Kanzaki M, Shibata H, Mogami H, Kojima I
Department of Cell Biology, Gunma University, Maebashi, Japan.
J Biol Chem. 1995 Jun 2;270(22):13099-104. doi: 10.1074/jbc.270.22.13099.
CD20 is a transmembrane protein that functions as a Ca(2+)-permeable cation channel (Bubien, J. K., Zhou, L. J., Bell, P. D., Frizzel, R. A., and Tedder, T. F. (1993) J. Cell Biol. 121, 1121-1132) and is involved in growth regulation of B lymphocytes. In order to further investigate the role of calcium entry in cell cycle progression, we introduced the cDNA encoding a Ca(2+)-permeable cation channel, CD20, into Balb/c 3T3 cells. Balb/c 3T3 cells transfected with a vector containing cDNA encoding CD20 expressed the CD20 protein, which was detected by assaying the binding of a monoclonal antibody against CD20. Calcium-permeable cation channel activity was detected in CD20-expressing cells by whole cell patch clamp recording and microfluorometric determination of the cytoplasmic Ca2+ concentration using fura-2. The expression of CD20 induced significant alterations in the responses of the cells to insulin-like growth factor-I (IGF-I). IGF-I induced DNA synthesis by control cells only when they had been pretreated with both platelet-derived growth factor (PDGF) and epidermal growth factor (EGF). In contrast, DNA synthesis by 30% of the quiescent CD20-expressing cells was initiated in response to IGF-I in the absence of priming with PDGF and EGF. When control quiescent cells were primed with PDGF and EGF, the addition of IGF-I led to the initiation of DNA synthesis after 14 h or more, whereas it induced DNA synthesis by CD20-expressing cells primed with PDGF and EGF 4 h earlier. The IGF-induced DNA synthesis was dependent on extracellular Ca2+, and expression of CD20 reduced the concentration of extracellular Ca2+ required for it. Furthermore, DNA synthesis by approximately 25% of the CD20-expressing cells was initiated after priming with PDGF and EGF, even in the absence of the progression factor IGF-I. These results indicate that CD20 expressed in Balb/c 3T3 cells functions as a constitutively active Ca(2+)-permeable cation channel and that expression of CD20 accelerates G1 progression in a Ca(2+)-dependent manner.
CD20是一种跨膜蛋白，作为一种Ca(2+)通透阳离子通道发挥作用（布比恩，J.K.，周，L.J.，贝尔，P.D.，弗里泽尔，R.A.，和特德，T.F.（1993年）《细胞生物学杂志》121卷，1121 - 1132页），并参与B淋巴细胞的生长调节。为了进一步研究钙离子内流在细胞周期进程中的作用，我们将编码Ca(2+)通透阳离子通道CD20的cDNA导入Balb/c 3T3细胞。用含有编码CD20的cDNA的载体转染的Balb/c 3T3细胞表达CD20蛋白，通过检测抗CD20单克隆抗体的结合来检测该蛋白。通过全细胞膜片钳记录以及使用fura - 2对细胞质Ca2+浓度进行微荧光测定，在表达CD20的细胞中检测到了Ca(2+)通透阳离子通道活性。CD20的表达诱导细胞对胰岛素样生长因子 - I（IGF - I）的反应发生显著改变。IGF - I仅在对照细胞预先用血小板衍生生长因子（PDGF）和表皮生长因子（EGF）预处理后才诱导其DNA合成。相比之下，30%的静止的表达CD20的细胞在没有用PDGF和EGF预处理的情况下，对IGF - I作出反应启动了DNA合成。当对照静止细胞用PDGF和EGF预处理后，添加IGF - I在14小时或更长时间后导致DNA合成启动，而它在4小时前就诱导了用PDGF和EGF预处理的表达CD20的细胞进行DNA合成。IGF诱导的DNA合成依赖于细胞外Ca2+，并且CD20的表达降低了其所需的细胞外Ca2+浓度。此外，即使在没有进展因子IGF - I的情况下，约25%的表达CD20的细胞在用PDGF和EGF预处理后也启动了DNA合成。这些结果表明，在Balb/c 3T3细胞中表达的CD20作为一种组成性激活的Ca(2+)通透阳离子通道发挥作用，并且CD20的表达以Ca(2+)依赖方式加速G1期进程。
