Department of Anatomy and Cell Biology, University of Western Ontario, London, Ontario N6A 5C1, Canada.
J Biol Chem. 2011 Aug 5;286(31):27639-53. doi: 10.1074/jbc.M111.260711. Epub 2011 Jun 9.
Pannexin1 (Panx1) is an integral membrane protein comprised of three species as follows: an unglycosylated core-Gly0, a high mannose-Gly1, and a complex glycosylated Gly2 species. Although Panx1 channels mediate several cellular responses, the domain regulating its oligomerization and cell surface trafficking and the mechanisms governing its internalization and degradation have not been identified. This study characterizes the role of the Panx1 C-tail domain by truncating the polypeptide at residue 307 and expressing the mutant in BICR-M1R(k) and HEK-293T cells. Enzymatic digestion and immunolabeling assays revealed that the Panx1(T307)-RFP was glycosylated primarily to the high mannose species consistent with its retention in the endoplasmic reticulum. Co-expression of Panx1(T307)-RFP with Panx1 followed by co-immunoprecipitation assays revealed that the mutant and Panx1 could interact, whereas biotinylation assays showed that this interaction inhibited Panx1 from maturing into the Gly2 species and reaching the cell surface. Additional inhibitor studies indicated that the degradation of the mutant was via proteasomes, whereas Panx1 was degraded by lysosomes. Analysis of the pathways important in Panx1 internalization revealed partial co-distribution of Panx1 with many molecular constituents of the endocytic machinery that include clathrin, AP2, dynamin II, caveolin-1, and caveolin-2. However, co-immunoprecipitation assays together with the disruption of lipid rafts by methyl-β-cyclodextrin suggest that Panx1 does not engage this endocytic machinery. Furthermore, dominant-negative and pharmacological studies revealed that Panx1 internalization was dynamin II-independent. Collectively, these results indicate that the oligomerization and trafficking of Panx1 are regulated by the C-terminal domain, whereas internalization of long lived Panx1 channels occurs in a manner that is distinct from classical endocytic pathways.
连接蛋白 1（Panx1）是一种完整的膜蛋白，由以下三种形式组成：未糖基化的核心-Gly0、高甘露糖-Gly1 和复杂糖基化的 Gly2 形式。尽管 Panx1 通道介导多种细胞反应，但调节其寡聚化和细胞表面运输的结构域以及控制其内化和降解的机制尚未确定。本研究通过在残基 307 处截断多肽来表征 Panx1 C 端结构域的作用，并在 BICR-M1R(k) 和 HEK-293T 细胞中表达突变体。酶消化和免疫标记分析表明，Panx1(T307)-RFP 主要糖基化为高甘露糖形式，与它在内质网中的保留一致。Panx1(T307)-RFP 与 Panx1 的共表达以及随后的共免疫沉淀分析表明，突变体和 Panx1 可以相互作用，而生物素化分析表明这种相互作用抑制了 Panx1 成熟为 Gly2 形式并到达细胞表面。进一步的抑制剂研究表明，突变体的降解是通过蛋白酶体进行的，而 Panx1 的降解是通过溶酶体进行的。对 Panx1 内化过程中重要途径的分析表明，Panx1 与许多内吞机制的分子成分部分共分布，包括网格蛋白、AP2、dynamin II、 caveolin-1 和 caveolin-2。然而，共免疫沉淀分析以及甲基-β-环糊精对脂筏的破坏表明，Panx1 不参与这种内吞机制。此外，显性负和药理学研究表明，Panx1 的内化与 dynamin II 无关。总之，这些结果表明 Panx1 的寡聚化和运输受 C 端结构域调节，而长寿命 Panx1 通道的内化是以与经典内吞途径不同的方式发生的。
