Department of Cancer Prevention, Institute for Cancer Research, Norwegian Radium Hospital, Oslo University Hospital, Montebello, 0310 Oslo, and Centre for Cancer Biomedicine, Faculty of Medicine, University of Oslo, Oslo, Norway.
J Cell Sci. 2012 Sep 1;125(Pt 17):3966-76. doi: 10.1242/jcs.093500. Epub 2012 May 23.
Gap junctions consist of arrays of intercellular channels that enable adjacent cells to communicate both electrically and metabolically. Gap junction channels are made of a family of integral membrane proteins called connexins, of which the best-studied member is connexin43. Gap junctions are dynamic plasma membrane domains, and connexin43 has a high turnover rate in most tissue types. However, the mechanisms involved in the regulation of connexin43 endocytosis and transport to lysosomes are still poorly understood. Here, we demonstrate by live-cell imaging analysis that treatment of cells with 12-O-tetradecanoylphorbol 13-acetate (TPA) induces endocytosis of subdomains of connexin43 gap junctions. The internalized, connexin43-enriched vesicles were found to fuse with early endosomes, which was followed by transport of connexin43 to the lumen of early endosomes. The HECT E3 ubiquitin ligase smad ubiquitination regulatory factor-2 (Smurf2) was found to be recruited to connexin43 gap junctions in response to TPA treatment. Depletion of Smurf2 by small interfering RNA resulted in enhanced levels of connexin43 gap junctions between adjacent cells and increased gap junction intercellular communication. Smurf2 depletion also counteracted the TPA-induced endocytosis and degradation of connexin43. Collectively, these data identify Smurf2 as a novel regulator of connexin43 gap junctions.
缝隙连接由细胞间通道的阵列组成，使相邻细胞能够进行电和代谢通讯。缝隙连接通道由一组称为连接蛋白的整合膜蛋白组成，其中研究最充分的成员是连接蛋白 43。缝隙连接是动态的质膜域，连接蛋白 43 在大多数组织类型中具有高周转率。然而，调节连接蛋白 43 内吞作用和向溶酶体运输的机制仍知之甚少。在这里，我们通过活细胞成像分析表明，用 12-O-十四烷酰佛波醇 13-乙酸酯（TPA）处理细胞会诱导连接蛋白 43 缝隙连接亚域的内吞作用。发现内化的、富含连接蛋白 43 的小泡与早期内体融合，随后连接蛋白 43 被转运到早期内体的腔中。发现 HECT E3 泛素连接酶 smad 泛素化调节因子 2（Smurf2）在 TPA 处理后被募集到连接蛋白 43 缝隙连接。通过小干扰 RNA 耗尽 Smurf2 会导致相邻细胞之间的连接蛋白 43 缝隙连接水平升高，并增加缝隙连接细胞间通讯。Smurf2 耗尽也会抵消 TPA 诱导的连接蛋白 43 内吞作用和降解。总的来说，这些数据表明 Smurf2 是连接蛋白 43 缝隙连接的新型调节剂。
