Nair Anil K, Menon K M J
Departments of Biological Chemistry and Obstetrics/Gynecology, University of Michigan Medical Center, Ann Arbor, Michigan 48109-0617, USA.
J Biol Chem. 2004 Apr 9;279(15):14937-44. doi: 10.1074/jbc.M309484200. Epub 2004 Jan 28.
Post-transcriptional mechanisms play a major role in regulating luteinizing hormone (LH) receptor mRNA expression in the ovary. An ovarian cytosolic protein that we have identified in rats and humans, which binds to a polypyrimidine-rich bipartitate sequence in the coding region of LHR mRNA, acts as a trans-acting factor in this process. In the present study, we isolated and characterized this LH receptor mRNA-binding protein (LRBP) from rat ovary. LRBP was purified to homogeneity by cation exchange chromatography followed by Northwestern analysis and subsequent elution of the single protein band from SDS-polyacrylamide gel. Purified LRBP was subjected to N-terminal microsequencing followed by homology search, which revealed its identity as mevalonate kinase. Purified rat mevalonate kinase antibody recognized the gel-purified LRBP on Western blots performed with one- and two-dimensional SDS-polyacrylamide gels. When recombinant mevalonate kinase produced in human embryonic kidney cells (293 cells) was tested, it showed all of the characteristics of LRBP with respect to specificity of LHR mRNA binding sequence, as examined by gel mobility shift analysis. Inhibition of LHR mRNA binding activity of mevalonate kinase in the presence of ATP and mevalonate indicates that the RNA recognition site of mevalonate kinase might involve the ATP/mevalonate binding region of the protein. Treatment of 293 cells with mevastatin to deplete cellular mevalonate resulted in an increase in LHR mRNA binding activity of mevalonate kinase. Collectively, the data support the novel function of rat mevalonate kinase as a LHR mRNA-binding protein in the post-transcriptional regulation of LH receptor expression in the ovary.
转录后机制在调节卵巢中促黄体生成素（LH）受体mRNA表达方面发挥着重要作用。我们在大鼠和人类中鉴定出一种卵巢胞质蛋白，它与LHR mRNA编码区富含多聚嘧啶的双联体序列结合，在此过程中作为反式作用因子。在本研究中，我们从大鼠卵巢中分离并鉴定了这种LH受体mRNA结合蛋白（LRBP）。通过阳离子交换色谱法将LRBP纯化至同质，随后进行蛋白质印迹分析，并从SDS-聚丙烯酰胺凝胶中洗脱单一蛋白条带。对纯化的LRBP进行N端微量测序，随后进行同源性搜索，结果显示其为甲羟戊酸激酶。在用一维和二维SDS-聚丙烯酰胺凝胶进行的蛋白质印迹实验中，纯化的大鼠甲羟戊酸激酶抗体能够识别凝胶纯化的LRBP。当检测在人胚肾细胞（293细胞）中产生的重组甲羟戊酸激酶时，通过凝胶迁移率变动分析发现，它在LHR mRNA结合序列特异性方面表现出LRBP的所有特征。在ATP和甲羟戊酸存在的情况下，甲羟戊酸激酶的LHR mRNA结合活性受到抑制，这表明甲羟戊酸激酶的RNA识别位点可能涉及该蛋白的ATP/甲羟戊酸结合区域。用美伐他汀处理293细胞以耗尽细胞内甲羟戊酸，导致甲羟戊酸激酶的LHR mRNA结合活性增加。总体而言，这些数据支持大鼠甲羟戊酸激酶作为LHR mRNA结合蛋白在卵巢中LH受体表达的转录后调控中的新功能。
