Bielinska M, Rzymkiewicz D, Boime I
Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110.
Mol Endocrinol. 1994 Jul;8(7):919-28. doi: 10.1210/mend.8.7.7984153.
LH is a dimeric glycoprotein hormone that is stored in the anterior pituitary and is released in response to GnRH, while the placental hormone, human CG (hCG), sharing the same alpha-subunit and a related beta-subunit, is secreted constitutively. In search of a determinant that allows sorting of LH into a regulated secretory pathway, the genes encoding the common alpha- and LH/CG beta-subunits were expressed in the GH3 rat pituitary tumor cell line, which contains a regulated secretory pathway. Steady state labeling and subsequent chase experiments showed that not only LH but also hCG can be sorted to a regulated secretory pathway; after an initial period of constitutive secretion, the mature forms of both hormones containing processed oligosaccharides were stored intracellularly, and their release was stimulated by either forskolin or KCl depolarization. In Chinese hamster ovary cells, which lack a regulated pathway and are devoid of storage granules, only hormones containing unprocessed N-linked oligosaccharides were found. In GH3 cells the LH beta-subunit was partially retained in an endoglycosidase H-sensitive form, presumably in the endoplasmic reticulum; the enzyme-resistant fraction was secreted through a regulated secretory pathway. A large fraction of the hCG beta-subunit was released constitutively, although some mature hCG beta-subunit accumulated in secretory granules and was released by forskolin. The common alpha-subunit was secreted constitutively with little intracellular accumulation of the mature forms. We conclude that the LH beta-subunit contains sufficient information to direct LH to a regulated pathway, and alpha:LH beta assembly is not a prerequisite for this targeting. The sorting of hCG to a regulated pathway in GH3 cells presumably reflects a structural similarity between LH and hCG. In addition, we have shown that GH3 cells can recognize the N-linked oligosaccharides on the gonadotropin subunits as substrates for sulfation.
促黄体生成素（LH）是一种二聚体糖蛋白激素，储存于垂体前叶，在促性腺激素释放激素（GnRH）的刺激下释放；而胎盘激素人绒毛膜促性腺激素（hCG），虽与LH共享相同的α亚基和一个相关的β亚基，但却是持续分泌的。为寻找一种能使LH进入调节性分泌途径的决定因素，编码共同α亚基和LH/CGβ亚基的基因在GH3大鼠垂体肿瘤细胞系中表达，该细胞系含有调节性分泌途径。稳态标记及随后的追踪实验表明，不仅LH，hCG也能被分选至调节性分泌途径；在初始的持续分泌期后，两种含有加工后寡糖的成熟激素形式被储存于细胞内，且它们的释放可被福司可林或氯化钾去极化所刺激。在中国仓鼠卵巢细胞中，该细胞缺乏调节性途径且没有储存颗粒，仅发现了含有未加工的N - 连接寡糖的激素。在GH3细胞中，LHβ亚基部分以对内切糖苷酶H敏感的形式保留，推测位于内质网中；抗酶部分则通过调节性分泌途径分泌。大部分hCGβ亚基持续释放，尽管一些成熟的hCGβ亚基积累于分泌颗粒中并被福司可林释放。共同α亚基持续分泌，成熟形式在细胞内几乎没有积累。我们得出结论，LHβ亚基包含将LH导向调节性途径的足够信息，且α:LHβ组装并非这种靶向的先决条件。hCG在GH3细胞中被分选至调节性途径大概反映了LH和hCG之间的结构相似性。此外，我们已表明GH3细胞可将促性腺激素亚基上的N - 连接寡糖识别为硫酸化的底物。
