Harrison S M, Barnard R, Ho K Y, Rajkovic I, Waters M J
Department of Physiology and Pharmacology, University of Queensland, St. Lucia, Australia.
Endocrinology. 1995 Feb;136(2):651-9. doi: 10.1210/endo.136.2.7835299.
In humans and rabbits, the circulating GH binding protein (GHBP) is released from the GH receptor by cleavage at a site proximal to the cell surface. There is evidence that GHBP status is predictive of GH responsiveness, presumably because it reflects GH receptor status. This assumes that GHBP release is not a regulated step. Here we report a model for study of GHBP release that provides some insight into this question. Human HepG2 cells were stably transfected with rabbit GH receptor and shown to be responsive to nonprimate (bovine) GH, indicating functionality of the transfected receptor. These cells released GHBP of the expected size, and this release could be increased by incubation with a phorbol ester, which stimulated receptor synthesis through the cytomegalovirus promoter. We surveyed a wide range of protease inhibitors both with and without streptolysin-O permeabilization, with the intention of defining the endogenous protease. Of 16 inhibitors, only benzamidine proved an effective inhibitor of release, indicating the existence of a novel protease. We could increase GHBP release with a membrane impermeable thiol blocker, suggesting activation of a membrane protease. We examined the ability of IGF-1, insulin, dexamethasone, sex steroids, and T4 to influence GHBP release. Although these agents are known to be effective in the parent hepatoma line, none were effective in modulating GHBP release, although GH itself decreased release by around 30% as assessed with a ligand immunofunctional assay. We conclude that GHBP release appears to be constitutive in this model and driven by receptor availability. This is consistent with an in vivo situation where circulating GHBP provides an index of hepatic receptor expression.
在人类和兔子中，循环中的生长激素结合蛋白（GHBP）是通过在细胞表面近端的一个位点进行切割而从生长激素受体上释放出来的。有证据表明，GHBP状态可预测生长激素的反应性，大概是因为它反映了生长激素受体的状态。这假定GHBP的释放不是一个受调控的步骤。在此，我们报告了一个用于研究GHBP释放的模型，该模型为这个问题提供了一些见解。人肝癌细胞系HepG2稳定转染了兔生长激素受体，并被证明对非灵长类（牛）生长激素有反应，这表明转染受体具有功能。这些细胞释放出预期大小的GHBP，并且通过与佛波酯一起孵育可以增加这种释放，佛波酯通过巨细胞病毒启动子刺激受体合成。我们在有和没有链球菌溶血素-O通透处理的情况下，对多种蛋白酶抑制剂进行了检测，目的是确定内源性蛋白酶。在16种抑制剂中，只有苯甲脒被证明是释放的有效抑制剂，这表明存在一种新型蛋白酶。我们可以用一种膜不透性的巯基阻断剂增加GHBP的释放，这表明一种膜蛋白酶被激活。我们研究了胰岛素样生长因子-1、胰岛素、地塞米松、性类固醇和甲状腺素影响GHBP释放的能力。尽管已知这些物质在亲本肝癌细胞系中有效，但没有一种能有效调节GHBP的释放，不过用配体免疫功能测定法评估，生长激素本身可使释放减少约30%。我们得出结论，在这个模型中，GHBP的释放似乎是组成性的，并受受体可用性驱动。这与体内情况一致，即循环中的GHBP提供了肝脏受体表达的一个指标。
