTGAAATGGCAGAGAAGGAGAAAGAGAAGATTGAACGGCGAACGGAGGAGCTGATGGAGAGGCTGAAGCAGATCGAGGAACAGACTAAGAACGCTCAGCAAGAACTGGAAGA

1101

ACAGACCCGTAGGGCTCTGGAACTTGAGCAGGAACGGAAGCGTGCCCAGAGCGAGGCTGAAAAGCTGGCCAAGGAGCGTCAAGAAGCTGAAGAGGCCAAGGAGGCCTTGCTGCAGGCCTC

K

K

Q T

R

R

E

R

M

A

L

A

E

E

X

L

E

E

X

E

K

Q E

R

I

K

E R

R

E

A Q

E

K S

E

E

A

L

E

M

E K

L

R

A

K Q

L

E

K

I

E

R Q E

E A

Q T E

E

K A

K K

A E

A

L

L

L Q

A

S

1221

CCGGGACCAGAAAAAGACTCAGGAACAGCTGGCCTTGGMAATGGCAGAGCTGACAGCTCGMATCTCCCAGCTGGAGATGGCCCGACAGAAGAAGGAGAGTGAGGCTGTGGAGTGGCAGCA

1341

GAAGGCCCAGATGGTACAGGAAGACTTGGAGAAGACCCGTGCTGAGCTGikGACTGCCATGAGTACACCTCATGTGGCAGAGCCTGCTGAGAATGAGCAGGATGAGCAGGATGAGAATGG

R

K

Q Q

K

M

K

V

T

Q

0o

R

E

L

D

T.

E

L K

A

K

T

R

M

A

A

E

E

L

L

T

K

A

R

A N

T

SO

T

S

T

P

TH

M

V

A

A

R

E

P

O K

A

E

K N

E

E

E

S

Q

D

A E

V

0

E D

W E

0 0 N

G.

287 327 367 407 447

487

GGCAGAGGCTAGTGCTGACCTACGGGCTGATGCTATGGCCAAGGACCGCAGTGAGGAGGAACGTACCACTGAGGCAGAGAAGAATGAGCGTGTGCAGAAGCACCTGAAGGCCCTCACTTC A

1581

D

A

127 167

N

I K N K K G S CATCTCTTTCAATGATAAGA I S F N D K K

1461

7

GCGTGTGACCACCATGGATGCAGAGCTGGAGTTTGCCATCCAGCCCAACACCACCGGGAAGCAGCTATTTGACCAGGTGGTGAAAACTATTGGCTTGAGGGAAGTTTGGTTCTTTGG TC T R V T T_ M D A F. r F A T 0 P N T T G K °0 L F D V V K T I C T. R E V W F F G L

E

A

S

A

D

L

R

A

D

A

M

A K

D

R

S

E

E

E

R

T

T

E

A

E

K N

E

R

V Q

K

H

L

K

A

L

T

S

527

GGAGCTGGCCAATGCCAGAGATGAGTCCAAGAAGACTGCCAATGACATGATCCATGCTGAGAACATGCGACTGGGCCGAGACAAATACAAGACCCTGCGCCAGATCCGGCAGGGCAACAC E

L

A

N

A

R

D

E

S

K

K

T

A

N

D

M

I

H

A

E

N R

N

L

G

R

D

K

Y

K

T

L

R Q

I

R Q G

N

T

567

1701 CAAGCAGCGCATTGACGAATTTGAGTCTATGTAATGGGCACCCAGCCTCTAGGGACCCCTCCTCCCTTTTTCCTTGTCCCCACACTCCTACACCTAACTCACCTAACTCATACTGTGCTG K Q R I D E F E S M 1821 GAGCCACTAACTAGAGCAGCCCTGGAGTCATGCCAAGCATTTAATGTAGCCATGGGACCAAACCTAGCCCCTTAGCCCCCACCCACTTCCCTGGGCAAATGAATGGCTCACTATGGTGCC 1941 AATGGAACCTCCTTTCTCTTCTCTGTTCCATTGAATCTGTATGGCTAGAATATCCTACTTCTCCAGCCTAGAGGTACTTTCCACTTGATTTTGCAAATGCCCTTACACTTACTGTTGTCC 2061 TATGGGAGTCAAGTGTGGAGTAGGTTGGMAGCTAGCTCCCCTCCTCTCCCCTCCACTGTCTTCTTCAGGTCCTGAGATTACACGGTGGAGTGTATGCGGTCTAGGAATGAGACAGGACCT 2181 AGATATC TTCT CCAGGGATGTCAACTGACCTAAAATTTGCCCTCCCATCCCGTTTAGAGTTATTTAGGCTTTGTAACGATTGGGGGAATAAAAGATGTTCAGTCATTTTTGTTTCTACC 2301 TCCCAGATCGGATCTGTTGCAAACTCAGCCTCAATAAGCCTTGTCGTTGACTTTAGGGACTCAATTTCTCCCCAGGGTGGATGGGGGAAATGGTGCCTTCAAGACCTTCACCAAACATAC 2421 TAGAAGGGCATTGGCCATTCTATTGTGGCAAGGCTGAGTAGAAGATCCTACCCCAATTCCTTGTAGGAGTATAGGCCGGTCTAAAGTGAGCTCTATGGGCAGATCTACCCCTTACTTATT 2541 ATTCCAGATCT GCAGTCAC TTCG TGGGATCTGC CCCTCCCTGCTTCAATACCCAAATCCTCTCCAGCTATAACAGTAGGGATGAGTACCCAAAAGC TCAGCCAGC CC CATCAGGACT CT T 2661 GTGAAAAGAGAGGATATGTTCACACCTAGCGTCAGTATTTTCCCTGCTAGGGGTTTTAGGTCTCTTCCCCTCTCAGAGCTACTTGGGCCATAGCTCCTGCTCCACAGC CATCCCAGCC-TT 2781 GGCATCTAGAGCTTGATGCCACTAGGCTCAACTAGGGAGTGAGTGCAAAAAGCTGAGTATGGTGAGAGAAGCCTGTGCCCTGATCCAAGTTTACTCAACCCTCTCAGGTGACCAAAATCC 2901 CCTTCTCATCACTCCCCTCAAAGAGGTGACTGGGCCCTGCCTCTGTTTGACAAACCTCTAACCCAGGTCTTGACACCAGCTGTTCTGTCCCTTGGAGCTGTAAACCAGAGAGCTGCTGGG 3021 GGATTCTGGCCTAGTCCCTTCCACACCCCCACCCCTTGCTCTCAACCCAGGAGCATCCACCTCCTTCTCTGTCTCATGTGTGCTCTTCTTCTTTCTACAGTATTATGTACTCTACTGATA 3141 TCTAAATATTGATTTCTGCCTTCCTTGCTAATGCACCATTAGAAGATATTAGTCTTGGGGCAGGATGATTTTGGCCTCATTACTTTACCACCCCCACACCTGGAAAGCATATACTATATT 3261 ACAAAATGACATTTTGCCAAAATTATTAATATAAGAAGCTTTCAGTATTAGTGATGTCATCTGTCACTATAGGTCATACAATCCATTCTTAAAGTACTTGTTATTTGTTTTTATTATTAC 3381 TGTTTGTCTTCTCCCCAGGGTTCAGTCCCTCAAGGGGCCATCCTGTCCCACCATGCAGTGCCCCCTAGCTTAGAGCCTCCCTCAATTCCCCCTGGCCACCACCCCCCACTCTGTGCCTGA 3501 3621 3741 CAGTGCCAATAAAATTTAGGTGACTTCAAAAAAAAAAAA

B 50

100

200

150

5.0

KD Hydrophilicity

1

r rVv

'

T

-

250

_AC

300

350

400

450

500

550

/3

A

-5. 0

CF Turns AA

LA

A

A

--Ulv

CF Alpha Helices

v

LJ-

CF Beta Sheets

Glycosyl. Sites 0

50

100

150

200

250

300

350

400

450

500

550

FIG. 3. Primary and secondary structure prediction of human moesin. (A) Nucleotide sequence of the cDNA and deduced amino acid Underlined are residues verified by N-terminal sequencing of the calf moesin and peptides of HL-60 moesin. (B) Hydrophilicity plot of moesin based on the program of Kyte and Doolittle (KD) and secondary structure predictions by Chou-Fasman (CF) analysis. Potential glycosylation sites are also indicated. sequence.

in the presence of 1% NP-40 apparently shows partial coprecipitation of the two proteins (see Fig. 5). This may be due to weak crossreactivity of the antisera because of the close structural relationship of the proteins. Isolation and Characterization of cDNA. The relationship of three clones from screening a random-primed HL60 cDNA library with mAb 38/87 and 6 of the 14 additional clones obtained by probe hybridization of the same library is shown in Fig. 2. All isolates were analyzed by restriction mapping and showed consistent overlaps, indicating the presence of only one mRNA with no hint of alternative splicing. Previously obtained N-terminal peptide sequencing data of moesin (see Fig. 3A) and Northern blotting (see Fig. 6) implied that we were unable to isolate a full-length cDNA from that source. We finally probe-screened a size-selected (3.0- to 4.5-kb) oligo(dT)-primed HL-60 cDNA library to obtain clone UIII, which is identical to the complete cDNA (Fig. 2). The

nucleotide sequence of moesin cDNA (Fig. 3A) consists of a 100-nucleotide 5' untranslated region followed by a 1731nucleotide open reading frame, thus coding for 577 residues, and a 3' untranslated region of 2145 nucleotides. The translation initiation site (GCCGCCACCATGC) is identical to the consensus for eukaryotic mRNA (25). The same ATG codon has been described as the initiation site for ezrin (2). Interestingly, a sequence 20 nucleotides downstream contains a second in-frame initiation site (GTGTGACCACCATGG), which has been postulated as the primary initiation site for cytovillin (3). In our protein chemical studies, however, we did not find indications for a protein lacking the N-terminal 11 residues. The polyadenylylation signal (AATAAA) is found 14 nucleotides downstream from what is apparently the beginning of the poly(A) tail. Primary Structure of Moesin. Human moesin cDNA encodes a unique protein of 577 amino acids with a pI of 6.35

8300

Proc. Natl. Acad. Sci. USA 88 (1991)

Cell Biology: Lankes and Furthmayr

been found in other proteins known to bind heparin (26), and they may represent heparin binding sites in moesin. Two cysteines at positions 117 and 284 are probably not linked via a cystine bond, as, at least at the level of analysis by SDS/ PAGE, the reduced and nonreduced proteins show identical electrophoretic motility. Finally, two possible N-glycosylation

E

moesin ezrin

moesin ezrin

moesin ezrin moesin ezrin

10 30 50 PKTISVRVTTMDAELEFAIQPNTTGKQLFDQVVKTIGLREVWFFGLQYQDTKGFSTWLKL Y

P N

H V N

P

70 90 110 NKKVTAQDVRKESPLLFKFRAKFYPEDVSEELIQDITQRLFFLQVKEGILNDDIYCPPET D S E N Q A S E K 130 150 170

AVLLASYAVQSKYGDFNKEVHKSGYLAGDKLLPQRVLEQHKLNKDQWEERIQVWHEEHRG G

A F

SSER I

Y

MD

TR

D

A

190 210 230 MLREDAVLEYLKIAQDLEMYGVNYFSIKNKKGSELWLGVDALGLNIYEQNDRLTPKIGFP I

KDN M

TD

E

KD K

270

250

290

moesin ezrin

WSEIRNISFNDKKFVIKPIDKKAPDFVFYAPRLRINKRILALCMGNHELYMRRRKPDTIE Q

moesin ezrin

310 330 350 VQQMKAQAREEKHQKQMERAMLENEKKKREMAEKEKEKIEREKEELMERLKQIEEQTKKA R TV R L QQ T QMM L QDY K

moesin

370 390 410 QQELEEQTRRALELEQERKRAQSEAEKLAKERQEAEEAKEALLQASRDQKKTQEQLALEM

ezrin

ER

moesin

470 450 AELTARISQLEMARQKKESEAVEWQQKAQMVQEDLEKTRAELKTAMSTPHVAEPAENE..

ezrin

S Y

IQ 430

E

Q

E

K AL

RR

E

D VE

E ERQAV

R EAD MA LR

HR KEA D

V

KE

A L

HLV TA PPPP PVY PV

510

490

I S

530

moesin

.... QDEQDENGAEA ...SADLRADAMAKDRSEEERTTEAEKNERVQKHLKALTSELANA

ezrin

PTGY E SSEGIRD N K I SYHV ESLQDE 550 570 RDESKKTANDMIHAENMRLGRDKYKTLRQIRQGNTKQRIDEFESM

moesin ezrin

A N R H

I

N

Q

RQ VT S

SQ

AL

FIG. 4. Homologies of moesin with other proteins. (A-D) Graphic representation of amino acid sequence comparison of human moesin (vertical) with itself (A), human ezrin (B), human band 4.1 (C), and residues 150-730 of mouse talin (D) (all horizontal). (E) Optimal alignment of human moesin to human ezrin, with only the differences noted in ezrin.

(Fig. 3A). We have confirmed the HL-60 cDNA derived peptide sequence by amino acid sequence analysis of the lower band of purified HL-60 moesin (residues 1-15) and human placental moesin (residues 1-15, 53-59, and 413-434). Furthermore, the 38 N-terminal amino acids match the bovine sequence with the exception of position 5 (bovine moesin has serine). Although there are stretches of hydrophobic residues (starting at residues 120 and 213), moesin contains neither an amphipathic helix nor a signal peptide nor a transmembrane domain (Fig. 3B). The predicted secondary structure displays characteristics that allow dividing the protein into three structural domains (Fig. 3C). The N-terminal 300 amino acids are relatively more hydrophobic and contain all but 1 of 13 tyrosine residues. Amino acids 300-550 include an abundance of charged amino acids and most likely form an a-helical segment. They are followed by a small nonhelical C-terminal tail. The sequence features multiple clusters of up to four basic residues (BBBB) and regions of the general structure (XBXBXBX), (XBBXBX), and (XBXBBX). Such clusters have

sites are present. Homologies of moesin to itself are found within the a-helical region, but we could not attribute them to internal repeats as has been observed for ezrin (2). They are rather due to an abundance of charged residues. Moesin shows a 72% amino acid identity with ezrin (Fig. 4A) and homologies to protein 4.1 and talin. Whereas the homology to ezrin covers the entire length of the protein, with the highest variability seen in the a-helical portion, protein 4.1 and talin are most closely related to moesin in their respective N-terminal domain (Fig. 4B). A striking heptaproline sequence, residues 469-475 in ezrin, is not observed in moesin (Fig. 4A). In Vitro Translation. Because of the considerable discrepancy of calculated molecular mass (67,820 Da) and that determined from SDS/PAGE (-78,000 Da) we have translated full-length cDNA-derived RNA in vitro and found comigration of the immunoprecipitated product with that of cultured HL-60 cells on SDS/PAGE, representing the lower band of moesin. The reduced mobility may be due to the high amount of charged amino acids in the helical region of the protein. Antibodies against ezrin do not react with the in vitro translation product (Fig. 5). mRNA Expression in Cells and Tissues. By immunofluorescence and Western blotting analysis of cells and tissue we became aware of the widespread distribution of moesin. mAb 38/87 reacted with a 78-kDa double band of various intensities on Western blots of a variety of bovine tissue lysates. Similarly, we were able to detect the 4.2-kb moesin mRNA in Northern blots of a wide variety of cells and tissues (Fig. 6). In contrast to ezrin these include heart, skeletal muscle, and a comparatively strong signal in spleen. Induction of HL-60 cells by various agents (dimethyl sulfoxide, phorbol 12-tetradecanoate 13-acetate, and dibutyryl cAMP) did not change the level of expression or the size of the mRNA. A single band of 4.2 kb was found not only in human, but also in rat, mouse, and calf (data not shown).

DISCUSSION The results presented in this paper allow for the molecular characterization of moesin as another member of the ezrinprotein 4.1-talin family of proteins. In spite of the high degree of identity of ezrin and moesin, we were able to elicit specific antibodies in rabbits that allow us to clearly distinguish these two proteins on Western blots of a variety of mammalian tissues. Analysis of the two 78-kDa bands of moesin by peptide mapping shows their near identity, with minor differences probably attributable to posttranslational modifications, if not to technical limitations of the method. This is confirmed by the fact that neither in library screening nor in Northern blotting have we found indications for a related gene or an alternatively spliced form, as has been described for protein 4.1 (27). Previous studies (2) have shown that ezrin mRNA is detected as a transcript of 3.2 kb, with three other RNA species of unknown nature also hybridizing, in kidney, intestine, skin, and lung, but low in heart, brain, and spleen and not at all in testes, skeletal muscle, or liver. In contrast, moesin mRNA is found as a single 4.2-kb transcript in muscle, heart, spleen, and all other organs and cultured cells studied. Considering the structural relationship to ezrin, protein 4.1, and taln, and the functions that have been assigned to these proteins, a similar role might be assumed for moesin. The highly homologous N-terminal portion of protein 4.1 has been assigned an important role in the phosphatidylinositol phos-

Cell Biology: Lankes and Furthmayr 1

2

4

3

5

kDa

- 180 - 116

_ _ --

84

FIG. 5. Immunoprecipitation of

[355]methionine-labeled proteins. HL-60 total cell lysate in 1% NP40

- 58 - 48.5

(lanes 1, 4, and 5) and in vitro translation products of moesin mRNA transcribed from clone UIRI (lanes 2 and 3) were precipitated with rabbit anti-moesin 90-7 (lanes 1, 2, and 5) and anti-ezrin 90-3 (lanes 3 and 4).

- 36.5 loop

phate-regulated binding to glycophorin in human erythrocytes (12, 28). In talin the homologous domain might be responsible for the binding to integrins (5). The helical rodlike structure is also characteristic for ezrin, protein 4.1, and talin. This region has been shown to be important in binding of the spectrin/ actin complex to protein 4.1 (29) and is thought to also mediate the interaction of vinculin with talin (5). It is important to point out that homology of this helical segment to the other proteins of the family exists mainly with respect to the predicted secondary structure, while similarity of the primary structure is limited. One might also speculate that this could provide the molecular basis for specificity of binding to an as-yetunidentified protein. Moreover, the structural differences and resulting selective binding may be critically important for the different subcellular localizations found for ezrin and moesin in tissue (ref. 1; M. Amieva, W.T.L., and H.F., unpublished results). Immunofluorescence studies of cultured cells, such as primary cultures of aortic smooth muscle cells, A431, or phorbol 12-tetradecanoate 13-acetate-induced HL-60 cells, indicate that both ezrin and moesin are distributed diffusely on or near the cell membrane with significant preference for retraction fibers, blebs, microspikes, filipodia, and lamellipodia (refs. 1, 6, 7, and 30; M. Amieva, W.T.L., and H.F., unpublished observations). These cell surface protrusions have been related to exploration (31), attachment and cell movement in vitro (32), and events in epithelial-mesenchymal transformations during development (33). The cDNA data presented here do not favor the model of moesin as a typical receptor molecule. They leave unaccounted for why moesin is found on the surface of cells in culture and why antibodies to moesin exhibit biological effects (1). Our previous findings can be explained as a phenomenon caused by the high surface activity ofcultured cells. A working hypothesis is that cells bud off or shed part of the aforementioned membrane protrusions such as microspikes and filipodia, which then release their contents (34), allowing intracellular proteins to bind to the cell surface. This mechanism, with 1

2

3

4

5

6

7

_-_u

q

8

9

.

10 -4.2kb

- 78 kDa

*

.

FIG. 6. Northern and Western blot analysis of moesin mRNA and protein in bovine tissues and HL-60 cells. Lanes: 1, intestine; 2, brain; 3, heart; 4, skin; 5, kidney; 6, muscle; 7, lung; 8, liver; 9, spleen; 10, HL-60 poly(A)+ RNA. (Upper) The cDNA fragment hybridizes to a single mRNA 4.2 kb in size in every tissue. (Lower) mAb 38/87 detects a double band at 78 kDa in the tissues. The asterisk indicates that no complementary Western blot of that tissue was available.

Proc. Nati. Acad. Sci. USA 88 (1991)

8301

intriguing functional implications, is being discussed for a number of other extracellular proteins without a signal or transmembrane sequence, including heparin-binding fibroblast growth factor, interleukin 1 (17, 35), and annexin 1 (34). More detailed studies on the functions of ezrin and moesin, their changes in cellular distribution, and the role of phosphorylation and binding to other cellular components and heparan sulfate will be useful in understanding basic cellular processes. The cloning of moesin now enables us to address these questions by employing mutated proteins and their expression in eukaryotic cells. We thank T. Leto and H. Malech for the HL60 A Zap expression library, P. Murphy for the size-selected A Uni-Zap library, T. Hunter for the ezrin cDNA clone, A. Bretscher for a sample of anti-ezrin antibodies, R. Schwartz-Albiez for mAbs to moesin, B. Wolf and S. Mische for amino acid sequence analysis, and B. Dsupin for technical assistance. This work was supported by the U.S. Public Health Service. W.T.L. is the recipient of a Boehringer-Ingelheim Foundation Fellowship. 1. Lankes, W., Griesmacher, A., Grfnwald, J., Schwartz-Albiez, R. & Keller, R. (1988) Biochem. J. 251, 831-842. 2. Gould, K. L., Bretscher, A., Esch, F. S. & Hunter, T. (1989) EMBO J. 8, 4133-4142. 3. Turunen, O., Winqvist, R., Pakkanen, R., Grzeschik, K., Wahlstr6m, T. & Vaheri, A. (1989) J. Biol. Chem. 264, 16727-16732. 4. Conboy, J., Kan, Y. W., Shohet, S. B. & Mohandas, N. (1986) Proc. Nat!. Acad. Sci. USA 83, 9512-9516. 5. Rees, D. J. G., Ades, S. E., Singer, S. J. & Hynes, R. 0. (1990) Nature (London) 347, 685-689. 6. Bretscher, A. (1983) J. Cell Biol. 97, 425-432. 7. Bretscher, A. (1989) J. Cell Biol. 108, 921-930. 8. Gould, K. L., Cooper, J. A., Bretscher, A. & Hunter, T. (1986) J. Cell Biol. 102, 660-669. 9. Birgbauer, E. & Solomon, F. (1989) J. Cell Biol. 109, 1609-1620. 10. Pakkanen, R., Hedman, K., Turunen, O., Wahistrom, T. & Vaheri, A. (1987) J. Histochem. Cytochem. 35, 809-816. 11. Forte, J. G., Hanzel, D. K., Okamoto, C., Chow, D. & Urushidani, T.

(1990) J. Intern. Med. Suppl. 732, 7-26. 12. Anderson, R. A. & Marchesi, V. T. (1985) Nature (London) 318, 295298. 13. Burridge, K. & Mangeat, P. (1984) Nature (London) 308, 744-746. 14. Saku, T. & Furthmayr, H. (1989) J. Biol. Chem. 264, 3514-3523. 15. L4emmli, U. K. (1970) Nature (London) 227, 680-685. 16. fowbin, H., Staehelin, T. & Gordon, J. (1979) Proc. Natl. Acad. Sci. USA 76, 4350-4354. 17. Muesch, A., Hartmann, E., Rohde, K., Rubartelli, A., Sitia, R. & Rapoport, T. A. (1990) Trends Biochem. Sci. 15, 86-87. 18. Lomax, K. J., Leto, T. J., Nunoi, H., Gallin, J. I. & Malech, H. L. (1989) Science 245, 409-412. 19. Huynh, T. V., Young, R. A. & Davis, R. W. (1985) in DNA Cloning: A Practical Approach, ed. Glover, D. M. (IRL, Oxford), Vol. 1, pp. 49-78. 20. flenikoff, S. (1984) Gene 28, 351-359. 21. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Nat!. Acad. Sci. USA 74, 5463-5467. 22. Devereux, J., Haeberli, P. & Smithies, 0. (1984) Nucleic Acids Res. 12, 387-395. 23. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J. & Rutter, W. J. (1979) Biochemistry 18, 5294-5301. 24. Schwab, M., Alito, K., Varmus, H. E., Bishop, J. M. & George, D. (1983) Nature (London) 303, 497-501. 25. Kozak, M. (1984) Nucleic Acids Res. 12, 857-872. 26. Cardin, A. D. & Weintraub, H. J. R. (1989) Arteriosclerosis 9, 21-32. 27. Tang, K. T., Qin, Z., Leto, T., Marchesi, V. T. & Benz, E. (1990) J. Cell Biol. 110, 617-624. 28. Leto, T. L., Correas, I., Tobe, T., Anderson, R. A. & Home, W. C. (1986) in Membrane Skeletons and Cytoskeletal Membrane Associations, eds. Bennett, V., Cohen, C. M., Lux, S. E. & Palek, J. (Liss, New York), pp. 201-209. 29. Correas, I., Leto, T. L., Speicher, D. W. & Marchesi, V. T. (1986) J. Biol. Chem. 261, 3310-3315. 30. Pakkanen, R. (1988) J. Cell. Biochem. 38, 65-75. 31. Albrecht-Buehler, G. in Cell Motility, eds. Goldman, R., Pollard, T. & Rosenbaum, J. (Cold Spring Harbor Lab., Cold Spring Harbor, NY), Book A, pp. 247-264. 32. Taylor, A. C. & Robbins, E. (1963) Dev. Biol. 7, 660-673. 33. Hay, E. D. (1989) Cell Motil. Cytoskeleton 14, 455-457. 34. Christmas, P., Callaway, J., Fallon, J., Jones, J. & Haigler, H. T. (1991) J. Biol. Chem. 266, 2499-2507. 35. Rubartelli, A., Cozzolino, F., Talio, M. & Sitia, R. (1990) EMBO J. 9, 1503-1510.