Variants of the carboxyl-terminal KDEL sequence direct intracellular retention.
Variants of the carboxyl-terminal KDEL sequence direct intracellular retention.
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DOI：
10.1016/s0021-9258(19)39273-7
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发表时间：
1990-04
期刊：
影响因子：
--
通讯作者：
D. Andres;I. Dickerson;J. Dixon
中科院分区：
文献类型：
--
作者：
D. Andres;I. Dickerson;J. Dixon
存在于内质网腔中的可溶性蛋白质共享一个共同的羧基末端四肽 Lys-Asp-Glu-Leu (KDEL)。将四肽添加到正常分泌的蛋白质中对于引起在内质网中的保留是必要且充分的。为了表征 KDEL 信号中的关键残基，在 AtT-20 细胞系中表达了编码具有 4 个氨基酸羧基末端延伸 KDEL 的原神经肽 Y (pro-NPY) 或一系列 KDEL 变体的 cDNA。 AtT-20 细胞是一种小鼠垂体前叶促肾上腺皮质激素细胞系，合成、加工和分泌促 ACTH 前体/内啡肽前体。由于 AtT-20 细胞中的翻译后加工已被广泛表征，因此它提供了一个模型系统，可以在其中比较外源肽前体（NPY 前体）和内源前体（ACTH 前体/内啡肽）的加工。使用在 COOH 末端编码 KDEL、DKEL、RDEL、KNEL、KDQL 或 KDEA 的 pro-NPY 的改变的 cDNA 来生成稳定的 AtT-20 细胞系。使用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳、胰蛋白酶肽图谱和放射测序研究了前 NPY 加工成神经肽 Y 和羧基末端肽的过程。将四肽 KDEL、DKEL、RDEL 或 KNEL 添加到神经肽 Y 前体（一种通常由神经元细胞加工和分泌的肽激素）的 COOH 末端，导致 AtT-20 细胞中未加工的激素原完全保留在细胞内。然而，当在 AtT-20 细胞中表达时，KDQL 和 KDEA 延伸的 pro-NPY 分子会像野生型 pro-NPY 一样被加工和分泌。在基础条件和刺激条件下，这些细胞系中proNPY衍生肽的分泌与内源性ACTH原/内啡肽衍生产物的分泌平行。这些诱变研究表明，KDEL 保留信号的变体可以指导细胞内保留。
Soluble proteins which reside in the lumen of the endoplasmic reticulum share a common carboxyl-terminal tetrapeptide Lys-Asp-Glu-Leu (KDEL). Addition of the tetrapeptide to a normally secreted protein is both necessary and sufficient to cause retention in the endoplasmic reticulum. In order to characterize the critical residues in the KDEL signal, cDNAs encoding proneuropeptide Y (pro-NPY) with the 4-amino acid carboxyl-terminal extension KDEL or a series of KDEL variants were expressed in the AtT-20 cell line. AtT-20 cells, a mouse anterior pituitary corticotrope cell line, synthesize, process, and secrete the pro-ACTH/endorphin precursor. Since post-translational processing in AtT-20 cells has been extensively characterized, it provides a model system in which the processing of a foreign peptide precursor (pro-NPY) and the endogenous precursor (pro-ACTH/endorphin) can be compared. Altered cDNAs encoding pro-NPY with KDEL, DKEL, RDEL, KNEL, KDQL, or KDEA at the COOH terminus were used to generate stable AtT-20 cell lines. The processing of pro-NPY to neuropeptide Y and the carboxyl-terminal peptide was studied using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, tryptic peptide mapping, and radiosequencing. Addition of the tetrapeptides KDEL, DKEL, RDEL, or KNEL to the COOH terminus of the neuropeptide Y precursor, a peptide hormone normally processed and secreted from neuronal cells, caused complete intracellular retention of the unprocessed prohormone in AtT-20 cells. However, KDQL and KDEA-extended pro-NPY molecules were processed and secreted like wild-type pro-NPY when expressed in AtT-20 cells. The secretion of proNPY-derived peptides in these cell lines paralleled secretion of endogenous pro-ACTH/endorphin-derived products under both basal and stimulated conditions. These mutagenesis studies demonstrate that variants of the KDEL retention signal can direct intracellular retention.