Abstract
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.
Highlights
The interaction of luteinizing hormone (LH)1 with its cell surface receptors controls reproductive functions including steroidogenesis in the gonad [1]
We have shown that the decline in cell surface luteinizing hormone receptor (LHR) expression seen after human chorionic gonadotropin (hCG) administration is paralleled by a specific loss of all four LHR mRNA transcripts (6.7, 4.4, 2.6, and 1.8 kb) in the ovary [9]
LH Receptor mRNA Coding Region-binding Protein purification were purchased from Roche Applied Science. [␣-32P]UTP was obtained from PerkinElmer Life Sciences. mMessage mMachine Kit and MAXIscript Kit were the products of Ambion (Austin, TX)
Summary
The interaction of luteinizing hormone (LH) with its cell surface receptors controls reproductive functions including steroidogenesis in the gonad [1]. We have shown that the decline in cell surface LHR expression seen after hCG administration is paralleled by a specific loss of all four LHR mRNA transcripts (6.7, 4.4, 2.6, and 1.8 kb) in the ovary [9]. MRNA halflives are influenced by the interaction of various cytoplasmic proteins (trans-acting factors) with regulatory regions (cis-acting elements) in the mRNA, forming ribonucleoprotein (RNP) complexes [10]. C-Fos, c-Myc, tropoelastin, thymidylate synthase, and dihydrofolate reductase are some of the mRNAs containing regulatory elements in the coding region for trans-acting factors [17,18,19,20,21,22,23,24]. We have purified the LHR mRNA-binding protein from rat ovaries, established its identity as mevalonate kinase by N-terminal microsequencing, and subsequently cloned and characterized the expressed protein in 293 cells. Depletion of cellular levels of mevalonate resulted in increased binding of LRBP to LH receptor mRNA
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