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. 1998 Feb 23;140(4):885-95.
doi: 10.1083/jcb.140.4.885.

Ezrin/radixin/moesin (ERM) proteins bind to a positively charged amino acid cluster in the juxta-membrane cytoplasmic domain of CD44, CD43, and ICAM-2

S Yonemura  1 , M Hirao, Y Doi, N Takahashi, T Kondo, S Tsukita, S Tsukita
Affiliations

Ezrin/radixin/moesin (ERM) proteins bind to a positively charged amino acid cluster in the juxta-membrane cytoplasmic domain of CD44, CD43, and ICAM-2

S Yonemura et al. J Cell Biol. .

Abstract

CD44 has been identified as a membrane-binding partner for ezrin/radixin/moesin (ERM) proteins, plasma membrane/actin filament cross-linkers. ERM proteins, however, are not necessarily colocalized with CD44 in tissues, but with CD43 and ICAM-2 in some types of cells. We found that glutathione-S-transferase fusion proteins with the cytoplasmic domain of CD43 and ICAM-2, as well as CD44, bound to moesin in vitro. The regions responsible for the in vitro binding of CD43 and CD44 to moesin were narrowed down to their juxta-membrane 20-30-amino acid sequences in the cytoplasmic domain. These sequences and the cytoplasmic domain of ICAM-2 (28 amino acids) were all characterized by the positively charged amino acid clusters. When E-cadherin chimeric molecules bearing these positively charged amino acid clusters of CD44, CD43, or ICAM-2 were expressed in mouse L fibroblasts, they were co-concentrated with ERM proteins at microvilli, whereas those lacking these clusters were diffusely distributed on the cell surface. The specific binding of ERM proteins to the juxta-membrane positively charged amino acid clusters of CD44, CD43, and ICAM-2 was confirmed by immunoprecipitation and site-directed mutagenesis. From these findings, we conclude that ERM proteins bind to integral membrane proteins bearing a positively charged amino acid cluster in their juxta-membrane cytoplasmic domain.

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Figures

Figure 1
Figure 1
Structure of GST fusion proteins (a) and E-cadherin chimeric proteins (b) of entire or truncated cytoplasmic domain of CD44, CD43, or ICAM-2. Details are described in Materials and Methods.
Figure 8
Figure 8
Amino acid sequences of the juxta-membrane cytoplasmic domains of CD43 and CD44 and the entire cytoplasmic domain of ICAM-2 that are responsible for their ERM binding and ERM colocalization (Fig. 7). Positively charged amino acid residues are presented in bold letters. By in vitro mutagenesis, positively charged amino acid clusters, which were underlined in CD44, CD43, and ICAM-2, were substituted with noncharged amino acids to construct E-44/KKK:QIN, E-43/KRR:NGG, and E-ICAM-2/RRR:GGA, respectively. These sequence data are available from EMBL/GenBank/DDBJ under accession number Y00090 (rat CD43), X66081 (mouse CD44), and X6549/S46669 (mouse ICAM-2).
Figure 7
Figure 7
Comparison of the results obtained from in vitro binding studies with transfection studies. Hatched squares represent the regions in cytoplasmic domains of CD44, CD43, and ICAM-2, which are responsible for their in vitro–direct binding to moesin (Fig. 3). In transfection experiments, E-cadherin chimeric molecules containing cytoplasmic regions represented as thick lines were co-concentrated with moesin at microvilli, whereas those containing regions represented by broken lines were diffusely distributed on the plasma membrane.
Figure 3
Figure 3
Comparison of the moesin-binding ability among various truncated cytoplasmic domains of CD44 (a) and CD43 (b). GST fusion proteins (Fig. 1 a) or GST were incubated with moesin at 150 mM KCl. Relative binding ability of each GST fusion protein to moesin was calculated as explained in Fig. 2. Relative-binding abilities of G-44/1–31 and G-43 were defined as 1 in a and b, respectively. In CD44, a.a.1–19 and a.a.1–31 (thick lines, bottom, a) bound to moesin with similar affinity to the whole cytoplasmic domain of CD44 at 40 mM KCl, whereas a.a.19–70 as well as a.a.1–70 showed very low affinity to moesin (broken lines, bottom, a), indicating that a.a.1–19 are responsible for moesin binding of CD44 (Fig. 2) and that a.a.19–70 are inhibitory for moesin binding of CD44. In CD43, a.a.1–31 and a.a.62– 124 strongly bound to moesin with similar affinity to the whole cytoplasmic domain of CD43 (thick lines, bottom, b), a.a.1–39 and a.a.38–124 were rather weakly associated with moesin (thin lines, bottom, b), and a.a.1–64 and a.a.78–124 showed very low affinity to moesin (broken lines, bottom, b). These findings indicate that at least in vitro both a.a.1–31 and a.a.62–78 appear to be responsible for moesin binding of CD43 (Fig. 2), and that the other region is inhibitory for moesin binding of CD43.
Figure 2
Figure 2
Association of moesin with the cytoplasmic domains of CD44, CD43, and ICAM-2. GST (GST) or GST fusion proteins with the entire cytoplasmic domain of E-cadherin (G-E-cad), occludin (G-Oc), CD43 (G-43), CD44 (G-44), or ICAM-2 (G-ICAM-2) were bound to Glutathione-Sepharose beads, and incubated with purified recombinant moesin at physiological (150 mM KCl) or low ionic strength (40 mM KCl). After washing, GST or GST fusion proteins were eluted together with their binding proteins from the beads with a buffer-containing glutathione. Proteins in the glutathione eluate were separated by SDS-PAGE followed by Coomassie brilliant blue staining to densitometrically estimate the amount of GST or GST fusion proteins in each eluate (GST fusion), or followed by immunoblotting with antimoesin mAb M22 to densitometrically estimate the amount of moesin bound to GST or GST fusion proteins (Bound Moesin). Relative binding ability of GST fusion proteins to moesin (Relative Binding of Moesin) was calculated by comparing the amount of bound moesin in each eluate containing a constant amount of nondegraded GST fusion protein with that in G-43 (150 mM KCl) eluate, with a careful attention to the effects of degradation products on the binding data. Values represent relative binding abilities averaged from three experiments ± SEM.
Figure 4
Figure 4
Immunofluorescence localization of E-cadherin chimeric molecules with entire and truncated cytoplasmic domains of CD44 (Fig. 1 b). L cells transiently expressing E-44 (a and b), E-44/1–19 (c and d) or E-44/ 20–70 (e and f) were doubly stained with anti–E-cadherin antibody (a, c, and e) and antimoesin antibody (b, d, and f). Both E-44 and E-44/1–19 were precisely co-concentrated with moesin at microvilli (arrows), whereas E-44/20–70 was diffusely distributed on cell surface, indicating that the ERM-colocalization signal resides in a.a.1–19 of the cytoplasmic domain of CD44. Bar, 20 μm.
Figure 5
Figure 5
Immunofluorescence localization of E-cadherin chimeric molecules with the truncated cytoplasmic domain of CD43 (a and b; E-43/ 1–31) or the entire cytoplasmic domain of ICAM-2 (c and d; E-ICAM-2) (Fig. 1 b). L cells transiently expressing each protein were doubly stained with anti–E-cadherin antibody (a and c) and antimoesin antibody (b and d). Both chimeric proteins were precisely co-concentrated with moesin at microvilli (arrows). Bars, 20 μm.
Figure 6
Figure 6
Coimmunoprecipitation of ERM proteins with E-cadherin chimeric protein with the whole cytoplasmic domain of CD43 (E-43). Cultured L cell transfectants expressing E-43 or E-43/1–9, 49–124 were solubilized with a lysis buffer containing 0.1% Nonidet P-40, and then immunoprecipitated with anti– E-cadherin mAb. The immunoprecipitate was separated by SDS-PAGE followed by immunoblotting with anti-ERM protein pAb (TK89; Anti-ERM) or anti–E-cadherin mAb (Anti–E-cad). TK89 recognized ezrin (E)/radixin (R) as well as moesin (M). ERM proteins were coimmunoprecipitated with E-43, but not with E-43/1–9,49–124.
Figure 9
Figure 9
Moesin-binding abilities of site-directed mutants of the cytoplasmic domains of CD44, CD43, and ICAM-2. GST fusion proteins with the entire cytoplasmic domains of CD43 (G-43) and the site-directed mutants of CD43, CD44, and ICAM-2 (G-43/ KRR:NGG, G-44/KKK:QIN, and G-ICAM-2/RRR:GGA; Fig. 8) were incubated with moesin at 150 mM KCl (for G-43/KRR: NGG and G-ICAM-2/RRR:GGA) or at 40 mM KCl (for G-44/ KKK:QIN). Relative-binding ability of each GST fusion protein to moesin was calculated as explained in Fig. 2. Relative-binding ability of G-43 was defined as 1. As compared to G-43, the site-directed mutants appeared to lose their moesin-binding abilities.
Figure 10
Figure 10
Immunofluorescence localization of site- directed mutants of E-cadherin chimeric molecules with entire cytoplasmic domains of CD44, CD43, and ICAM-2. KKK, KRR, and RRR in the juxta-membrane regions of CD44, CD43, and ICAM-2 were substituted with noncharged amino acids, respectively (E-44/KKK:QIN, E-43/ KRR:NGG, and E-ICAM-2/ RRR:GGA) (Fig. 8). L cells transiently expressing these mutants were doubly stained with anti–E-cadherin antibody (a, c, and e) and antimoesin antibody (b, d, and f). These mutants were diffusely distributed on the cell surface and were never concentrated at microvilli. Bar, 20 μm.
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References

    1. Algrain M, Turunen O, Vaheri A, Louvard D, Arpin M. Ezrin contains cytoskeleton and membrane binding domains accounting for its proposed role as a membrane-cytoskeletal linker. J Cell Biol. 1993;120:129–140. - PMC - PubMed
    1. Amieva MR, Wilgenbuns KK, Furthmayr H. Radixin is a component of hepatocyte microvilli in situ. Exp Cell Res. 1994;210:140–144. - PubMed
    1. Andréoli C, Martin M, Le-Borgne R, Reggio H, Mangeat P. Ezrin has properties to self-associate at the plasma membrane. J Cell Sci. 1994;107:2509–2521. - PubMed
    1. Arpin M, Algrain M, Louvard D. Membrane-actin microfilament connections: an increasing diversity of players related to band 4.1. Curr Opin Cell Biol. 1994;6:136–141. - PubMed
    1. Bennett V. The spectrin-actin junction of erythrocyte membrane skeletons. Biochem Biophys Acta. 1989;988:107–121. - PubMed

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