Department of Biological Sciences, Lehigh University, 111 Research Drive, Bethlehem, PA 18015, USA.
Int J Mol Sci. 2023 Aug 16;24(16):12851. doi: 10.3390/ijms241612851.
Proteins fated to be internalized by clathrin-mediated endocytosis require an endocytic motif, where AP-2 or another adaptor protein can bind and recruit clathrin. Tyrosine and di-leucine-based sorting signals are such canonical motifs. Connexin 43 (Cx43) has three canonical tyrosine-based endocytic motifs, two of which have been previously shown to recruit clathrin and mediate its endocytosis. In addition, di-leucine-based motifs have been characterized in the Cx32 C-terminal domain and shown to mediate its endocytosis. Here, we examined the amino acid sequences of all 21 human connexins to identify endocytic motifs across the connexin gene family. We find that although there is limited conservation of endocytic motifs between connexins, 14 of the 21 human connexins contain one or more canonical tyrosine or di-leucine-based endocytic motif in their C-terminal or intracellular loop domain. Three connexins contain non-canonical (modified) di-leucine motifs. However, four connexins (Cx25, Cx26, Cx31, and Cx40.1) do not harbor any recognizable endocytic motif. Interestingly, live cell time-lapse imaging of different GFP-tagged connexins that either contain or do not contain recognizable endocytic motifs readily undergo endocytosis, forming clearly identifiable annular gap junctions when expressed in HeLa cells. How connexins without defined endocytic motifs are endocytosed is currently not known. Our results demonstrate that an array of endocytic motifs exists in the connexin gene family. Further analysis will establish whether the sites we identified in this in silico analysis are legitimate endocytic motifs.
被网格蛋白包被内吞作用所内化的蛋白质需要一个内吞作用的基序，衔接蛋白或其他衔接蛋白可以结合并募集网格蛋白。酪氨酸和双亮氨酸基序是这种典型的基序。连接蛋白 43（Cx43）有三个典型的基于酪氨酸的内吞作用基序，其中两个以前已经被证明可以募集网格蛋白并介导其内吞作用。此外，在 Cx32 C 末端结构域中已经描述了基于双亮氨酸的基序，并证明其介导了内吞作用。在这里，我们检查了所有 21 个人类连接蛋白的氨基酸序列，以鉴定连接蛋白家族中的内吞作用基序。我们发现，尽管连接蛋白之间的内吞作用基序的保守性有限，但在 21 个人类连接蛋白中的 14 个中，其 C 末端或细胞内环结构域中含有一个或多个典型的酪氨酸或双亮氨酸基序的内吞作用基序。三个连接蛋白含有非典型（修饰）的双亮氨酸基序。然而，四个连接蛋白（Cx25、Cx26、Cx31 和 Cx40.1）不含有任何可识别的内吞作用基序。有趣的是，活细胞时间 lapse 成像显示，不同的 GFP 标记的连接蛋白，要么含有可识别的内吞作用基序，要么不含有可识别的内吞作用基序，很容易发生内吞作用，当在 HeLa 细胞中表达时，形成清晰可识别的环形缝隙连接。目前还不知道没有定义的内吞作用基序的连接蛋白是如何被内吞的。我们的结果表明，一系列内吞作用基序存在于连接蛋白基因家族中。进一步的分析将确定我们在这个计算机分析中确定的位点是否是合法的内吞作用基序。
