Dickerson I M, Dixon J E, Mains R E
Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
J Biol Chem. 1987 Oct 5;262(28):13646-53.
An expression vector was constructed that placed the cDNA for human neuropeptide Y (NPY) under the control of the mouse metallothionein promoter and was used to transfect the AtT-20 mouse anterior pituitary corticotrope cell line. AtT-20 cells normally process the pro-ACTH/endorphin precursor but do not produce detectable levels of NPY. The resulting AtT-20/NPY cell line (Mt.NPY1a) was used to study the ability of the corticotrope cells to synthesize, process, and secrete the foreign proNPY-related peptide products. The stable cell line created contains approximately 40 copies of proNPY cDNA per cell. NPY mRNA levels and proNPY synthesis were increased at least 35-fold when maximally induced with cadmium; proNPY synthesis was also induced by glucocorticoids. Upon induction the NPY secretion rate was equimolar to that of the endogenous peptides. ProNPY, NPY, and the COOH-terminal peptide produced by this cell line had molecular weight and amino acid-labeling pattern predicted from cDNA sequence data and from previous isolation of NPY-related molecules from NPY-producing cells. The structures of secreted proNPY, NPY, and COOH-terminal peptide, as well as determination of the site of proteolytic cleavage between NPY and the COOH-terminal peptide, were determined by tryptic mapping and Edman degradation of secreted biosynthetically labeled peptide products. The proNPY molecule appears to be processed in the same pathway responsible for cleavage of the endogenous pro-ACTH/endorphin precursor. Secretion of proNPY-derived peptides paralleled secretion of endogenous pro-ACTH/endorphin-derived products, under both basal and stimulated conditions. With induction proNPY expression there is a dose-dependent inhibition of both proNPY and pro-ACTH/endorphin proteolytic processing.
构建了一个表达载体，该载体将人神经肽Y（NPY）的cDNA置于小鼠金属硫蛋白启动子的控制之下，并用于转染AtT - 20小鼠垂体前叶促肾上腺皮质激素细胞系。AtT - 20细胞通常加工促肾上腺皮质激素/内啡肽前体，但不产生可检测水平的NPY。所得的AtT - 20/NPY细胞系（Mt.NPY1a）用于研究促肾上腺皮质激素细胞合成、加工和分泌外源促NPY相关肽产物的能力。所创建的稳定细胞系每个细胞含有约40个促NPY cDNA拷贝。在用镉最大诱导时，NPY mRNA水平和促NPY合成增加了至少35倍；糖皮质激素也可诱导促NPY合成。诱导后，NPY分泌率与内源性肽的分泌率等摩尔。该细胞系产生的促NPY、NPY和C末端肽具有根据cDNA序列数据以及先前从产生NPY的细胞中分离NPY相关分子所预测的分子量和氨基酸标记模式。通过对分泌的生物合成标记肽产物进行胰蛋白酶图谱分析和埃德曼降解，确定了分泌的促NPY、NPY和C末端肽的结构，以及NPY和C末端肽之间蛋白水解切割位点的确切位置。促NPY分子似乎是通过负责切割内源性促肾上腺皮质激素/内啡肽前体的相同途径进行加工的。在基础和刺激条件下，促NPY衍生肽的分泌与内源性促肾上腺皮质激素/内啡肽衍生产物的分泌平行。随着促NPY表达的诱导，促NPY和促肾上腺皮质激素/内啡肽的蛋白水解加工均受到剂量依赖性抑制。
