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(224) NEURONAL PROTEIN MICROTUBULE-ASSOCIATED PROTEIN 2D IS A FUNCTIONAL A-KINASE ANCHORING PROTEIN IN OVARIAN GRANULOSA AND LUTEAL CELLS. Salvador, Lisa1, Carr, Daniel2, Hunzicker-Dunn, Mary1, 1 Northwestern University Medical School, Chicago, IL2 Veterans Affairs Medical Center, Portland, OR ABSTRACT- A kinase anchoring proteins (AKAPs) function to localize cAMP-dependent protein kinase (PKA) to specific locations within the cell. They are functionally identified by their ability to bind the type II regulatory subunits (RII) of PKA in an overlay assay. We showed that an AKAP at 80 kDa is induced by follicle stimulating hormone (FSH) as rat ovarian granulosa cells mature from a preantral (PA) to a preovulatory (PO) phenotype. We recently identified AKAP 80 as the microtubule-associated protein, MAP 2D, based on co-purification and coincident induction at 80 kDa of both RII binding activity in the RII overlay assay as well as MAP 2D immunoreactivity. In the following studies we expand on earlier experiments to explore the hypothesis that MAP 2D plays an important role in the signal transduction pathways that function in granulosa cell differentiation. Treatment of PA granulosa cells with antisense oligonucleotides to MAP 2D prior to FSH treatment caused a profound decrease in the appearance of MAP 2D protein as well as inhibited the acute stimulation of CREB phosphorylation by human chorionic gonadotropin (hCG). This result indicates that MAP 2D may play a role in acute responses to LH receptor activation. To further characterize the expression of MAP 2D we examined the protein and mRNA expression of MAP 2D in both granulosa cells obtained from PA follicles as well as in the intact animal. MAP 2D expression was studied in both granulosa cells treated with FSH for varying time periods and then with an ovulatory concentration of hCG at 72 h post FSH, MAP 2D expression was also studied in immature rats treated sequentially with pregnant mare serum gonadotropin (PMSG), an FSH analog, for 48 h then with hCG to induce ovulation and corpus luteum formation. In granulosa cells, both mRNA and protein expression of MAP 2D was induced by FSH by 48 h and remained high after acute treatment with hCG. In the intact rat, we found that the expression of MAP 2D protein was induced in response to PMSG by 48 h and that the expression increased after hCG treatment with formation of the corpus luteum. MAP 2D expression begins to decrease by 6 days post-hCG injection, a time when the corpus luteum begins to regress. These results suggest a possible role for MAP 2D in not only preovulatory granulosa cells but also in the corpus luteum. Supported by HD21921. KEY WORDS: A-Kinase Anchoring Protein, Signal Transduction, Follicle-Stimulating Hormone, Luteinizing Hormone |
