Clapp C, Weiner R I
Reproductive Endocrinology Center, University of California, San Francisco 94143.
Endocrinology. 1992 Mar;130(3):1380-6. doi: 10.1210/endo.130.3.1311239.
A 16-kilodalton N-terminal fragment of PRL (16K PRL) is formed by enzymatic cleavage of intact 23-kilodalton PRL (23K PRL) in the pituitary gland and in target tissues for PRL. 16K PRL inhibits the growth of capillary endothelial cells, while intact PRL was inactive suggesting that 16K PRL acted via a receptor other than the PRL receptor. To analyze whether this inhibitory effect could be mediated through an specific 16K PRL receptor, we characterized the binding of 16K PRL to membrane preparations of bovine brain capillary endothelial (BBE) cells. 16K PRL was generated by the proteolysis of rat 23K PRL with a particulate fraction from rat mammary gland homogenates and purified by gel filtration. The specific binding of [125I]16K PRL to BBE cell membranes was high affinity (Kd = 9.9 nM), saturable (Bmax = 4.8 pmol/mg protein), and reversible. In competition studies for [125I]16K PRL binding, 16K PRL was most potent, while little displacement was observed with high concentrations of 23K PRLs, growth hormones, and basic fibroblast growth factor. Blockade of reformation of disulfide bonds by carbamidomethylation of 16K PRL, a procedure which increases the biological activity of the molecule, increased its binding affinity (Kd = 0.9 nM). Cross-linking experiments identified a 52,000 and a 32,000 mol wt protein as the major 16K PRL binding species. These data demonstrate the presence of specific, high affinity, saturable binding sites for 16K PRL on BBE cell membranes and support biological findings that 16K PRL inhibits capillary endothelial cell proliferation, through a novel, high affinity receptor.
催乳素（PRL）的16千道尔顿N端片段（16K PRL）是由垂体腺和PRL靶组织中完整的23千道尔顿PRL（23K PRL）经酶切形成的。16K PRL可抑制毛细血管内皮细胞的生长，而完整的PRL则无此活性，这表明16K PRL是通过PRL受体以外的受体发挥作用的。为了分析这种抑制作用是否可通过特异性的16K PRL受体介导，我们对16K PRL与牛脑毛细血管内皮（BBE）细胞膜制剂的结合进行了表征。16K PRL是通过用大鼠乳腺匀浆的颗粒部分对大鼠23K PRL进行蛋白水解产生的，并通过凝胶过滤进行纯化。[125I]16K PRL与BBE细胞膜的特异性结合具有高亲和力（Kd = 9.9 nM）、可饱和性（Bmax = 4.8 pmol/mg蛋白）且可逆。在[125I]16K PRL结合的竞争研究中，16K PRL最有效，而高浓度的23K PRL、生长激素和碱性成纤维细胞生长因子几乎没有观察到置换作用。通过对16K PRL进行氨基甲酰甲基化来阻断二硫键的重新形成，该过程可增加分子的生物活性，同时增加了其结合亲和力（Kd = 0.9 nM）。交联实验确定了分子量为52,000和32,000的蛋白质为主要的16K PRL结合物种。这些数据证明了BBE细胞膜上存在16K PRL的特异性、高亲和力、可饱和结合位点，并支持了16K PRL通过一种新型高亲和力受体抑制毛细血管内皮细胞增殖的生物学发现。
