Development of recombinant human prolactin receptor antagonists by molecular mimicry of the phosphorylated hormone
Development of recombinant human prolactin receptor antagonists by molecular mimicry of the phosphorylated hormone
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DOI：
10.1210/en.139.2.609
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发表时间：
1998-02-01
期刊：
影响因子：
4.8
通讯作者：
Walker, AM
中科院分区：
文献类型：
--
作者：
Chen, TJ;Kuo, CB;Walker, AM
先前的研究表明，天然磷酸化的PRL在两种不同的PRL反应细胞系中拮抗未经修饰的PRL的促生长作用。在这项研究中，我们的目的是通过用一个相当大的、带负电荷的氨基酸(谷氨酸或天冬氨酸)代替正常磷酸化的丝氨酸[人PRL中的丝氨酸179]来产生一种模拟磷酸化的PRL的分子。此外，由于磷酸化对生物活性的显著影响，我们研究了未经修饰的丝氨酸在促进PRL生长活性中的重要性。从美国典型培养库中获得hPRL互补DNA，定向亚克隆入pt7-SCII。使用脱氧尿嘧啶核苷方法进行突变。在大肠杆菌BL21(DE3)中表达的蛋白质主要以包涵体形式存在。用NB2生物测定法对每个丝氨酸179突变体的激动剂和拮抗剂活性进行了评价。与标准hPRL相比，重组野生型在NB2检测中更具活性，这既证明了这些细胞的制剂中没有或低水平的内毒素污染，也证明了分子的适当折叠。天冬氨酸和谷氨酸突变体没有内在的激动剂活性，但都拮抗野生型催乳素的促生长活性，其中天冬氨酸突变体被证明是一个非常有效的拮抗剂。每英里200皮克的天冬氨酸突变体对400pg/ml野生型PRL的生长反应否定了75%。当丝氨酸179突变为丙氨酸或缬氨酸时，分别产生了生物活性为野生型PRL的0%和14%的突变PRL。这些结果表明，1)对磷酸化激素的分子模拟确实产生了PRL拮抗剂，2)179位的丝氨酸对PRL的促生长活性是至关重要的。天冬氨酸突变体现在可以用来研究催乳素生理学的许多方面。
Previous studies have demonstrated that naturally phosphorylated PRL antagonizes the growth-promoting effects of unmodified PRL in two different PRL-responsive cell lines. In this study our aim was to produce a molecular mimic of phosphorylated PRL by substituting a fairly bulky, negatively charged amino acid (glutamate or aspartate) for the normally phosphorylated serine [serine 179 in human PRL (hPRL)]. In addition, because of the marked effect of phosphorylation on biological activity, we investigated the importance of the unmodified serine in the growth-promoting activity of PRL. hPRL complementary DNA was obtained from the American Type Culture Collection and subcloned into pT7-SCII after site-directed. mutagenesis using the deoxyuridine approach. Proteins were expressed in Escherichia coli BL21 (DE3) and were primarily found in inclusion bodies. Agonist and antagonist activities of each serine 179 mutant were assessed using the Nb2 bioassay. Compared with standard hPRL, the recombinant wild-type was more active in the Nb2 assay, attesting to both the absence, or low level, of endotoxin contamination in preparations from these cells and the appropriate folding of the molecule. The aspartate and glutamate mutants had no intrinsic agonist activity, but both antagonized the growth-promoting activity of wild-type PRL, with the aspartate mutant proving to be a very effective antagonist. Two hundred picograms per mi of the aspartate mutant negated 75% of the growth response to 400 pg/ml wild-type PRL. When serine 179 was mutated to alanine or valine, mutant PRLs with 0% and 14% of the biological activity of wild-type PRL, respectively, were produced. These results demonstrate 1) that molecular mimicry of the phosphorylated hormone does produce a PRL antagonist, and 2) that the serine at position 179 is crucial to the growth-promoting activity of PRL. The aspartate mutant can now be used to study many aspects of the physiology of PRL.