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内容記述 |
Band4.1 protein is one of peripheral membrane proteins of erythrocyte membranes, which directly binds to glycophorin C, an integral membrane protein, at its N-terminal half. Recently, the amino acid sequence of the N-terminal half of this protein was found to be conserved in the N-terminal end of several distinct proteins, pointing to the existence of the band4.1 superfamily. In addition to band4.1 protein, this band4.1 superfamily includes ezrin, radixin, moesin, talin and protein-tyrosine-phosphatases (PTPHI and PTPMEG). Furthermore, the tumor suppressor protein for neurofibromatosis 2 named 'merlin' is also reportedly included in this superfamily. For a better understanding of the structure and functions of these superfamily members, I first tried to clarify the more detail picture of this superfamily using polymerase chain reaction (PCR) methods (Chapter I), and next to examine the physiological functions of ezrin/radixin/moesin by the use of antisense oligonucleotides (Chapter II). <br /> In the Chapter I , in order to examine the structural diversity of this superfamily members, using the PCR method with synthesized mixed primers, I have attempted to list up as many members of the band4.1 superfamlly as possible expressing in mouse teratocarcinoma F9 cells and the mouse brain tissue. In total, 14 distinct types of cDNA clones were obtained; 8 clones were identical to the corresponding parts of cDNAs for the so far identified members, while 6 clones appeared to encode novel members (NBL 1-6 :Novel βand4.1-like proteins). Sequence analyses of these clones revealed that the band4.1 superfamily can be subdivided into 6 gene families such as band4.1 protein, ERM (ezrin/ radixln/ moesin), talin, PTPHI (PTPH1/ PTPMEG/ NBL1-3), and NBL4 (NBL4/ NBL5) and merlin (merlin/ NBL6) families. The existence of the NBL4 family was first recognised here. I screened F9 cell cDNA Iibrary and obtained a full length (2.5-kb) cDNA encoding an NBL4. NBL4 cDNA contains an open reading frame of 554 amino acids. Its N-terminal half segment is more homologous to those of the PTPases and band4.1 protein than the other superfamily members. Its band4.1 homology domain bears a putative myristoylation site and phosphorylation sites for A-kinase and protein-tyrosine kinases, suggesting its possible involvement in the regulation of cellular events just beneath the plasma membrane. In this study, I describe initial characterization of these new members and discuss the evolution of the band4.1 superfamily. <br /> In the Chapter II, to examine the functions of ERM family members, antisense oligonucleotides to the N-terminal sequences of ERM family members were constructed and added to cultures of mouse epithelial cells (MTD-1A cells) and thymoma cells (L5178Y), which coexpress all the members. Immunoblotting revealed that each antisense oligonucleotides selectively suppressed the expression of the corresponding member to the undetectable level. Immunofluorescence microscopy of these ezrin, radixin or moesm "null" cells showed that all the ERM family members are colocalized at cell-to-cell adherens junctions, microvilli and cleavage furrows where actin filaments were densely associated with plasma membranes. The ezrin/radixin/moesin antisense oligonucleotides mixture induced the destruction of both cell-cell and cell-substrate adhesion and the disappearance of microvilli. Ezrin or radixin antisense oligonucleotides singly affected the initial step of the formation of both cell-cell and cell-substrate adheslon, but showed no effects on the microvilli structures. In sharp contrast, moesin antisense oligonucleotide did not singly show any effect on cell-cell and cell-substrate adhesion, whereas it partly affected the microvilli structure. These data indicate that ezrin and radixin can be functionally substituted for each other, that moesin has some synergetic functional interaction with ezrin and radixin, and that these ERM family members are directly involved in cell-cell and cell-substrate adhesion as well as microvilli formation. |
要旨・審査要旨 / Abstract, Screening Result (298.8 kB)
