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Characterization of Estrogen Receptor-ß (ERß) Messenger Ribonucleic Acid and Protein Expression in Rat Granulosa Cells¹

Graduate Center for Toxicology (M.L.O., O.-K.P.-S.) and Department of Physiology (K.P., Y.I., O.-K.P.-S.), University of Kentucky, Lexington, Kentucky 40536

Address all correspondence and requests for reprints to: Dr. Ok-Kyong Park-Sarge, Department of Physiology, University of Kentucky, Lexington, Kentucky 40536-0084. E-mail: OKPS{at}pop.uky.edu

We have examined estrogen-responsiveness of ovarian granulosa cells by focusing on estrogen receptor (ER) expression. Estrogen responsiveness was determined by examining the effect of 17ß-estradiol (1–10 nM) on luciferase reporter activity in rat granulosa cells transfected with an ERE-luciferase construct. The results demonstrate an estrogen-induced (approximately 3-fold) increase in luciferase reporter activity, indicating that granulosa cells contain functional estrogen response element (ERE)-binding transcriptional activators. Gel mobility shift assays in combination with ER antibodies show that ERß is the predominant ERE-binding protein in granulosa cells. Western blotting results show that granulosa cells contain ERß-immunoreactive protein(s) migrating at a size substantially larger than the recombinant protein generated from the originally proposed 485 amino acid open-reading frame. This size discrepancy is not due to granulosa cell expression of ERß isoforms with insertions within the coding region because RT-PCR assays revealed products with sizes expected for ERß, ERßB, and 3 isoforms. This size discrepancy appears to be due to usage of a well-conserved, upstream in-frame translation initiation codon (ATG436) leading to a 530 amino acid open reading frame. ERß messenger RNA (mRNA) characterization using 5'-rapid amplification of complementary DNA ends (5'-RACE) show the presence of two different (P1- and P2-) 5'-ends of rat ERß mRNA encoding the full-length ERß protein. The generation of the P2-specific exon is likely due to initiation of transcription from an alternative promoter. Both P1- and P2-specific exon-containing ERß mRNAs are expressed in granulosa cells, and they are rapidly down-regulated by the cAMP-mediated intracellular signaling pathway in cultured granulosa cells. Taken together, our results show that rat granulosa cells produce two different 3',5'-cAMP-regulated ERß mRNA species and that these mRNA species are capable of encoding the full-length ERß protein.

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