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• W2026771277 abstract "A-kinase anchoring proteins (AKAPs) function to target protein kinase A (PKA) to specific locations within the cell. AKAPs are functionally identified by their ability to bind the type II regulatory subunits (RII) of PKA in an in vitro overlay assay. We previously showed that follicle-stimulating hormone (FSH) induces the expression of an 80-kDa AKAP (AKAP 80) in ovarian granulosa cells as they mature from a preantral to a preovulatory phenotype. In this report, we identify AKAP 80 as microtubule-associated protein 2D (MAP2D), a low molecular weight splice variant of the neuronal MAP2 protein. MAP2D is induced in granulosa cells by dexamethasone and by FSH in a time-dependent manner that mimics that of AKAP 80, and immunoprecipitation of MAP2D depletes extracts of AKAP 80. MAP2D is the only MAP2 protein present in ovaries and is localized to granulosa cells of preovulatory follicles and to luteal cells. MAP2D is concentrated at the Golgi apparatus along with RI and RII and, based on coimmunoprecipitation results, appears to bind both RI and RII in granulosa cells. Reduced expression of MAP2D resulting from treatment of granulosa cells with antisense oligonucleotides to MAP2 inhibited the phosphorylation of cAMP-response element-binding protein. These results suggest that this classic neuronal RII AKAP is a dual RI/RII AKAP that performs unique functions in ovarian granulosa cells that contribute to the preovulatory phenotype. A-kinase anchoring proteins (AKAPs) function to target protein kinase A (PKA) to specific locations within the cell. AKAPs are functionally identified by their ability to bind the type II regulatory subunits (RII) of PKA in an in vitro overlay assay. We previously showed that follicle-stimulating hormone (FSH) induces the expression of an 80-kDa AKAP (AKAP 80) in ovarian granulosa cells as they mature from a preantral to a preovulatory phenotype. In this report, we identify AKAP 80 as microtubule-associated protein 2D (MAP2D), a low molecular weight splice variant of the neuronal MAP2 protein. MAP2D is induced in granulosa cells by dexamethasone and by FSH in a time-dependent manner that mimics that of AKAP 80, and immunoprecipitation of MAP2D depletes extracts of AKAP 80. MAP2D is the only MAP2 protein present in ovaries and is localized to granulosa cells of preovulatory follicles and to luteal cells. MAP2D is concentrated at the Golgi apparatus along with RI and RII and, based on coimmunoprecipitation results, appears to bind both RI and RII in granulosa cells. Reduced expression of MAP2D resulting from treatment of granulosa cells with antisense oligonucleotides to MAP2 inhibited the phosphorylation of cAMP-response element-binding protein. These results suggest that this classic neuronal RII AKAP is a dual RI/RII AKAP that performs unique functions in ovarian granulosa cells that contribute to the preovulatory phenotype. Ovarian follicles house the oocyte and, upon maturation, produce steroid and protein hormones that regulate uterine receptivity and the reproductive axis. Follicles exist in a relatively dormant, preantral (PA) 1The abbreviations used are: PA, preantral; AKAP, A-kinase anchoring protein; C subunit, catalytic subunit of PKA; CREB, cAMP-response element-binding protein; DEX, dexamethasone; DSP, dithiobis[succinimidylpropionate]; ERK, extracellular signal-regulated kinase; FSH, follicle-stimulating hormone; hCG, human chorionic gonadotropin; LH, luteinizing hormone; MAP2, microtubule-associated protein 2; NI, nonimmune; PKA, protein kinase A; PO, preovulatory; PMSG, pregnant mares serum gonadotropin; R subunit, PKA regulatory subunit; HA, hemagglutinin; DTT, dithiothreitol; PBS, phosphate-buffered saline. state until they are recruited to grow and differentiate to a preovulatory (PO) phenotype by the pituitary hormone follicle-stimulating hormone (FSH) (1McGee E.A. Hsueh A.J. Endocr. Rev. 2000; 21: 200-214Crossref PubMed Scopus (1220) Google Scholar, 2Richards J.S. Physiol. Rev. 1980; 60: 51-89Crossref PubMed Scopus (697) Google Scholar). Maturation of follicles to a PO phenotype involves not only proliferation but also differentiation of the enclosed granulosa cells. FSH triggers these events by binding to its G-protein-coupled receptor, located exclusively on granulosa cells in female mammals, and activating adenylyl cyclase, which converts ATP to cAMP. cAMP then acts as a second messenger primarily by activating protein kinase A (PKA) (3Robinson-White A. Stratakis C.A. Ann. N. Y. Acad. Sci. 2002; 968: 256-270Crossref PubMed Scopus (59) Google Scholar). PKA is a tetrameric enzyme that consists of a dimeric regulatory (R) subunit and two catalytic subunits (4Taylor S.S. J. Biol. Chem. 1989; 264: 8443-8446Abstract Full Text PDF PubMed Google Scholar). Upon binding of cAMP to the R subunits, a conformational change occurs that allows for dissociation of the active catalytic subunits, which can then phosphorylate neighboring substrates. Two classes of PKA holoenzymes, PKA I and PKA II, exist based on the association of two possible RI subunits (RIα and RIβ) or two possible RII subunits (RIIα and RIIβ) with four possible catalytic subunits (Cα, Cβ1, Cβ2, and Cγ) (5Dell'Acqua M.L. Scott J.D. J. Biol. Chem. 1997; 272: 12881-12884Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). In rat granulosa cells of PA and PO follicles, PKA IIα and PKA IIβ are the predominant PKA isoforms present, whereas less than 5% of PKA holoenzyme activity is contributed by PKA Iα (6Hunzicker-Dunn M. Lorenzini N.A. Lynch L.L. West D.E. J. Biol. Chem. 1985; 260: 13360-13369Abstract Full Text PDF PubMed Google Scholar, 7Hunzicker-Dunn M. Maizels E.T. Kern L.C. Ekstrom R.C. Constantinou A.I. Mol. Endocrinol. 1989; 3: 780-789Crossref PubMed Scopus (6) Google Scholar, 8Carr D.W. Cutler Jr., R.E. Cottom J.E. Salvador L.M. Fraser I.D.C. Scott J.D. Hunzicker-Dunn M. Biochem. J. 1999; 344: 613-623Crossref PubMed Scopus (33) Google Scholar). The specificity of PKA action is accomplished by the targeting of PKA to specific cellular locales by virtue of its binding to a growing family of A-kinase anchoring proteins (AKAPs). Most known AKAPs anchor RII and exhibit at least a 100-fold lower affinity for RI (9Michel J.J. Scott J.D. Annu. Rev. Pharmacol. Toxicol. 2002; 42: 235-257Crossref PubMed Scopus (288) Google Scholar). RII subunits of PKA bind with nanomolar affinity to AKAPs (5Dell'Acqua M.L. Scott J.D. J. Biol. Chem. 1997; 272: 12881-12884Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 10Carr D.W. Hausken Z.E. Fraser I.D.C. Stofko-Hahn R.E. Scott J.D. J. Biol. Chem. 1992; 267: 13376-13382Abstract Full Text PDF PubMed Google Scholar). The domain on the AKAP responsible for RII binding comprises an amphipathic helix that binds to the N termini of the RII dimer (11Carr D.W. Stofko-Hahn R.E. Fraser I.D.C. Bishop S.M. Acott T.S. Brennan R.G. Scott J.D. J. Biol. Chem. 1991; 266: 14188-14192Abstract Full Text PDF PubMed Google Scholar). A growing number of “dual” AKAPs have been identified, although they still exhibit higher affinity for RII over RI (12Reinton N. Collas P. Haugen T.B. Skalhegg B.S. Hansson V. Jahnsen T. Tasken K. Dev. Biol. 2000; 223: 194-204Crossref PubMed Scopus (92) Google Scholar, 13Li H. Adamik R. Pacheco-Rodriguez G. Moss J. Vaughan M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1627-1632Crossref PubMed Scopus (71) Google Scholar, 14Huang L.J. Wang L. Ma Y. Durick K. Perkins G. Deerinck T.J. Ellisman M.H. Taylor S.S. J. Cell Biol. 1999; 145: 951-959Crossref PubMed Scopus (143) Google Scholar, 15Huang L.J. Durick K. Weiner J.A. Chun J. Taylor S.S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11184-11189Crossref PubMed Scopus (202) Google Scholar). Recent reports, however, indicate that some AKAPs can preferentially bind RI (16Gronholm M. Vossebein L. Carlson C.R. Kuja-Panula J. Teesalu T. Alfthan K. Vaheri A. Rauvala H. Herberg F.W. Tasken K. Carpen O. J. Biol. Chem. 2003; 278: 41167-41172Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 17Angelo R. Rubin C.S. J. Biol. Chem. 1998; 273: 14633-14643Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 18Miki K. Eddy E.M. J. Biol. Chem. 1998; 273: 34384-34390Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 19Li H. Degenhardt B. Tobin D. Yao Z.X. Tasken K. Papadopoulos V. Mol. Endocrinol. 2001; 15: 2211-2228Crossref PubMed Scopus (140) Google Scholar). AKAPs anchor PKA to specific cellular locations, such as the actin cytoskeleton (20Li Y. Ndubuka C. Rubin C.S. J. Biol. Chem. 1996; 271: 16862-16869Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 21Dong F. Feldmesser M. Casadevall A. Rubin C.S. J. Biol. Chem. 1998; 273: 6533-6541Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), plasma membrane (22Dodge K. Scott J.D. FEBS Lett. 2000; 476: 58-61Crossref PubMed Scopus (117) Google Scholar), mitochondria (23Lin R.-Y. Moss S.R. Rubin C.S. J. Biol. Chem. 1995; 270: 27804-27811Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 24Ginsberg M.D. Feliciello A. Jones J.K. Avvedimento E.V. Gottesman M.E. J. Mol. Biol. 2003; 327: 885-897Crossref PubMed Scopus (85) Google Scholar), Golgi apparatus (25Keryer G. Rios R.M. Landmark B.F. Skalhegg B. Lohmann S.M. Bornens M. Exp. Cell Res. 1993; 204: 230-240Crossref PubMed Scopus (57) Google Scholar), centrosome (26Diviani D. Langeberg L.K. Doxsey S.J. Scott J.D. Curr. Biol. 2000; 10: 417-420Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar), and nuclear envelope (27Steen R.L. Beullens M. Landsverk H.B. Bollen M. Collas P. J. Cell Sci. 2003; 116: 2237-2246Crossref PubMed Scopus (54) Google Scholar). The localization of PKA to distinct regions within the cell is generally thought to allow for both specific and efficient substrate phosphorylation in response to a specific stimulus (28Pawson T. Scott J.D. Science. 1997; 278: 2075-2080Crossref PubMed Scopus (1900) Google Scholar). FSH receptor signaling in PA granulosa cells stimulates the PKA-dependent phosphorylation of a number of signaling intermediates including histone H3 (29DeManno D.A. Cottom J.E. Kline M.P. Peters C.A. Maizels E.T. Hunzicker-Dunn M. Mol. Endocr. 1999; 13: 91-105Crossref PubMed Scopus (83) Google Scholar), cAMP-response element-binding protein (CREB) (30Mukherjee A. Park-Sarge O.K. Mayo K.E. Endocrinology. 1996; 137: 3234-3245Crossref PubMed Scopus (108) Google Scholar, 31Pei L. Dodson R. Schoderbek W.E. Maurer R.A. Mayo K.E. Mol. Endocr. 1991; 5: 521-534Crossref PubMed Scopus (117) Google Scholar), and an extracellular regulated kinase (ERK)-protein-tyrosine phosphatase that leads to ERK activation (32Cottom J. Salvador L.M. Maizels E.T. Reierstad S. Park Y. Carr D.W. Davare M.A. Hell J.W. Palmer S.S. Dent P. Kawakatsu H. Ogata M. Hunzicker-Dunn M. J. Biol. Chem. 2003; 278: 7167-7179Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). In addition, FSH receptor activation induces the transcription of a number of genes, including those for the luteinizing hormone (LH) receptor and inhibin-α as well as the P450 aromatase and side chain cleavage steroidogenic enzymes (33Richards J.S. Endocr. Rev. 1994; 15: 725-751Crossref PubMed Scopus (760) Google Scholar, 34Hsueh A.J.W. Adashi E.Y. Jones P.B.C. Welsh Jr., T.H. Endocr. Rev. 1984; 5: 76-110Crossref PubMed Scopus (875) Google Scholar). On the other hand, in granulosa cells of the PO follicle, LH receptor signaling causes an up-regulation in genes that encode for progesterone receptor and cyclooxgenase-2 while at the same time causing a down-regulation in genes that encode for the LH and FSH receptors, inhibin-α, and aromatase proteins (33Richards J.S. Endocr. Rev. 1994; 15: 725-751Crossref PubMed Scopus (760) Google Scholar, 35Richards J.S. Fitzpatrick S.L. Clemens J.W. Morris J.K. Alliston T. Sirois J. Rec. Prog. Horm. Res. 1995; 50: 223-254PubMed Google Scholar). Like FSH receptor signaling, LH receptor signaling also stimulates the PKA-dependent phosphorylation of key substrates such as histone H3, CREB, and an unidentified substrate upstream of ERK that leads to the activation of ERK (36Salvador L.M. Maizels E. Hales D.B. Miyamoto E. Yamamoto H. Hunzicker-Dunn M. Endocrinol. 2002; 143: 2986-2994Crossref PubMed Scopus (0) Google Scholar). The fact that PKA plays a predominant role in the pleotrophic signaling events regulated by these hormones in PA versus PO granulosa cells led us to hypothesize that these cells may express different complements of AKAPs to localize PKA to distinct subcellular environments. In this report, we demonstrate that an 80-kDa AKAP that we previously showed is induced in ovarian granulosa cells by FSH (8Carr D.W. Cutler Jr., R.E. Cottom J.E. Salvador L.M. Fraser I.D.C. Scott J.D. Hunzicker-Dunn M. Biochem. J. 1999; 344: 613-623Crossref PubMed Scopus (33) Google Scholar, 37Carr D.W. DeManno D.A. Atwood A. Hunzicker-Dunn M. Scott J.D. J. Biol. Chem. 1993; 268: 20729-20732Abstract Full Text PDF PubMed Google Scholar) is microtubule-associated protein 2D (MAP2D). MAP2 is a microtubule- and microfilament-binding protein localized primarily to dendrites and to nonneuronal glial cells (38Matus A. J. Cell Sci. (Suppl.). 1991; 15: 61-67Crossref PubMed Google Scholar, 39Doll T. Meichsner M. Riederer B.M. Honegger P. Matus A. J. Cell Sci. 1993; 106: 633-639Crossref PubMed Google Scholar). Most of the reports on MAP2 focus on its role in neurite outgrowth and dendrite development in the brain (40Lim R.W. Halpain S. J. Biol. Chem. 2000; 275: 20578-20587Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). MAP2 functions in the brain to stabilize microtubules, to stimulate microtubule assembly, and to regulate cell shape (41Sharma N. Kress Y. Shafit-Zagardo B. Cell Motil. Cytoskeleton. 1994; 27: 234-247Crossref PubMed Scopus (56) Google Scholar, 42Ferhat L. Represa A. Bernard A. Ben-Ari Y. Khrestchatisky M. J. Cell Sci. 1996; 109: 1095-1103PubMed Google Scholar). The MAP2 gene encodes high and low molecular weight protein isoforms that are generated by alternative splicing (43Sanchez C. Diaz-Nido J. Avila J. Prog. Neurobiol. 2000; 61: 133-168Crossref PubMed Scopus (409) Google Scholar). MAP2A and MAP2B are high molecular mass isoforms (270 and 280 kDa, respectively), whereas MAP2C and MAP2D are low molecular mass isoforms (70 and 80 kDa, respectively) (44Ferhat L. Represa A. Ferhat W. Ben-Ari Y. Khrestchatisky M. Eur. J. Neurosci. 1998; 10: 161-171Crossref PubMed Scopus (16) Google Scholar). The low molecular weight isoforms contain N- and C-terminal regions of the high molecular weight isoforms but lack the large central domain found in high molecular weight MAP2 isoforms. Each isoform contains at least three imperfect microtubule-binding domains in their C termini. MAP2D contains an additional 93-base pair insert, which comprises the fourth microtubule-binding domain that is absent from MAP2C (45Ferhat L. Bernard A. Ribas d.P. Ben-Ari Y. Khrestchatisky M. Neurochem. Int. 1994; 25: 327-338Crossref PubMed Scopus (29) Google Scholar, 46Ferhat L. Ben-Ari Y. Khrestchatisky M. Comptes Rendus l'Academie Sciences Ser. 3 Sci. Vie. 1994; 317: 304-309PubMed Google Scholar). MAP2 was the first protein to be recognized as an AKAP (47Theurkauf W.E. Vallee R.B. J. Biol. Chem. 1982; 257: 3284-3290Abstract Full Text PDF PubMed Google Scholar), and the RII-binding region was localized to amino acids 83-113 in the N-terminal region of the protein and is conserved among all MAP2 isoforms (48Rubino H.M. Dammerman M. Shafit-Zagardo B. Erlichman J. Neuron. 1989; 3: 631-638Abstract Full Text PDF PubMed Scopus (100) Google Scholar). Our results show that the neuronal protein MAP2D is induced by FSH and localized to the granulosa cells of the PO follicle and that its expression is maintained in luteal cells following ovulation and corpus luteum formation. MAP2D appears to anchor both RI and RII to the Golgi apparatus in granulosa cells of PO follicles, and, based on antisense oligonucleotide experiments, MAP2D appears to participate in acute LH receptor signaling events. The function of MAP2D in ovarian granulosa cells is thus expected to be quite distinct from its established neuronal roles. Materials—The following were purchased: ovine FSH (oFSH-20) from Dr. A. F. Parlow of the NIDDK, National Institutes of Health, National Hormone and Pituitary Program (Harbor-UCLA Medical Center, Torrance, CA); Profasi® hCG from Serono Laboratories Inc. (Randolph, MA); [γ-32P]ATP, ammonium salt (3000 Ci/mmol), and [32P]orthophosphate (∼9000 Ci/mmol) from PerkinElmer Life Sciences; [2,8-3H]cAMP sodium salt (15-40 Ci/mmol) from ICN Chemical and Radioisotope Division (Costa Mesa, CA); DEAE-cellulose (DE-52) and P-81 cellulose phosphate paper from Whatman (Clifton, NJ); ECL reagents, rainbow molecular weight markers, and Hybond-C nitrocellulose membranes from Amersham Biosciences; SDS-PAGE reagents from Bio-Rad; X-Omat AR film from Eastman Kodak Co.; all culture media from Invitrogen; brefeldin A from LC Laboratories (San Diego, CA); MAP2D scrambled and antisense oligonucleotides from Integrated DNA Technologies (Coralville, IA); MAP2D and glyceraldehyde-3-phosphate dehydrogenase PCR primers from Northwestern University Biotechnology Laboratory (Chicago, IL); DNase and reverse transcriptase-PCR reagents and buffers from Promega (Madison, WI); actin phalloidin from Molecular Probes, Inc. (Eugene, OR); and protein A+G-agarose from Santa Cruz Biotechnologies, Inc. (Santa Cruz, CA). All other biochemical reagents were purchased from Sigma, unless otherwise indicated. Antibodies—Inhibin-α antibody was kindly provided by Dr. Wiley Vale of the Salk Institute for Biological Studies (San Diego, CA). The following were purchased: anti-MAP2 mouse monoclonal and anti-γ-tubulin from Sigma; anti-cyclin D2 polyclonal from Santa Cruz Biotechnologies; anti-PKA RI mouse monoclonal and anti-PKA RIIβ mouse monoclonal from BD Biosciences; anti-PKA RII (RIIα and -β) goat polyclonal and anti-phospho-CREB (S133) from Upstate Biotechnology, Inc. (Lake Placid, NY); goat anti-mouse rhodamine, goat anti-rabbit fluorescein, goat anti-rabbit rhodamine, donkey anti-goat rhodamine, donkey anti-mouse fluorescein, and goat anti-mouse fluorescein secondary antibodies from Jackson Immunochemicals (West Grove, PA); anti-Gm 130-fluorescein conjugate from BD Biosciences (Palo Alto, CA), and anti-hemagglutinin (HA) protein of human influenza virus from Roche Applied Science. Primary Granulosa Cell Cultures—Sprague-Dawley rats were obtained at 15-18 days of age (Sasco, Baltimore, MD) and maintained in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Rats at 21-24 days of age were injected subcutaneously with either 1.5 mg/ml estrogen for 3 days to obtain PA ovaries or 10 IU of pregnant mares serum gonadotropin (PMSG) to obtain PO ovaries (harvested 48-50 h postinjection). Ovaries were trimmed to remove the bursa, fat, and oviducts and incubated for 15-30 min at 37 °C in 6 mm EGTA in Dulbecco's modified Eagle's medium/Ham's F-12 medium. Ovaries were then incubated for 5-20 min in 0.5 m sucrose in Dulbecco's modified Eagle's medium/Ham's F-12 medium. Granulosa cells from PA or PO ovaries were expressed by penetration of PA or large PO follicles, respectively, with a 30-gauge needle. The cells were plated at a density of 5-7 × 106 in serum-free Dulbecco's modified Eagle's medium/Ham's F-12 medium on plates coated with 0.5 μg/ml fibronectin (Invitrogen) and cultured as previously described (29DeManno D.A. Cottom J.E. Kline M.P. Peters C.A. Maizels E.T. Hunzicker-Dunn M. Mol. Endocr. 1999; 13: 91-105Crossref PubMed Scopus (83) Google Scholar, 37Carr D.W. DeManno D.A. Atwood A. Hunzicker-Dunn M. Scott J.D. J. Biol. Chem. 1993; 268: 20729-20732Abstract Full Text PDF PubMed Google Scholar). Cells were treated the next day, as indicated under “Results.” Detergent-soluble Ovarian Extracts—21-24-Day-old Sprague-Dawley rats (Sasco) were not treated or were injected subcutaneously with 25 IU PMSG 48 h prior to injection with 25 IU hCG, as specified under “Results.” Ovaries were harvested at various time points after injections; cooled to 4 °C in an iced 10 mm potassium phosphate buffer, pH 7.0; dissected free of bursa, fat, and oviducts; weighed; and homogenized (5:1 ratio of homogenization buffer/wet weight) in buffer A (10 mm potassium phosphate, pH 7.0, 1 mm EDTA, 5 mm EGTA, 10 mm MgCl2, 50 mm β-glycerol phosphate, 1 mm sodium orthovanadate, 2 mm dithiothreitol (DTT), 40 μg/ml phenylmethylsulfonyl fluoride, 10 μg/ml E64, 0.5% Nonidet P-40, 0.1% deoxycholate) using 15-20 strokes with a ground glass homogenizer. A supernatant fraction was obtained by centrifugation at 10,000 × g for 10 min at 4 °C. Protein concentrations were determined by the Lowry protein assay (49Lowry O.W. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) using bovine serum albumin as the standard. Electrophoresis and Western Blotting—Total cell extracts were made by harvesting granulosa cells into 300 μl of SDS-PAGE sample buffer (50Hunzicker-Dunn M. J. Biol. Chem. 1981; 256: 12185-12193Abstract Full Text PDF PubMed Google Scholar) and boiled. Protein concentrations were controlled by plating equal numbers of cells in each dish for each experiment followed by loading equal volumes onto an SDS-PAGE gel. These cells do not divide under serum-free conditions. The proteins in the cell extracts were separated by SDS-PAGE (51Rudolph S.A. Krueger B.K. Adv. Cyclic Nucleotide Res. 1979; 10: 107-133PubMed Google Scholar), electrotransferred to Hybond C-extra nitrocellulose overnight at 4 °C, and stained for protein loading using Ponceau S. The nitrocellulose blots were incubated with primary antibody at 4 °C overnight. Antigen-antibody complexes were detected using ECL. DEAE-cellulose Chromatography and Sucrose Density Gradient Centrifugation—DEAE-cellulose chromatography was conducted as previously described (52Hunzicker-Dunn M. Cutler Jr., R.E. Maizels E.T. DeManno D.A. Lamm M.L.G. Erlichman J. Sanwal B.D. LaBarbera A.R. J. Biol. Chem. 1991; 266: 7166-7175Abstract Full Text PDF PubMed Google Scholar), collecting fractions (∼0.75 ml) in tubes containing 50 μl of a concentrated mixture of protease inhibitors (buffer B) at the indicated final concentrations: 10 mm benzamidine, 2 mm DTT, 5 μg/ml pepstatin, 10 μg/ml leupeptin, 5 μg/ml aprotinin, 40 μg/ml phenylmethylsulfonyl fluoride, 5 μg/ml soybean trypsin inhibitor, 10 μg/ml E-64. Protein kinase activity in DEAE fractions was determined in the presence of 0.5 μm cAMP and 71.5 μm Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) as substrate (8Carr D.W. Cutler Jr., R.E. Cottom J.E. Salvador L.M. Fraser I.D.C. Scott J.D. Hunzicker-Dunn M. Biochem. J. 1999; 344: 613-623Crossref PubMed Scopus (33) Google Scholar). Total [3H]cAMP binding sites on R subunits were determined by incubating sample with 0.3 μm [3H]cAMP for 30 min at 30 °C (52Hunzicker-Dunn M. Cutler Jr., R.E. Maizels E.T. DeManno D.A. Lamm M.L.G. Erlichman J. Sanwal B.D. LaBarbera A.R. J. Biol. Chem. 1991; 266: 7166-7175Abstract Full Text PDF PubMed Google Scholar). When indicated, DEAE-cellulose fractions (collected in the absence of DTT) were concentrated and subjected to sucrose density gradient centrifugation, as previously described (52Hunzicker-Dunn M. Cutler Jr., R.E. Maizels E.T. DeManno D.A. Lamm M.L.G. Erlichman J. Sanwal B.D. LaBarbera A.R. J. Biol. Chem. 1991; 266: 7166-7175Abstract Full Text PDF PubMed Google Scholar), in the presence of protease and phosphatase inhibitors. Following centrifugation, fractions were mixed with SDS-sample buffer, boiled, and subjected to SDS-PAGE and Western blotting. RII Overlay Assay and cAMP-agarose Affinity Chromatography—Recombinant murine RIIα was expressed in Escherichia coli and purified by affinity chromatography on cAMP-Sepharose (53Scott J.D. Stofko R.E. McDonald J.R. Comer J.D. Vitalis E.A. Mangili J.A. J. Biol. Chem. 1990; 265: 21561-21566Abstract Full Text PDF PubMed Google Scholar). For solid phase RII overlay assays, proteins were separated by SDS-PAGE and electrotransferred to Immobilon-P polyvinylidene difluoride (Millipore Corp.), and blots were probed with 0.5 μg of recombinant RIIα phosphorylated using [γ-32P]ATP with the catalytic subunit of PKA and then subjected to autoradiography (54Coghlan V.M. Langeberg L.K. Fernandex A. Lamb N.J.C. Scott J.D. J. Biol. Chem. 1994; 269: 7658-7665Abstract Full Text PDF PubMed Google Scholar). For cAMP-agarose affinity chromatography, pooled and concentrated DEAE-cellulose fractions were added to cAMP-agarose (0.2-ml packed volume; Sigma) equilibrated in buffer C (10 mm Hepes, pH 7.0, 1 mm EDTA, 5 mm EGTA, 10 mm MgCl2, 2 mm DTT, 10 μg/ml pepstatin, 10 μg/ml leupeptin, 5 μg/ml aprotinin, 10 μg/ml soybean trypsin inhibitor, 40 μg/ml phenylmethylsulfonyl fluoride, 1 mm sodium vanadate, 10 μm isobutylmethylxanthine, 20 mm benzamidine, and 10 μg/ml E-64) and incubated overnight at 4 °C on a Nutator (8Carr D.W. Cutler Jr., R.E. Cottom J.E. Salvador L.M. Fraser I.D.C. Scott J.D. Hunzicker-Dunn M. Biochem. J. 1999; 344: 613-623Crossref PubMed Scopus (33) Google Scholar). The flow-through fraction (∼1.0 ml) was collected by centrifugation at 5000 × g for 5 min, an aliquot (0.1 ml) was diluted to 0.3 ml, mixed with 0.15 ml of 3-fold SDS-PAGE sample buffer, and boiled. The cAMP-agarose pellet was then washed three times by sequential centrifugations with a total of 3 ml of buffer C, three times with buffer C containing 1 m NaCl, and three times with buffer C containing 0.3 m NaCl, collecting an aliquot (0.3 ml) of the final 1.0-ml wash. cAMP-agarose was then incubated at room temperature for 30 min with 0.3 ml of 5 μm Ht31 peptide (residues 493-515) in buffer C containing 0.3 m NaCl, collecting eluate from agarose using a syringe followed by boiling after the addition of 3-fold SDS-PAGE sample buffer (0.15 ml). Finally, cAMP-agarose was incubated at room temperature for 30 min with 0.3 ml of 75 mm cAMP in buffer C containing 0.3 m NaCl, collecting eluate as described above. RNA Isolation and Reverse Transcriptase-PCR—RNA was collected by lysing cultured granulosa cells in 1 ml of Trizol® reagent (Invitrogen) per 3 ml of medium according to the manufacturer's instructions. RNA (5 μg) was treated with DNase I (Promega) for 15 min at room temperature and heat-inactivated at 65 °C for 10 min. The reverse transcription reaction was performed using 2.5 μg of DNase-treated RNA and avian myeloblastosis virus reverse transcriptase (Promega) as described previously (55Park Y. Freedman B.F. Lee E.J. Jameson L.J. Diabetologia. 2003; 46: 365-377Crossref PubMed Scopus (260) Google Scholar). PCR was performed using 500 ng of DNA using primers for MAP2 (forward, 5′-CAC AAG GAT CAG CCT GCA GCT CTG-3′; reverse, 5′-CAC CTG GCC TGT GAC GGA TGT TCT-3′) that generate a 756-base pair PCR product (45Ferhat L. Bernard A. Ribas d.P. Ben-Ari Y. Khrestchatisky M. Neurochem. Int. 1994; 25: 327-338Crossref PubMed Scopus (29) Google Scholar) or primers for glyceraldehyde-3-phosphate dehydrogenase (forward, 5′-CCCTTCATTGACCTCAACTA-3′; reverse, 5′-CCAAAGTTGTCATGGATGAC-3′) that generate a 350-bp PCR product (56Yu R.N. Ito M. Jameson J.L. Mol. Endocrinol. 1998; 12: 1010-1022Crossref PubMed Scopus (113) Google Scholar). The PCR consisted of 24 cycles of 94 °C denaturation, 55 °C annealing, and 72 °C elongation steps. The reactions were analyzed on a 1.5% agarose gel containing ethidium bromide. Immunofluorescence and Immunohistochemistry—For immunofluorescence analysis, granulosa cells from PA follicles were cultured on fibronectin-coated glass coverslips and treated as indicated under “Results.” The cells were washed in phosphate-buffered saline (PBS), fixed for 10 min in 4% formaldehyde in PBS, and permeabilized with 0.1% Triton X-100. Coverslips were then washed three times with PBS and blocked for 1 h in 1% bovine serum albumin in PBS. Coverslips were incubated overnight at 4 °C in a humidified chamber with primary antibody at a 1:200 dilution (unless otherwise indicated). Coverslips were then washed three times with PBS, incubated for 2 h at 37 °C with secondary antibody conjugated to rhodamine or flourescein, as indicated, and washed three times with PBS. Coverslips were then allowed to dry and mounted onto glass slides using Vectashield® mounting medium (Vector Laboratories, Burlingame, CA). The slides were analyzed using a Zeiss LSM510 laser-scanning microscope. For immunohistochemistry, whole ovaries were harvested in 4% paraformaldehyde, paraffin-embedded, sectioned at 4 μm onto glass slides, and subjected to enzyme-induced epitope retrieval immunohistochemistry according to the protocol developed by Northwestern University Robert H. Lurie Cancer Center Pathology Core (Chicago IL). Immunoprecipitation—For immunoprecipitation of MAP2 from ovarian and brain extracts (see Fig. 3), extracts were prepared in buffer A and incubated with 10 μl of anti-MAP2 antibody and 30 μl of protein A+G-agarose for 4 h at 4 °C on a Nutator. The agarose was then washed with buffer D (20 mm Hepes, 150 mm NaCl, 10% glycerol, and 0.1% Triton-X). Proteins bound to the agarose were eluted by boiling in 50 μl of SDS-PAGE sample buffer. For PKA R subunit immunoprecipitations from ovarian extracts and DEAE fractions, samples (containing 500-800 μg of protein in ovarian extracts or 1 ml from pooled DEAE fractions collected in the absence of DTT) were incubated with 1 μm dithiobis[succinimidylpropionate] (DSP) (Pierce) in dimethyl sulfoxide for 15 min at room temperature. The cross-linking reaction was stopped by adding 1 m Tris-HCl, pH 7.5, to a final concentration of 25 mm and incubating samples at room temperature for 15 min. Antibody (10 μl of anti-MAP2 (Sigma), 25 μl of anti-RI (BD" @default.
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• W2026771277 date "2004-06-01" @default.
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• W2026771277 title "Neuronal Microtubule-associated Protein 2D Is a Dual A-kinase Anchoring Protein Expressed in Rat Ovarian Granulosa Cells" @default.
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• W2026771277 doi "https://doi.org/10.1074/jbc.m402980200" @default.
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