Characterization of the gap junction protein, connexin45.
Characterization of the gap junction protein, connexin45.
复制标题
间隙连接蛋白 connexin45 的表征。
DOI：
10.1007/bf00232672
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发表时间：
1994
期刊：
影响因子：
--
通讯作者：
Beyer,EC
中科院分区：
文献类型：
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作者：
Laing,JG;Westphale,EM;Engelmann,GL;Beyer,EC
Connexin45是一种形成通道的缝隙连接蛋白，具有独特的特性。RNA印迹显示，connecin45在包括WB、SK Hepl、BHK、A7r5、CLEM和BWEM细胞在内的许多细胞系中都有表达。我们在BWEM细胞中进一步研究了Connexin45，使用了特异性亲和纯化的抗体，针对合成的代表其序列中285-298个氨基酸的肽段。免疫荧光实验表明，BWEM细胞同时表达connexin43和connexin45，并且这些连接蛋白具有共定位。Connexin45多肽由代谢标记为[35S]-蛋氨酸的BWEM细胞免疫沉淀而成，主要由48 kD多肽组成。从代谢标记为[32P]-正磷酸的BWEM细胞免疫沉淀中，Connexin45和connexin43含有放射性磷酸盐。这个磷酸盐标签通过碱性磷酸酶消化从连接蛋白45中去除。促瘤剂12- o - tetradecanoylphorbol13 -acetate （TPA）处理BWEM细胞可抑制微注射路西法黄的细胞间传代。虽然TPA处理在这些细胞中诱导了连接蛋白43的磷酸化，但它降低了连接蛋白45的表达。此外，TPA处理后表达的connexin45没有磷酸化。这些结果表明，改变蛋白磷酸化的处理可能通过不同的机制调节BWEM细胞中的connexin43和connexin45。
Connexin45 is a gap junction protein which forms channels with unique characteristics. RNA blots demonstrated that connexin45 is expressed in a number of cell lines including WB, SK Hepl, BHK, A7r5, CLEM, and BWEM cells. Connexin45 was further studied in BWEM cells using specific affinity-purified antibodies directed against a synthetic peptide representing amino acids 285–298 of its sequence. Immunofluorescence experiments demonstrated that the BWEM cells expressed both connexin43 and connexin45 and that these connexins colocalized. Connexin45 polypeptide, immunoprecipitated from BWEM cells metabolically labeled with [35S]-methionine, consisted of a predominant 48 kD polypeptide. Connexin45 and connexin43 contained radioactive phosphate when immunoprecipitated from BWEM cells metabolically labeled with [32P]-orthophosphoric acid. This phosphate label was removed from connexin45 by alkaline phosphatase digestion. Treatment of BWEM cells with the tumor promoting agent 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibited intercellular passage of microinjected Lucifer yellow. While TPA treatment induced phosphorylation of connexin43 in these cells, it reduced the expression of connexin45. Furthermore, the connexin45 expressed after TPA treatment was not phosphorylated. These results suggest that treatments which alter protein phosphorylation may regulate connexin43 and connexin45 in BWEM cells by different mechanisms.