Isolation and Characterization of a Novel trans-Factor for Luteinizing Hormone Receptor mRNA from Ovary

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 SDSpolyacrylamide gels. When recombinant mevalonate kinase produced in human embryonic kidney cells ( The interaction of luteinizing hormone (LH) 1 with its cell surface receptors controls reproductive functions including steroidogenesis in the gonad (1). In the ovary, luteinizing hormone receptor (LHR), a member of G s -protein coupled receptors, regulates ovarian function mainly through increased production of cAMP (2-4). The cell surface expression of LHR varies during different stages of the ovarian cycle. Its expression in granulosa cells and luteal cells is greatly decreased by an endogenous preovulatory LH surge or by the administration of a pharmacological dose of human chorionic gonadotropin (hCG), a placental counterpart of LH (5-8). 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). Furthermore, the loss of LHR mRNA does not result from decreased transcription but occurs post-transcriptionally with a 3-fold decrease in half-life (8).Regulation of mRNA turnover is one of the major control mechanisms of gene expression in all organisms. 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). The formation and disruption of RNP complexes in response to various cellular stimuli mainly controls the turnover of cytoplasmic mRNA. Studies have indicated the presence of cis-acting regulatory elements in the 5Ј-untranslated region, coding region, and 3Ј-untranslated region of mRNA (10). A number of cytoplasmic trans-acting factors, some of which shuttle between the nucleus and cytoplasm, have been identified as mRNA-stabilizing, destabilizing, or translational repressor proteins (11-16). 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 (17-24).We have identified a LHR mRNA-binding protein in rat and hu...

Regulation of Luteinizing Hormone/Chorionic Gonadotropin Receptor Messenger Ribonucleic Acid Expression in the Rat Ovary: Relationship to Cholesterol Metabolism

“…Amino-terminal analysis and MS-MALDI of the gel purified LHR mRNA-binding protein (LRBP) established its identity as mevalonate kinase (Mvk) (9). The binding characteristics of the recombinant Mvk for LHR mRNA recognition are similar to those seen for the endogenous LRBP isolated from rat ovary (4,7,9).…”

“…The identity of LRBP was established as Mvk, an enzyme involved in cholesterol biosynthesis (9). Furthermore, both ATP and mevalonate competed for the binding of Mvk to LHR mRNA, suggesting that the catalytic site of Mvk might participate in the binding of LHR mRNA either directly or indirectly (9).…”

“…Previous studies from our laboratory demonstrated that the loss of LHR mRNA during hCG-induced down-regulation was due to accelerated mRNA decay, and a transacting protein was involved in this process (4,(7)(8)(9). As indicated in the introduction, the protein has been identified as Mvk, which binds to a cytidine-rich region of LHR mRNA, and the binding was shown to be regulated by hCG (4,8).…”

“…A specific RNA-binding protein was identified in the rat ovary, which was found to bind to a cytidine-rich region of LHR mRNA (4,7) and accelerate its degradation in vitro (8). Amino-terminal analysis and MS-MALDI of the gel purified LHR mRNA-binding protein (LRBP) established its identity as mevalonate kinase (Mvk) (9). The binding characteristics of the recombinant Mvk for LHR mRNA recognition are similar to those seen for the endogenous LRBP isolated from rat ovary (4,7,9).…”

“…1A). The Nterminal amino acids of purified LRBP had been determined previously (MLSEVLLVSA) (9). Based on this sequence, a GSP1 was designed.…”