Radixin Is a Novel Member Ofthe Band 4.1 Family

Radixin Is a Novel Member ofthe Band 4.1 Family Noriko Funayama,*t Akira Nagafuchi,* Naruki Sato,* Sachiko Tsukita,* and Shoichiro Tsukita* * Department ofInformation Physiology, National Institute for Physiological Sciences, Okazaki, Aichi 444; and t Department of Biochemistry, Faculty ofMedicine, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, Japan

Abstract. Radixin is an actin barbed-end capping pro- demonstrated that mouse radixin shares 75.3% identity tein which is highly concentrated in the undercoat of with human ezrin, which was reported to be a mem- the cell-to-cell adherens junction and the cleavage fur- ber of the band 4.1 family. We then isolated the row in the interphase and mitotic phase, respectively cDNA encoding mouse ezrin. Sequence analysis and (Tsukita, Sa., Y Hieda, and Sh. Tsukita. 1989a. J. Cell Northern blot analysis revealed that radixin and ezrin Downloaded from http://rupress.org/jcb/article-pdf/115/4/1039/1062193/1039.pdf by guest on 24 September 2021 Biol. 108:2369-2382 ; Sato, N., S. Yonemura, T. are similar but distinct (74.9% identity), leading us to Obinata, Sa. Tsukita, and Sh. Tsukita. 1991. J. Cell conclude that radixin is a novel member of the band Biol. 113 :321-330). To further understand the struc- 4.1 family. In erythrocytes the band 4.1 protein acts as ture and functions of the radixin molecule, we isolated a key protein in the association of short actin filaments and sequenced the cDNA clones encoding mouse with a plasma membrane protein (glycophorin), to- radixin. Direct peptide sequencing of radixin and im- gether with spectrin. Therefore, the sequence similar- munological analyses with antiserum to a fusion pro- ity between radixin and band 4.1 protein described in tein were performed to confirm that the protein en- this study favors the idea that radixin plays a crucial coded by these clones is identical to radixin. The role in the association of the barbed ends of actin fila- composite cDNA is 4,241 nucleotides long and codes ments with the plasma membrane in the cell-to-cell for a 583-amino acid polypeptide with a calculated adherens junction and the cleavage furrow. molecular mass of 68.5 kD. Sequence analysis has

HE cell-to-cell adherens junction (AJ)' is a special- win et al., 1986; Geiger, 1979 ; Geiger et al., 1984; Laza- ized region of the plasma membrane, where cadherin rides and Burridge, 1975). Recently, we developed an isola- molecules (uvomorulin, L-CAM, A-CAM, etc.) work tion method for AJ from rat liver to open the way for the Tas adhesion molecules, and actin filaments are densely as- systematic analysis of the molecular organization of its un- sociated with plasma membrane through a well-developed dercoat (Tsukita and Tsukita, 1989). From the isolated AJ, plasmalemmal undercoat (Farquhar and Palade, 1963; Gal- three novel undercoat-constitutive proteins-tenuin, radixin, lin et al., 1987; Geiger, 1983; Ringwald et al., 1987; Staehe- and the 102-kD cadherin-associated protein (CAP102)- lin, 1974 ; Takeichi, 1988, 1990; Volk and Geiger, 1984). It have been purified and characterized (Nagafuchi et al., 1991; is now widely accepted that this type ofjunction is important Tsukita et al., 1989a,b). Most recently, taking advantage of for the formation and maintenance of tissues as well as for the isolated AJ, it was shown that specific protooncogenic the transformation and metastasis of cancer cells. Recently, tyrosine kinases of src family are highly enriched in AJ (Tsu- the cell adhesion function of cadherin molecules was kita et al., 1991). reported to be regulated through the association of some cy- The manner of association of actin filaments with the toplasmic proteins with the cytoplasmic domain of cadherin plasma membrane in AJ in general (end-to-membrane or molecules (Nagafuchi and Takeichi, 1988, 1989; Ozawa et side-to-membrane fashion) remains to be clarified . How- al., 1989) . Therefore, to understand the functions of AJ in ever, it is clear that, at least in AJ of cardiac muscle cells, molecular terms, it is prerequisite to clarify the molecular all actin filaments are bound to the undercoat in an end-to- architecture of the undercoat of AJ, i.e., the molecular link- membrane fashion by their barbed ends. Therefore, one of age from cadherin molecules to actin filaments. Recent the interesting approaches to the molecular linkage between studies have revealed that the molecular organization of the cadherin and actin filaments from the actin side appears to undercoat of AJ is not as simple as had been expected (Tsu- be to identify a barbed-end capping protein in the AJ kita et al ., 1990) . Through immunohistochemical studies undercoat-constitutive proteins. Among the constituents of many AJ undercoat-constitutive proteins such as a-actinin, the AJ undercoat so far reported, only radixin shows the abil- vinculin, filamin, and placoglobin have been identified (Co- ity to cap the barbed ends of actin filaments (Tsukita et al ., 1989a). Most recently, radixin was reported to be highly 1. Abbreviation used in this paper: AJ, adherens junction . concentrated at the cleavage furrow during cytokinesis (Sato

© The Rockefeller University Press, 0021-9525/91/11/1039/10 $2 .00 TheJournal of Cell Biology, Volume 115, Number4, November 1991 1039-1048 1039 et al ., 1991) . Considering that the most prominent feature Isolation ofRNA and Northern Blot Hybridization shared by AJ and the cleavage furrow is the tight association Total RNAs from cultured F9 cells were isolated according to the method of actin filaments with plasma membranes, it is tempting to for rapid isolation of total RNA frommammalian cells described by Mania- speculate that radixin may play a crucial role in directly or tis et al . (1989) . Approximately 8 i~g per well of RNA was subjected to elec- indirectly binding the barbed end of actin filaments to the trophoresis and blotted onto nitrocellulose membrane . RNA ladder plasma membrane . (Bethesda Research Laboratories, Bethesda, MD) was used as size marker. Mouse radixin cDNA R2 (1 .2 kb) and mouse ezrin cDNA (2 .5 kb) were In the present study, to clarify the structure and functions labeled with [a- 32P]dCTP using the Random Primer DNA Labeling Kit of radixin molecules in more detail, we have attempted to (Takara Shuzo Co ., LTD., Kyoto, Japan), and were used as probes. Hybrid- isolate and sequence cDNA encoding mouse radixin . The ization was carried out at high stringency (50% formamide/IOX Denhart's deduced amino acid sequence of radixin has led us to con- solution without BSA/5 x SSC/50 mM phosphate buffer [pH 6.5]/100 pg/pl boiled salmon sperm clude that radixin is homologous with but distinct from ezrin DNA) . which is reported to be localized immediately under the membrane of the microvilli (Bretscher, 1983 ; Gould et al ., Production ofAntiserum against Radixin 1986 ; Gould et al ., 1989 ; Pakkanen et al ., 1987; Thrunen Fusion Protein et al ., 1989), and that radixin is a novel member of a band The 765-bp Pst fragment of R2 (see Fig . 1 a) was subcloned in pBluescript, 4 .1 family . Webelieve this study will give us a clue to a better and the Bam HI-Eco RV fragment was then inserted into the Bam HI site understanding of the functions of radixin at AJ and at the of the pGEMEX-1 (Promega Biotech, Madison, WI) in frame, the expression vector based on the T7 expression system for high level gene expression cleavage furrow. in vivo . Fusion proteins were expressed in JM109 (DE3) synthesized according to the Promega Biotec (Madison, WI) procedure, and Escherichia coii lysate was separated by SDS-PAGE . The fusion protein was purified Downloaded from http://rupress.org/jcb/article-pdf/115/4/1039/1062193/1039.pdf by guest on 24 September 2021 Materials andMethods electrophoretically, and the antiserum to this purified protein was elicited in a rabbit. The affinity purification of the antibody was performed using Purification ofRadixin and Its Direct fusion proteins bound to nitrocellulose sheets . Peptide Sequencing Sequence Analysis The AJ was prepared from mouse liver by a combination of homogeniza- tion, sucrose density centrifugation, and NP-40 treatment as previously Nucleotide andamino acid sequences were analyzed with the program from described (Tsukita and Tsukita, 1989) . A low salt extract containing GENETYX software package (Software Development Co ., Japan) . undercoat-constitutive proteins was obtained by dialysis of the AJ fraction against the extraction solution (1 mM EGTA, 0.5 mM PMSF, 1 ;4g/ml Immunoblotting and Immunofiuorescence Microscopy leupeptin, 2 mM Tris-HCI [pH9.2]), followed by centrifugation at 100,000 g for 60 min . After electrophoresis of the extract obtained from 73 mice, SDS-PAGE was based on the discontinuous Tris-glycine system of Laemmli the band of radixin was removed with a razor blade. Such bands were (1970) . Immunoblotting was performed by one-dimensional electrophore- pooled, and radixin was eluted electrophoretically . The eluted protein was sis, followed by electrophoretic transfer to nitrocellulose sheets . Nitrocellu- precipitated by adding acetone, and the pellet was redissolved in 10 mM lose transfers were incubated with affinity-purified antiradixin fusion pro- Tris-HCI (pH 9 .5) . The lysyl endopeptidase (Wako Pure Chemical Indus- tein antibody. For antibody detection, a blotting detection kit (Amersham tries, LTD., Osaka, Japan) was added in an enzyme-to-substrate ratio of International, Amersham UK) was used . 1:100 (wt/wt) . A second aliquot of enzyme was added after digestion for Indirect immunofluorescence microscopy of frozen sections of mouse 20 h at 37°C, and cleavage was continued for an additional 20 h . The en- liver was performed as described previously (Tsukita et al ., 1989a) . The zyme digest was subjected directly to reverse-phase HPLC (Wakosi15c18- frozen sections were air dried and fixed in 95% ethanol at 4°C for 30 min 200 column), using a linear gradient of acetonitrile in 0.1% trifluoroacetic and in 100% acetone at room temperature for 1 min . acid at 1 % acetonitrile per min . Selected peptide fragments were sequenced using a protein sequencer (model 477A, Applied Biosystems, Foster City, CA) . Results cDNA Library Screening Isolation and Sequencing ofcDNA Encoding Two different XgtII expression libraries made from mouse F9 poly(A) + Mouse Radixin RNAs were used in the following procedures (Nagafuchi et al ., 1987) . In preparing these libraries, either a random mixture of hexanucleotides or We have previously obtained a polyclonal antibody specific oligo(dT) was used as primer for the first-strand synthesis . The initial cDNA for radixin (Tsukita et al ., 1989a) . Using this polyclonal an- clones, R2 and R3 (see Fig . 1 a), were isolated from a randomly primed tibody we screened -2 .4 x 10 6 plaques from a random library using a polyclonal antiradixin antibody (see Tsukita et al ., 1989a) according to the method described by Huynh et al . (1985), with the excep- primed Agtll cDNA library made from mouse teratocarci- tion that 5% skim milk in TBS, was used as a blocking solution, instead noma cells (F9 cells) as described in Materials and Methods, of 20% FCS in TBS . Then the 320-bp Sma I-Pvu II fragment of R2 was and cloned two positive phage recombinants : R2 (1 .2 kb) and radiolabeled with [a-32 P]dCTP Using this fragment as a probe, the RTl R3 (1 .85 kb) (Fig. 1 a) . Judging from their restriction maps, clone (Fig . 1 a) was isolated from the oligo(dT)-primed library. To obtain cDNA encoding mouse ezrin, we rescreened the oligo(dT)-primed library it was clear that these clones overlapped extensively, so both by DNA hybridization at low stringency with R2 as a probe. Two cDNA clones were sequenced . clones, T6 and T9, showing the same restriction map were isolated (see To isolate the rest of the radixin gene, -2 x 105 colonies Fig, 4) . of the oligo(dT) primed Agtl l cDNA library were screened by DNA hybridization with the radiolabeled Sma I-Pvu II DNA Sequencing fragment (320 bp) of R2 at high stringency. One positive clone (RT1) was isolated and then sequenced . Fig . 1 a shows All clones to be analyzed were subcloned into pBluescript SK(-) and se- the overall relationship of R2, R3, and RTl and the restric- quenced with the 7-deaza Sequenase Version 2 .0 kit (U.S . Biochemical Corp ., Cleveland, Ohio) . Long inserts were sequenced from nested deletion tion sites relevant for subcloning and sequencing . The com- subclones created using the Deletion Kit for Kilo-Sequence (Takara Shuzo plete nucleotide sequence and deduced amino acid se- Co ., LTD., Kyoto, Japan). Both strands of all clones were sequenced . quences of the cloned molecule are shown in Fig . 1 b. The

The Journal of Cell Biology, Volume 115, 1991 1040

0 0.5 10 Kb R2 1 1 1

RT1

b GGGCTCGCACCGCCGACTCTCCGCTCGTTCGCCGCCAGCCACGACGCCCCCGCCGAGCCGCGCCTCAGCCAGCCGGCGCGGGCAGGGAAGGAGCCACTCATTTTCGGGGCTATAATTGTT 120 TATAGAAGTGAAGAGTTTCTAAAGAAGGTAACAAACAAGAAAAAGAAAATGCCGAAGCCAATCAATGTAAGAGTAACTACAATGGACGCTGAGCTAGAATTrGCCATCCAGCCCAATACA 240 M P K P I N V R V T T M D A E L E F A I Q P N T 24

ACTGGCAAACAACTTTTTGACCAGG1TGTAAAAACAGTTGGCTTACGTGAGGTCTGG7T9TTTGGGCTGCAATATGTAGACAGCAAAGGATATTCTACATGGCTTAAATTGAATAAAAAG 360 T G K Q L F D Q V V K T V G L R E V W F F G L Q Y V D S K G Y S T W L K L N K K 64 Downloaded from http://rupress.org/jcb/article-pdf/115/4/1039/1062193/1039.pdf by guest on 24 September 2021 GTAACACAGCAAGATGTrAAAAAAGAAAATCCTTTACAGTTCAAGTTTAGAGCTAAATTCTTTCCTGAAGATG7TTCTGAGGAATTGATTCAAGAAATAACACAGAGACTTTTCTTCTTG 480 V T Q Q 0 V K K E N P L Q F K F R A K F F P E D V S E E L I Q E I T Q R L F F L 104

CAAGrrAAAGAAGCCATCTTAAATGATGAGATATATTGCCCACCAGAAACTGCAGTTCTTTTGGCTTCGTATGCTGTCCAAGCCAAGTATGGAGATTATAATAAAGAGATTCACAAACCA 600 Q V K E A I L N D E I Y C P P E T A V L L A S Y A V Q A K Y G D Y N K E I H K P 144

GGCTATCTGGCTAATGACAGACTCTTACCACAGCGTGTTTTGGAACAACACAAACTGACCAAAGAGCAGTGGGAGGAGAGAATACAGAACTGGCATGAGGAACACAGAGGGATGCTAAGG 720 G Y L A N D R L L P Q R V L E Q H K L T K E Q W E E R I Q N W H E E H R G M L R 184 GAAGATTCGATGATGGAATACrrGAAGATTGCGCAAGATCTAGAGATGTATGGTGTCAACTACTTTGAAATCAAGAATAAGAAGGGTACTGAGCTGTGGCTAGGTGTTGATGCCTTAGGC 840 E D S M M E Y L K I A Q D L E M Y G V N Y F E I K N K K G T E L W L G V D A L G 224

CTGAATAT7TATGAACATGATGACAAGTTAACACCTAAAATTGGTTTT000TGGAGTGAAATCAGAAATATTTCATTTAATGACAAAAAATTCGTTATAAAGCCAATTGACAAAAAGGCA 960 L N I Y E H D D K L T P K I G F P W S E I R N I S F N D K K F V I K P I D K K A 264 CCTGATTTTGTCTTCTATGCACCTCGTCTGAGAATCAATAAACGAATTTTGGCCTTATGTATGGGAAACCATGAACTGTACATGCGAAGGAGAAAGCCTGATACTATTGAAGTACAGCAG 1080 P D F V F Y A P R L R I N K R I L A L C M G N H E L Y M R R R K P D T I E V Q Q 304

ATGAAGGCTCAGGCCCGGGAGGTTTTGCATCAGAAGCAGCTAGAAAGGGCACAATTAGAGAATGAAAAGAAGAAACGAGAAATAGCAGAAAAGGAAAAGGAAAGAATAGAACGTGAAAAG 1200 M K A Q A R E V L H Q K Q L E R A Q L E N E K K K R E I A E K E K E R I E R E K 344

GAAGAGCTGATGGAGCGTCTGAGGCAGATCGAAGAGCAGACAGTGAAAGCCCAGAAAGAACTAGAAGAACAGACTCGGAAAGCTCTAGAACTGGAGCAGGAGCGCCAGCGAGCAAAAGAG 1320 E E L M E R L R Q I E E Q T V K A Q K E L E E Q T R K A L E L E Q E R Q R A K E 384

GAGGCAGAGCGGCTAGACAGGGAGCGCCGGGCTGCAGAAGAAGCCAAGTCTGCAATCGCCAAGCAAGCTGCTGACCAGATGAAGAACCAGGAGCAGCTGGCAGCAGAACTTGCCGAATTC 1440 E A E R L D R E R R A A E E A K S A I A K Q A A D Q M K N Q E Q L A A E L. A E F 424

ACTGCCAAGATTGCACTTTTAGAAGAAGCCAAGAAGAAAAAGGAAGAGGAAGCTACTGAGTGGCAGCACAAAGCTTTTGCAGCTCAGGAAGACTTGGAAAAGACCAAAGAAGAGCTAAAA 1560 T A K I A L L E .E A K K K K E E E A T E W Q H K A F A A Q E D L E K T K E E L K 464 ACTGTGATGTCTGCGCCACCTCCACCTCCCCCTCCTCCAGTCATCCCTCCCACGGAGAACGAGCATGACGAGCAGGACGAGAACAGTGCGGAGGCCAGCGCCGAGCTGTCCAGCGAGGGG 1680 T V M S A P P P P P P P P V I P P T E N E H D E Q D E N S A E A S A E L S S E G 504

GTGATGAACCACCGAAGCGAGGAGGAGTGGGTCACCGAAACCCAGAAGAATGAACGCGTGAAGAAGCAGCTCCAGGCATTGAGTTCAGAArrAGCCCAAGCTAGAGATGAAACCAAGAAG 1800 V M N H R S E E E W V T E T Q K N E R V K K Q L Q A L S S E L A Q A R D E T K K 544

ACACAGAACGATGTTCTTCACGCTGAGAATGTCAAGGCCGGCCGTGATAAGTACAAGACCCTTCGGCAGATTCGACAAGGCAATACGAAGCACGCGATCGATGAGTTTGAAGCCATGTGA 1920 T Q N D V L H A E N V K A G R D K Y K T L R Q I R Q G N T K H A I D E F E A M 583

GAGCTGTrCTTGTGATCCATGTCCTTCATAAGCTGAACCACCACCAGAGAAAAGCAGGCCTGTGCAGTGCAGATGCAATGGAACGCATCCCACCTTGCCAAAGCACTCATACCAGTCGAC 2040 TGTGCTCACTAGCAGGACACTGAAGGGTGTCCTTCATCATTAAGGCAGTGTGATGTCATGTTTGGTCATTTTTCT1TrGTCCAGGGAATGGATAATGACATTCTGTCACCCACCCTTTTG 2160 TACATTTTTCACTTTTTGTITGTTTCATCTTTTTCAGTTGAATATTAATACTAATGGCCACGTCTGACAAATGTGTGTATGTGCTTTGAGCACTGTGTACATCTTAGGATAATGGTGGAC 2280 A7TTCAGTGCATGTTTAGCTAATGAACTTAGTTTTCTTT000CTTTCCATGTTGTAGGCTACATACATCTCCATGTTCArrCTCATGTTTTTTGTCAGTGTGACCTTCTCATGCCCAGAC 2400 ATGATAGAGCTGTAATTCTGGTGTCACACGGATCACTTACTGGAGGTGTATAATATGAGATAGTGAGGTCAGACAGACCACAGACAAGACATGGGACTACATCATAAGATGATTAGAATG 2520 TTGGACATCGTAGTATGCAGCCNGTGGTTAGTTATTACAAAGATCCCTTAGTTACTGTATTGTTTGCTTTCCTTAAGTGCAGTCATCTCTGATAGCTTGAGTCCTTAAGTGCAGCCATCT 2640 CTGATAGCTTAGTGATGGTTTTCACGTCCTGTAGTCCTATGGTGGCTTTCCTTGCCTGTTACTrrAGAGTCTAGCCACTAGGAGTCGCrrCTAAGAAAATrATTGATGAAGTGGCC7TGT 2760 AAGTACATGGTCTCTGAAAAAAATTCAAGT7rrCTTGTTAGGATGTGGAATAGAAGATAACTTGGATGATAACTAATTCTAAGTTGTTGAA000GGAAAAAAACCCAAACCTTGGCCTTT 2880 CTGCAAGGGCAGCATATGGAAACATCAACTCTGAGAACAAAATACTACCTCACTCTTTACGTACGTGGTGACTGTTCGCACAGTCTGTGCTGCATGTCTGTCACCCATACGCAGTCTCAA 3000 TCATGGCCrrCACTGAGTGCTTTTGAGATCCCAGTGCTAATGTrrrAACTGCCTTTCTG7'rGAACTGTGGGGCTCTGATTTGTGTCTAGATTATTGTATATTCTCACTGGTATGCTGTTC 3120 CCTGGGGGGTGTTCTGTGTCCGACGCTGAGTGACCCTTTGTGAGAAAGACGTTGCCTTTGCAGTACATGAAGCATCCCATTCTTCGTTCTTGATGGGATAGGGATCGTTATTGTAGCATA 3240 TTrGGTTTCTAAATCACTGCTTATTTTTATTTGCAATCTCTAAAACAAAATTTTATTTCAATTACGTCTTACTGAAGAAGTCTTTCCAGCTAGAAAGATGrrCTGACACTGTCTGATCGT 3360 GAAGCACCTGAATTTACAGAAGATGCAGAAGCCTCGTTTTCTCTGCGTGGTTGAGCTACTTTCCATTTGGTATTATGCACTCAAACTTAACATTAATCTATTAAAATTGCCATTTTATTA 3480 AGCAATTTTCACGCACAGTAGATTCAACTTGTTT7ITGTGCTG I I I I L I'GATTTTTGCTGACTTAAAAAGGATTAATTTGGGCAGACATTATAAAAAGGAAAGGGTTAATATATTTTTGG 3600 ACTCTGTAGGACAAAAGAGAACTGGTTAACCTTGAAGTGACTGTTGTACAGTGGCTGTGCACGTGCTTCAGACTCATTTGCGAGGGAATGCTTTCTGTATGAGAGGGAGACGGTAGGCCT 3720 TACCACTCAGACCTGGACGTATAAGTGCTCACTTTTCCAACACATCACAGCGATAGTCCTTA7TGCTAAAGGACTTGGCTTCCCTCGGAGTTTTACTGATTTAT7T=CTCTTTGCAT 3840 TGTGTACATTATCATTGCTACATCAGTTGTTACAGTGTTGAAAGGCTACCAGTTATATTTGCTGTTAATAGTCTATTTGTAGATTAGGATTAAAGTGGGTTTAATCCATTTTTATAGCTG 3960 TATGAATTTTTCTAAACAGGAACAGCCATI'TGCAAAATGGGTTTTCATAGTGATTGCATCAATTATAACATTTATCATTAGCAGTTGAGAGCACATGTTCATATAGCAATGTAAAAACCT 4080 ACTAATGAGTATTTGGTAATTTCCCATTAGCATAATTTTATGTTGTACAAGTAAATTACAATTCCATCTCTAATCTTGGATAATACTGGTTGGTTAACAATAAAGTGACAAAAGCTCCCG 4200 CCTTGCAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 4241 Figure 1. Mouse radixin cDNA sequence. (a) Restriction map and cDNA fragments ofmouse radixin. Large and small open boxes indicate the coding region and the Sma I-Pvu 11 fragment used for the isolation of RTI, respectively. E, Eco RI; P, Pst I; Xb, Xba I. (b) Nucleotide sequence and deduced amino acid sequence ofmouse radixin. The underlined amino acid sequences perfectly match the sequences obtained from amino-terminal sequencing of lysyl-endopeptidase fragments of mouse liver radixin. The complete nucleotide sequence determined is 4,241 bases long including a poly(A)+ tail. The coding region is preceded by 168 nucleotides of 5'-untranslated sequence and followed by a 2,388-nucleotide-long 3'-untranslated region. These sequence data are available from EMBL/GenBank/DDBJ under accession number x60672.

Funayama et al . Primary Structure of Radixin 1041 taining short repeated similarities (Fig. 2 a). This is consis- a tent with the result of the secondary structure analysis of radixin, which predicts that residues 304-468 form an a helix (Fig. 2 b). To confirm that the protein encoded by the above, cloned cDNA is identical to radixin, direct peptide sequencing of radixin purified from mouse liver was carried out. After lysyl endopeptidase digestion, three fragments were isolated . Sequencing ofall of these fragments resulted inperfect matches with the amino acid sequence deduced from the cDNA sequence (Fig. 1 b). Furthermore, we generated fusion proteins encoded by the 765-bp Pst fragment of R2 and elicited a polyclonal antibody against this fusion protein in a rabbit. As shown in Fig. 3, this polyclonal antibody specifically recognized the 82-kD radixin band of F9 cells, mouse liver, and AJ fractions in immunoblot analysis, and clearly stained cell-to-cell AJ in the frozen sections ofmouse liver by immunofluorescence microscopy. Taken all together, we can conclude that the cDNA encoding mouse radixin is Downloaded from http://rupress.org/jcb/article-pdf/115/4/1039/1062193/1039.pdf by guest on 24 September 2021 successfully isolated and sequenced.

Isolation and Sequencing ofcDNA Encoding Mouse Ezrin C 3.0000 Sequence analysis with the above, cloned cDNA demon- strates that mouse radixin shares 75.3 % identity with human ezrin (cytovillin) (Gould etal., 1989 ; Tltrunen et al ., 1989). Since ezrin is reported to be localized in microvilli ofintesti- 0.0000 nal epithelial cells and not in the cell-to-cell adherens junctions, it is likely that radixin is not identical to ezrin (Bretscher, 1983). To verify this, we have isolated cDNA encoding "mouse" ezrin. For this purpose, R2 was used to x 5 -3.000 screen -3.2 10 colonies of an oligo(dT)-primed Xgtll cDNA library made frommouse F9 cells by DNA hybridiza- 100 200 390 400 506 tion at low stringency, and eight positive clones were ob- Figure 2. Internal similarities of radixin and secondary structure tained. Judging from restriction maps of the inserts of posi- predictions . (a) A diagonal plot of amino acid sequence of mouse tive clones, a single class of clones (e.g., T6 and T9 in Fig. radixin compared with itself. Comparison was made using the 4 a) was identified, which was distinct from cDNA encoding HARPLT2 program. The axes are labeled in amino acid residue radixin. Then, the longest of these cDNA clones (T9) was numbers. The dots correspond to midpoints of 50-residue spans sequenced. This cDNA is 2,701 nucleotides long, has a 95- with at least 10 identical residues between two proteins. The lines nucleotide-long Y-untranslated sequence, codes for a 586- parallel to the central diagonal represent internal similarities. Note amino acid polypeptide with a calculated molecular mass of that there is a region (residues 304-468) within the protein contain- 69.3 kD, and has an 848-nucleotide-long Y-untranslated se- ing short repeated similarities. (b) Open boxes indicate the regions quence (Fig. 4 b). The putativeprotein encoded by this cDNA which are predicted to form a helices. This prediction was obtained Robson method. Note that residues 304-468 are predicted clone is almost identical to human ezrin (96.2% identity). by the Furthermore, the Y-untranslated sequences of this cDNA to form an a helix. (c) Hydrophilicity plot for radixin by GENE- TYX software. The plot records the average hydrophilicity along were also highly homologous to those ofhuman ezrin cDNA the sequence over a window of 15 residues. Hydrophylic and hydro- (Fig. 5) . These data conclusively show that cDNA clone T9 phobic residues are in the upper and lower part ofthe frame, respec- (and also T6) encodes "mouse ezrin. tively. The axis is labeled in amino acid residue numbers. Comparison ofRadixin with Ezrin composite cDNA is 4,241 nucleotides long: 168 by of the Now that both cDNA clones encoding mouse radixin and Y-untranslated region, an ORF of 1,749 by encoding 583 mouse ezrin have been obtained, the primary structure ofthe amino acids, and 2,324 by of the Y-untranslated region with radixin molecule canbe directly compared with thatofezrin. a poly(A) tail. The calculated molecular mass ofthe protein Based on the alignment shown in Fig. 6 a, mouse radixin encoded by this cloned cDNA is 68.5 kD. This cDNA has shares 74.9 % identity with mouse ezrin. As compared to the no long hydrophobic stretches which could encode a signal carboxyl-terminal half of these molecules, amino acid sub- peptide or a transmembrane domain (Fig. 2 c), confirming stitutions in the amino-terminal halfare highly conservative ; that radixin is neither a secretory protein nor a transmem- 85.6% identity for the residues 1-311, and 43% identity for brane proteitr. (Tsukita et al., 1989a). A plot of the deduced the residues 312-000H terminus (Fig. 6 b). antino acid sequence of radixin against itself reveals that To detect mRNA encoding radixin and ezrin, Northern there is a region (residues 304-468) within the protein con- blot analysis with total mRNA obtained from mouse F9 cells

The Joumal of Cell Biology, Volume 115, 1991 1042 Downloaded from http://rupress.org/jcb/article-pdf/115/4/1039/1062193/1039.pdf by guest on 24 September 2021

Figure 3. Immunoblotting analysis (a) and indirect immunofluorescence staining (b) with the polyclonal antibody raised against the radixin fusion protein . (a) Coomassie blue-stained gels (lanes 1-3) and the corresponding immunoblot profiles with the antiradixin fusion protein antibody (lanes 1'-3') . (Lanes 1 and f) Low salt extract of isolated mouse AJ ; (lanes 2 and 2') F9 cells ; (lanes 3 and 3') mouse liver. Arrowheads indicate the major constituents of the undercoat of adherens AJ (400 [tenuin], 240, 235, 130 [vinculin], 100 [a-actinin], 82 [radixin], 70, 55, 50, and 43 kD [actin] from the top) . Only a single band corresponding to radixin (82 kD) is detected in each sample . A faint staining around 130 kD in lane 2' appears to correspond to a dimeric form of radixin, since radixin occasionally forms a dimer even in SDS-PAGE sample buffer (our unpublished data) . (b) Immunofluorescence staining of frozen sections ofmouse liver cells. Intense fluorescence appears on both sides of the bile canaliculi, resulting in a pair of parallel lines. Bar, 10 pm . was performed using R2 (see Fig . 1 a) and T9 (see Fig . 4 the existence of the homologous NHZ -terminal domain in a) as probes for radixin and ezrin, respectively. As shown in radixin molecule . Taking all these findings together, we can Fig . 7, the band at 4 .2 kb was detected by R2 (radixin), while conclude that radixin is a novel member of the band 4 .1 the band at 2 .7 kb was hybridized to T9 (ezrin) . 'Faking the family. distribution of the nonidentical residues between radixin and ezrin into consideration, we are led to conclude that these mRNAs are not derived from a single gene by an alternative Discussion splicing mechanism . We have isolated cDNA encoding mouse radixin . The sequence of radixin was determined from three overlapping Comparison ofRadixin with the Other Members clones encoding a protein of 583 amino acids . Identification ofthe Band 4.1 Family of these clones as radixin came from two pieces of evidence . Recently the existence of a novel family of submembranous First, the direct peptide sequencing of three lysyl endopep- cytoskeleton-associated proteins has been recognized . These tidase-digested fragments of radixin resulted in perfect include band 4.1 protein from red blood cells, ezrin (also matches with the amino acid sequence deduced from the called cytovillin or p81), and talin (Conboy et al ., 1986 ; cDNA sequence . Second, antiserum to a radixin fusion pro- Gould et al ., 1989 ; Rees et al ., 1990; Turunen et al ., 1989) . tein specifically recognized the mouse radixin molecule in Each member of this band 4 .1 family was reported to have immunoblotting analysis and clearly stained the cell-to-cell a homologous N112 -terminal domain followed by a domain AJ by immunofluorescence microscopy. Interestingly, se- rich in a helix and a highly charged COOH-terminal seg- quence analysis with this radixin cDNA has revealed that ment (Rees et al ., 1990) . Radixin shares these structural fea- mouse radixin shows a similarity to human ezrin . In addi- tures with these band 4 .1-like proteins . Fig . 8 clearly reveals tion, we have cloned cDNA encoding mouse ezrin and

Funayama et al . Primary Structure of Radixin 1043

P P P P I I I I

Tg o as kalb

b GGTCCCGACGCGTCTCCGCCGCCCTCGCCCTCCCCCTCGCCTCCGCCGTACAGCCGAATAGCCGAGGACCCACGCAGAGCCACCAACCAGCCAAGATOCCCAAGCCAATCAACGTCC000 120 M P K P I N V R V

TGACCACCATOOATGCCGAGCTGGAGTTTGCCATCCAGCCAAACACCACCGGGAAGCAGCTCTTTGATCAGGTGGTAAAGACGATTGGCCTCCGGGAAGTGTOOTACTTCOGCCTGCCGT 240 T T M D A E L E F A I Q P N T T G K Q L F D Q V V K T I G L R E V W Y F G L P Y

ATGTAGACAATAAAGGATTTCCTACCTGGCTGAAACTTGATAAAAAGGTCTCCGCACAGGAGGTCCGAAAGGAGAACCCTGTCCAGTTTAAATTCCGGGCCAAGTTCTACCCCOAAOACO 360 V D N K G F P T W L K L D K K V S A Q E V R K E N P V Q F K F R A K F Y P E D V

TGGCCGAGGAACTCATCCAGGACATCACACAGAAGCTCTTCTTCCTGCAAGTCAACGACGGGATCCTCAGCGACGAGATCTACTGCCCCCCAGAOACAGCCOTOCTCCTGGGCTCCTATG 480 A E E L I Q D I T Q K L F F L Q V K D G I L S D E I Y C P P E T A V L L G S Y A

CCGTTCAGGCCAAGTTCGGAGATTATAACAAGGAAATGCACAAGTCTGGGTACCTCAGCTCGGAGCGGCTGATCCCCCAGAGAGTCATGGACCAACACAAGCTCAGCAGOGACCAGTGGO $00 Downloaded from http://rupress.org/jcb/article-pdf/115/4/1039/1062193/1039.pdf by guest on 24 September 2021 V Q A K F G D Y N K E M H K S G Y L S S E R L I P Q R V M D Q H K L S R D ' Q W E

AGGACCGGATCCAGGTGTGGCACGCGGAACACCGAGGGATGCTCAAGGACAGTGCTATGCTGGAATACCTGAAGATTGCCCAGGACCTGGAAATGTATGGGATCAACTATTrCOAGATCA 720 D R I Q V W H A E H R G M L K D S A M L E Y L K I A Q D L E M Y G I N Y F E I K

AAAACAAGAAAGGAACAGACCTTTGGCTTGGAGTCGATGCCCTTGGACTTAACATTTATGAGAAAGATGACAAGTTGACCCCAAAGATCGGCTTCCCTTCGAGTGAGATCAGGAACATCT 840 N K K G T D L W L G V D A L G L N I Y E K D D K L T P K I G F P W S E I R N I S

CTTTCAACGACAAGAAGTTTGTCATTAAGCCCATCGACAAGAAGGCACCTGACTTTGTGTTCTACGCCCCGCGCCTGAGAATTAACAAGCGGATCCTGCAGCTCTGCATGGGGAACCATG 960 F N D K K F V I K P I D K K A P D F V F Y A P R L R I N K R I L Q L C M G N H E

AGCTGTACATGCGCCGCAGGAAGCCCGACACCATCGAGGTGCAGCAGATGAAGGCCCAGGCTCGGGAGGAGAAGCACCAGAAGCAGCTAGAGCGACAGCAGTTGGAAGCCGAGAAGAAGA 1080 L Y M R R R K P D T I E V Q Q M K A Q A R E E K H Q K Q L E R Q Q L E A E K K R

GGCGAGAGACGGTGGAGAGAGAAAAGGAGCAGATGCTCCGGGAGAAGGAGGAGCTGATGCTTCGGCTGCAGGACTACGAGCAGAAGACCAAGAGGGCGGAGAAAGAGCTCTCCGAGCAGA 1200 R E T V E R E K E Q M L R E K E E L M L R L Q D Y E Q K T K R A E K E L S E Q I

TTGAGAAGGCCCTCCAACTGGAGGAAGAGAGGAGGCGAGCCCAGGAGGAGGCTGAACGTCTGGAGGCCGACCGCATGGCCGCCCTGCGGGCCAAGGAAGAACTCGAGAGACAGGCGCAGG 1320 E K A L Q L E E E R R R A . Q E E A E R L E A D R M A A L R A K E E L E R Q A Q D

ATCAGATAAAGAGCCAGGAGCAGCTGGCTGCAGAGCTGGCAGAGTACACGGCCAAGATCGCACTGCTGGAGGAGGCGCGGAGGCGCAAGGAGGACGAGGTAGAAGAGTGGCAGCACCGGG 1440 Q I K S Q E Q L A A E L A E Y T A K I A L L E E A R R R K E D E V E E W Q H R A

CTAAAGAAGCCCAGGACGACCTGGTGAAGACCAAAGAGGAGCTGCACCTGGTGATGACGGCCCCACCGCCCCCACCGCCCCCAGTGTATGAGCCTGTGAATTACCACGTGCAGGAGGGAC 1560 K E A Q D D L V K T K E E L H L V M T A P P P P P P P V Y E P V N Y H V Q E G L

TGCAGGACGAGGGAGCAGAGCCTATGGGCTACAGTGCCGAGCTCTCCAGTGAGGGCATCCTGGATGACCGCAACGAGGAGAAGCGGATCACAGAGGCAGAGAAGAATGAGCGCGTGCAGC 1680 Q D E G A E P M G Y 5 A E L S S E G I L D D R N E E K R I T E A E K N E R V Q R

GGCAGCTGCTGACCCTGAGCAATGAGTTGTCCCAGGCCCGGGATGAGAACAAGAGGACCCACAATGACATCATCCACAACGAGAACATGCGGCAAGGCAGGGACAAGTATAAGACGCTGC 1800 Q L L T L S N E L S Q A R D E N. K R T H N D I I H N E N M R Q G R D K Y K T L R

GGCAAATCAGGCAGGGCAACACCAAGCAACGCATTGACGAGTTCGAGGCCATGTAGAGGCCAGGCTGGGACCAAGGGCAGAGGGCACCTCACTGCAGGCAGGTGTCACACTTGGCTCTTT 1920 Q I R Q G N T K Q R I D E F E A M

AGTTCTCTTAAGTTTAGACACCCCCrrGCTGTGTTCCAGTCCCTTAAAGAGCAGTTACGGGGCCTGCATTCTGCCCCGAGACCCAGTGGGCTCCTCCTTGGTTCCTTCTAATTGTATCAC 2040 ATAGTGCCAAACAGGTCAGATTTAATGACAGTTACCGAATCACTTCCTGTTTGGAGCAGGGATTCGGAGGGCTGGCCCTCATGAAGCAAGATTCATTGTCACTGGGACAGCACTGTGGCT 2160 CACGGGTGCCATACTTTTCTCTAGTTTTACAATGAGCTCAAATCGATTTTGTTCTTGATTTTTATGAAGGATCCATCTCTGTGTATTGAGGGGTAAAAATGATTTTGAAATTTGAGTCTA 2280 AAGCATGCCCCCACAGAGTCTCCTTCCTCCAGACCGCTGGCAGAGTCTCCAGGGGTCCTTCAGAGTGTACGGGTACAGGACTCTCCGATACAAAATTCTCATGCTTATCTGTTAGCATAC 2400 ATTGTTGGACTTATATATCTAATGATCTGCTATAGAGGATAGTATTTATATATAAGCCGATAATATGGGTTTGTAACATTAG=AAAAAAGGGAAAGTTTTGTTCTGTATATTTTGTT 2520 ACCTTTTACAGAATAAAAGAATTCAACACCAAGAACCATGTAACCGAGACACTTGATCTGACACAGGGGCAGTCGGGAAACCGATGACTGCAGTAATCACCACTGTACAAAAATGTTAGT 2640 GGGT'rrGTGCACGTAAAATGCACACTTCCGATTTCCTGTCAGTTTCTTATTTGAAAACCC 2701 Figure 4. Mouse ezrin cDNA sequence. (a) Restriction map and cDNA fragments of mouse ezrin. Open box indicates the coding region. E, Eco R1; P, Pst 1. (b) Nucleotide sequence and deduced amino acid sequence ofmouse radixin. The complete nucleotide sequence determined is 2,701 bases long. The coding region is preceded by 95 nucleotides of 5'-untranslated sequence and followed by a 848-nucleotide- long 3'-untranslated region. These sequence data are available from EMBL/GenBank/DDBJ under accession number x60671.

confirmed that radixin is distinct from ezrin although they undercoat-constitutive protein at the cell-to-substrate AJ, share 74.9 % amino acid sequence identity. Ezrin (also called was also reported to be a member of this band 4 .1 family cytovillin or p81) was originally identified as an undercoat- (Burridge and Connell, 1983 ; Burridge and Mangeat, 1984; constitutive protein of the microvilli plasma membrane Rees et al., 1990) . Our present data add radixin as a new (Bretscher, 1983; Pakkanen et al., 1987), and was reported member to the band 4 .1 family. to be a good substrate in vivo for some tyrosine kinases In various types of cells, there are some specialized (Bretscher, 1989; Gould et al ., 1986; Hunter and Cooper, regions of the cell surface which are tightly associated with 1981, 1983) . Recent sequence analysis of ezrin has revealed actin filaments: the cleavage furrow during cytokinesis, cell- that ezrin shows homology to the band 4.1 protein of erythro- to-cell AJ, cell-to-substrate AJ (focal contacts), ruffling cyte membranes (Conboy et al., 1986; Gould et al., 1989; membrane, and microvilli . According to the previous im- T1lrunen et al., 1989). Most recently, talin, a major munolocalization results, it may be safe to say that the first

The Joumal of Cell Biology, Volume 115, 1991 1044

Mouse Ezrin 750 1500 2250

750-

Ç L N W c i5ee 0 E

225 Downloaded from http://rupress.org/jcb/article-pdf/115/4/1039/1062193/1039.pdf by guest on 24 September 2021 300 Figure 7. Northern blot analysis of total Figure S. Comparison of nucleotide sequence of human ezrin RNA isolated from mouse F9 cells. Total cDNA with that of mouse ezrin cDNA . Diagonal plots were made RNA was probed at high stringency with using the HARPLT2 program . The dots correspond to midpoint of radixin R2 cDNA (R) and mouse ezrin 15-nucleotide spans with at least 12 identical nucleotides between cDNA (E) that covered almost the whole two cDNAs . The axes are labeled in nucleotide numbers. Note that length ofcoding region ofeach protein . The they are highly homologous not only within the coding regions but bars on the left indicate the position of RNA also in Y-noncoding regions (*) . markers of 9 .5, 7.5, 4 .4, 2 .4, and 1 .4 kb (from the top) . two of these regions contain high concentrations of radixin ; the third and fourth, talin ; and the fifth, ezrin (Bretscher, of ezrin, all of the previously published immunolocalization 1983 ; Burridge and Connell, 1983 ; Sato et al ., 1991 ; Tsukita workon ezrin and radixin shouldbe reevaluated carefully be- et al ., 1989a) . However, taking into consideration that the cause of the likelihood that antiezrin antibodies cross-react amino acid sequence ofradixin is highly homologous to that with radixin and vice versa . This is an important issue to re-

Figure 6. Comparison of amino acid sequence of mouse radixin with that ofmouse ezrin . (a) The sequences were aligned by the GENETYX program . Identities are indicated by dashes ; gaps, introduced to maximize alignment, are indicated by spaces . The mouse radixin and ezrin proteins are identical in 74 .9 % of their residues . The shadowed box indicates one of three regions whose amino acid sequences were directly determined from the purified radixin (see Fig . 1 b) . In this boxed region, the amino acid sequence in radixin differs from that in ezrin . (b) Diagonal plots at the amino acid sequence level . As compared to the C0011-terminal half of these molecules, the N112- terminal half is highly conservative in its amino acid sequence .

Funayama et al . Primary Structure of Radixin 1045 cessory proteins of human erythrocyte membranes, and associated with spectrin and actin in membrane skeletons (Bennett, 1989). In vitro assays so far clarified the following properties ofband 4.1 protein. (a) It participates in a ternary or higher order complex together with spectrin and actin. (b) lee It binds directly to spectrin and promotes spectrin binding to actin. (c) It does not interact directly with actin (Bennett, 1989; Correas et al., 1986; Fowler and Taylor, 1980; Oha- zee nian et al., 1984 ; Ungewickell et al ., 1979). In sharp con- trast, radixin specifically binds to the barbed ends of actin filaments (T§ukita et al., 1989a). As shown in Figs. 8 and 388 9, the amino acid sequence of an NH2-terminal domain of C band 4.1 protein is homologous to that of radixin, and the m binding site for a plasma membrane protein (glycophorin) is 488 mapped to this domain of band 4 .1 protein (Leto et al., 1986). Therefore, the rest part of each molecule (000H- terminal half) seems likely to have a bearing on the interaction ofeach with cytoskeletal proteins. Actually, the 000H- 588 terminal half of band 4.1 protein is reported to contain the spectrin-binding region (Correas et al., 1986) . The amino Downloaded from http://rupress.org/jcb/article-pdf/115/4/1039/1062193/1039.pdf by guest on 24 September 2021 acid sequence of this COOH-terminal half of band 4 .1 protein is not homologous to that ofradixin . This suggests that the COOH-terminal half of radixin is responsible for its barbed-end capping activity, although there are no obvious homologies between radixin and other barbed-end capping proteins such as gelsolin (Dieffenbach et al ., 1989), fragmin (Ampe and Vandekerckhove, 1987), and Cap-Z protein (Casella et al., 1989; Caldwell et al., 1989). Considering that the other members of the band 4 .1 family such as ezrin and talin have their respective COOH-terminal domains, it is likely that these proteins interact with cytoskeletons in distinct manners. No evidence has yet been obtained for any di- C rect interaction between actin filaments and ezrin. Tàlin was believed not to directly interact with actin filaments, but recently the direct interaction between talin and actin was reported (Muguruma et al ., 1990). The interaction of these band 4 .1-like proteins with actin filaments should be exam- ined under various conditions, for example, in the presence of phospholipids and/or other protein factors. The existence of an homologous NH2-terminal domain in proteins ofthe band 4 .1 family will give us a clue toward understanding their physiological functions. In band 4.1 protein, this domain contains a binding site for glycophorin A Figure 8. Comparison of amino acid sequence of mouse radixin or glycophorin C (also called glycoconnectin), one of the with that of the other members of the band 4.1 family. Diagonal major membrane proteins in erythrocytes (Anderson and plots of mouse radixin compared with human erythrocyte mem- Lovrien, 1984; Anderson and Marchesi, 1985; Bennett, brane protein 4.1 (band 4.1 protein) and the amino-terminal region 1989 ; Leto et al., 1986 ; Mueller and Morrison, 1981). As of mouse talin. Comparison was made using the HARPLT2 pro- shown in Fig. 6, this domain is highly homologous between gram. The dots correspond to midpoints of 50-residue spans with at least 10 identical residues between two proteins. The axes are la- radixin and ezrin, leading us to speculate that a single class beled in amino acid residue numbers. Note that the homologous of membrane protein may be present in cell-to-cell AJ, NH2-terminal domain exists in these members of the band 4.1 cleavage furrows, and microvilli as membrane-attachment family. sites for radixin and ezrin and that this membrane protein may have a similarity to glycophorin A or glycophorin C (glycoconnectin) . solve in near future. What we can say at present is that the In this study, we have shown that radixin is a new member band 4.1-like proteins play a pivotal role in cytokinesis, cell ofthe band 4.1 family. Recent studies have suggested the pos- adhesion, and cell motility by directly or indirectly linking sibility that in addition to band 4.1 protein, ezrin, talin, and actin filaments to plasma membranes. radixin, many other proteins may be included in the band 4 .1 To understand the physiological functions of these band family (Spiegel et al., 1984; Bretscher, 1989; Birgbauer and 4.1-like proteins in molecular terms, the following question Solomon, 1989). For example, a nonerythroid 4 .1 variant must be answered in future: How do these proteins interact was identified in lymphocytes and its cDNA was cloned and with actin filaments? Band 4.1 protein is one ofthe major ac- sequenced, indicating that mRNAs of both erythrocyte and

The Journal of Cell Biology, Volume 115, 1991 1046 Figure 9. Models of the structure of radixin, ezrin, erythrocyte band 4.1, lymphocyte band 4.1, and talin . The domain structure of radixin and ezrin is schematically drawn in detail . Both proteins are di- vided into four domains ; (a) a homologous domain between radixin and ezrin, (b) a domain rich in ot-helix, (c) a pro- line-rich domain, (d) a highly charged domain . Boundaries between each domain of radixin and ezrin are shown by the number of boundary residues. Nonerythroid band 4.1 variant was identified in lymphocytes, and both erythrocyte and lymphocyte band 4.1 proteins are known to be pro- Downloaded from http://rupress.org/jcb/article-pdf/115/4/1039/1062193/1039.pdf by guest on 24 September 2021 duced from a single gene by alternative splicing . Each protein has ahomologous domain (shaded box) in its NHZ-terminal region . In erythrocytes, this domain is responsible for the direct interaction of band 4.1 protein with glycophorin A or C (also called glycoconnectin) . lymphocyte band 4.1 proteins are produced from a single properties of both microtubule- and microfilament-associated protein . J. Cell Biol. 109 :1609-1620 . gene by alternative splicing (Tang et al ., 1988) (Fig . 9) . The Bretscher, A . 1983 . Purification of an 80,000-dalton protein that is a component relationship of radixin and ezrin to this variant in noneryth- of the isolated microvillus cytoskeleton, and its localization in nonmuscle cells remains to be clarified . The expression experi- cells . J. Cell Biol. 97 :425-432 . roid Bretscher, A . 1989 . Rapid phosphorylation and reorganization of ezrin and ments using cDNAs encoding the members ofband 4.1 fam- spectrin accompany morphological changes induced in A-431 cells by ily will lead us to a better understanding ofthe physiological epidermal growth factor . J. Cell Biol. 108 :921-930 . Burridge, K ., and L. Connell. 1983 . A new protein of adherens plaques and roles of these band 4 .1-like proteins . ruffling membranes . J. Cell Biol. 97 :359-367 . Burridge, K ., and P. Mangeat. 1984 . An interaction between vinculin and talin. to express our sincere appreciation to Professor M . Nature (Lond. ) . 308 :744-746 . We would like Caldwell, J . E ., J. A . Waddle, J . A . Cooper, J . A . Hollands, S . J . Casella, Muramatsu (University of Tokyo) and Dr. T . Yamakawa (The Tokyo Met- and J . F . Casella . 1989. cDNAs encoding the a subunit of cap Z, the actin- ropolitan Institute of Medical Science) for their encouragement throughout capping protein of the Z line of muscle. J. Biol. Chem . 264 :12648-12652 . this study . We also thank Miss N . Sekiguchi and Mrs . M . Sugiura (Na- Casella, J . F ., S . J . Casella, J . A . Hollands, J . E. Caldwell, and J . A . Cooper. 1989 . Isolation and characterization of cDNA encoding the a subunit of cap tional Institute for Physiological Sciences) for their expertise in protein se- Z, an actin-capping protein from the Z line of skeletal muscle . Proc . Natl. quencing . We are also grateful to the members of our lab for technical as- Acad. Sci . USA. 86 :5800-5804. sistance and helpful discussions . Conboy, J ., Y . W . Kan, S . B . Shohet, and N . Mohandas . 1986 . Molecular clon- This study was supported in part by a Grant-in-Aid for Cancer Research ing of protein 4 .1, a major structural element of the human erythrocyte mem- from the Ministry of Education, Science and Culture of Japan ; by a Grant- brane skeleton . Proc. Natl. Acad. Sci . USA . 83 :9512-9516 . Correas, I ., D. W . Speicher, and V . T . Marchesi . 1986 . Structure of the spec- in-Aid from the Ministry of Health and Welfare of Japan ; and by research trin-actin binding site of erythrocyte protein 4 .1 . J. Biol . Chem. 28 : grants from the Kowa Research Foundation to Shoichiro Tsukita . 13362-13366 . Cowin, P ., H .-P . Kapprell, W . W . Franke, J . Tamkun, and R . O . Hynes . 1986 . Received for publication 16 May 1991 and in revised form 8 July 1991 . Plakoglobin : a protein common to different kinds of intercellular adhering junctions . Cell. 46 :1063-1073 . Dieffenbach, C . W ., D . N . Sengupta, D . Krause, D . Sawzak, and R. H . Silver- References man . 1989 . Cloning of murine gelsolin and its regulation during differentia- tion of embryonal carcinoma cells . J. Biol . Chem. 264 :13281-13288 . Ampe, C ., and J . Vandekerckhove. 1987 . The F-actin capping proteins of Phys- Farquhar, M . G ., and G . E . Palade . 1963 . Junctiona l complexes in various arum polycephlum : cap42(a) is very similar, if not identical, to fragmin and epithelia. J. Cell Biol. 17 :375-409 . is structurally and functionally very homologous to gelsolin ; cap41(b) is Fowler, V ., and D . L . Taylor . 1980 . Spectrin plus band 4 .1 cross-link actin . Pysarum actin . EMBO (Eur. Mol. Biol . Organ.) J. 6 :4149-4157 . J. Cell Biol. 85 :361-376 . Anderson, R . A ., and R . E . Lovrien . 1984 . Glycophorin is linked by band 4 .1 Gallin, W . J ., B . C . Sorkin, G . M . Edelman, and B . A . Cunningham. 1987 . protein to the human erythrocyte membrane skeleton. Nature (Lond.) . Sequenc e analysis of a cDNA clone encoding the liver cell adhesion mole- 307 :655-658 . cule, L-CAM . Proc. Nall. Acad. Sci . USA. 84:2808-2812 . Anderson, R . A ., and V . T . Marchesi . 1985 . Regulation of the association of Geiger, B . 1979 . A 130k protein from chicken gizzard : its localization at the membrane skeletal protein 4 .1 with glycophorin by a polyphosphoinositide . termini of microfilament bundles in cultured chicken cells . Cell. 18 :193- Nature (Lond .) . 318:295-298 . 205 . Bennett, V . 1989 . The spectrin-actin junction of erythrocyte membrane skele- Geiger, B . 1983 . 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