Phosphorylation of chicken oviduct progesterone receptor by cAMP-dependent protein kinase.

Phosphorylation of immunopurified chicken oviduct progesterone receptor (PR) was studied in intact cells and under cell-free conditions. Cytosol PR was isolated by incubation with anti-PR monoclonal antibody alpha PR22 adsorbed to protein A-Sepharose and suspended in a reaction mixture containing 10 mM Mg2+, 0.1 mM [gamma-32P]ATP, and the catalytic subunit of cAMP-dependent protein kinase (cAMP-PK) from bovine heart. All three major proteins of avian PR (PR-A, 79 kDa; PR-B, 110 kDa; 90 kDa) incorporated 32P-radioactivity on serine residues. The phosphorylation reaction was inhibited by synthetic inhibitors of protein kinases, H-8 and 20-residue peptide IP20. A 40 degrees C preexposure of PR oligomer increased phosphorylation of the 90-kDa protein, known to be a heat-shock protein (hsp-90). The extent of the phosphorylation reaction was temperature-dependent as the 32P-incorporation into PR-A and PR-B increased gradually, showing a maximum at 37 degrees C. Multiple phosphopeptides (4-7) were resolved by two-dimensional electrophoresis chromatography following cleavage of 32P-labeled peptides with trypsin. Both A and B forms of receptor showed similar phosphorylation patterns with B receptor digestion exhibiting two to three additional peptides. Under physiological conditions, preincubation of oviduct mince with forskolin, a regulator of intracellular cAMP levels, caused a greater extent of phosphorylation of PR-A and PR-B proteins. The results of this study demonstrate that chicken oviduct PR is an excellent substrate for the action of cAMP-PK in vitro and that this enzyme may be a physiological regulator of progesterone action in the oviduct.     

Transformation and phosphorylation of purified molybdate-stabilized chicken oviduct progesterone receptor

The non-transformed, molybdate-stabilized chick oviduct cytosol progesterone receptor was purified approx. 7000-fold using biospecific affinity resin (NADAC-Sepharose), DEAE-Sephacel chromatography and gel filtration on Bio-Gel A-0.5m agarose. The purified preparation contained progesterone receptor which sedimented as a 7.9S molecule, had a Stokes' radius of 7.5 nm, was composed of three major peptides corresponding to Mr 108,000, 90,000 and 79,000. Upon removal of molybdate, the purified [3H]progesterone-receptor complex could be transformed from the 8S form to a 4S form by exposure to 23 degrees C or by an incubation with 10 mM ATP at 0 degrees C. The purified thermally transformed receptor could be adsorbed to columns of ATP-Sepharose. No cytosol factor(s) appeared to be required for the 8S to 4S transformation of purified receptor or for its subsequent binding to ATP-Sepharose. Incubation of purified non-transformed receptor preparation with [gamma-32P]ATP and cAMP-dependent protein kinase led to incorporation of radioactivity in all the three major peptides at serine residues. The results of this study show for the first time that purified 8S progesterone receptor can be phosphorylated in vitro by a cAMP-dependent protein kinase, and that it can be transformed to a 4S form by 0 degrees C incubation with 10 mM ATP.     

A protein kinase copurified with chick oviduct progesterone receptor

A magnesium-dependent protein kinase activity was copurified with both the molybdate-stabilized 8S form of the chick oviduct progesterone receptor (PR) and its B subunit. In each case, purification was performed by hormonal affinity chromatography followed by ion-exchange chromatography. The Km(app) values of the phosphorylation reaction for [gamma-32P]ATP and calf thymus histones were approximately 1.3 X 10(-5) M and approximately 1.6 X 10(-5) M, respectively, and only phosphorylated serine residues were found in protein substrates, including PR B subunit. Physicochemical parameters of the enzyme [pI approximately 5.3, Stokes radius approximately 7.2 nm, sedimentation coefficient (S20,w) approximately 5.6 S, and Mr approximately 200,000] were compared to those of purified forms of PR (B subunit, pI approximately 5.3, Stokes radius approximately 6.1 nm, and Mr approximately 110,000; 8S form, Stokes radius approximately 7.7 nm and Mr approximately 240,000). The results suggest that most of the protein kinase activity copurified with both oligomeric and monomeric forms of PR belongs to an enzyme distinct from currently known receptor components. Its physiological significance remains unknown.     

Effects of calcium on the chick oviduct progesterone receptor

The chick oviduct cytosol progesterone receptor can be transformed to a small form (Rs = 21A, S20,w:2.9) denoted "mero-receptor" by incubation in the presence of Ca2+ [8]. In the molybdate-free cytosol all the progestin binding components could be completely transformed to mero-form by 1 h treatment with 100 mM Ca2+ at 0 degrees C. If EDTA was secondarily added, the ligand was rapidly released. If molybdate (20 mM) containing cytosol was incubated with Ca2+, no radioactivity was found in the meroposition on the Agarose A 0.5 m column, but the bound steroid sedimented at 2.9 S in sucrose gradients containing Ca2+ (and no molybdate). When 20 nM molybdate was added to cytosol containing receptor activated by 0.3 M KCl, complete mero-transformation by Ca2+ was obtained also by the gel filtration criterion, indicating that molybdate does not inhibit the mero-transforming factor. Ligand-free progesterone receptor could also be completely converted to mero-form by endogenous cytosolic transforming factor and calcium. The transforming factor was completely inactivated, when cytosol was run through Agarose A 0.5 m gel. Mero-transformation was found to be irreversible. The purified progesterone receptor subunit 110 K (B) was partially converted to smaller forms by calcium alone (100 mM, 0 degrees C, 1 h) whereas addition of a small amount of cytosol allowed complete conversion to mero-form.     

Protein kinase activity of purified components of the chicken oviduct progesterone receptor

Preparations of the 90K and 110K components of the chick oviduct progesterone receptor (PR) purified to near homogeneity were tested for protein kinase activity. The 90K component was shown to incorporate radioactive phosphate from [γ-³²P]-ATP in the presence of Ca²⁺ but not of Mg²⁺ ions, while the 110K component was phosphorylated in the presence of Mg²⁺, but not of Ca²⁺. The enzymatic activity of the 90K polypeptide appeared selective, since added proteins (histones) did not become phosphorylated. However, all proteins present in the 110K preparations were phosphorylated in the presence of Mg²⁺. These data suggest that components of the chick oviduct PR display protein kinase activity.     

ZK98299, a novel antiprogesterone that does not interact with chicken oviduct progesterone receptor

Steroid antagonists, at receptor level, are valuable tools for elucidating the mechanism of steroid hormone action. We have examined and compared the interaction of avian and mammalian progesterone receptors with progestins; progesterone and R5020, and a newly synthesized antiprogesterone ZK98299. In the chicken oviduct cytosol, [3H]R5020 binding to macromolecule(s) could be eliminated with prior incubation of cytosol with excess radioinert steroids progesterone or R5020 but not ZK98299. Alternatively, [3H]ZK98299 binding in the chicken oviduct was not abolished in the presence of excess progesterone, R5020, or ZK98299. In the calf uterine cytosol, [3H]R5020 or [3H]ZK98299 binding was competeable with progesterone, R5020 and ZK98299 but not estradiol, DHT or cortisol. Furthermore, immunoprecipitation and protein A-Sepharose adsorption analysis revealed that in the calf uterine cytosol, the [3H]R5020-receptor complexes were recognized by anti-progesterone receptor monoclonal antibody PR6. This antibody, however, did not recognize [3H]ZK98299-receptor complexes. When phosphorylation of progesterone receptor was attempted in the chicken oviduct mince, presence of progesterone resulted in an increased phosphorylation of the known components A (79 kDa) and B (110 kDa) receptor proteins. Presence of ZK98299 neither enhanced the extent of phosphorylation of A and B proteins nor did it reverse the progesterone-dependent increase in the phosphorylation. The avian progesterone receptor, therefore, has unique steroid binding site(s) that exclude(s) interaction with ZK98299. The lack of immunorecognition of calf uterine [3H]ZK98299-receptor complexes, suggests that ZK98299 is either interacting with macromolecule(s) other than the progesterone receptor or with another site on the same protein. Alternatively, the antisteroid binds to the R5020 binding site but the complex adopts a conformation that is not recognized by the PRG antibodies.     

Kinetics of progesterone binding to the chick oviduct receptor protein

Kinetic parameters have been determined for the binding of progesterone to receptor proteins of the chick oviduct. Association and dissociation rate constants and the equilibrium constant have been determined as a function of temperature and ionic strength. Both the association and dissociation rate constants vary with temperature by about 2 orders of magnitude. However the equilibrium constant for the binding reaction does not change substantially over the temperature interval from 0 to 24°. The half-life of the complex under pseudo first-order conditions decreases from about 10 h at 0° to about 3 min at body temperature of the animal (42°). Arrhenius plots of the dissociation rate constant information show a linear plot over the temperature range studied indicating that the aggregation and the conformational changes accompanying this temperature range do not cause any apparent change in the hormone binding site.On the other hand glycerol has a considerable stabilizing effect on the receptors increasing the half-life at 24° from about 24 min to a matter of about 100 min as the glycerol concentration is increased to 40%. The hormone binding site can be disrupted irreversibly by high salt or urea concentrations in excess of one molar. Ionic strength over the range from 0–0.5 M has essentially no effect on any of the parameters measured. The half-life of the complexes decreases by only about 10–15% over this interval.The possible perturbation of the hormone binding sites accompanying binding of the proteins to ion exchange resins was studied. There is no detectable change in any of the kinetic parameters when the receptor protein is adsorbed to either DEAE cellulose or phosphocellulose. The hormone binding characteristics of the protein remain the same for up to 100 hours; only a single class of hormone binding site could be detected. Variations in half-life accompanying dissociation of hormone off the receptors was shown to be due merely to the presence of non specific binding in the preparation. The study concludes that the conformation of the hormone binding site is not strongly perturbed by the environment of the protein.     

Effects of delayed transfer and treatment with oestrogen on the transport of microspheres by the rat oviduct

Starch or dextran blue microspheres were transferred microsurgically to the infundibulum of the oviduct on Days 1, 2, or 3 of pregnancy of control and oestradiol-treated rats. The animals were killed a few hours to several days after transfer to assess the number and distribution of ova and microspheres in the tract. After transfer on Day 1 of pregnancy, microspheres and eggs crossed the ampullary-isthmic junction (AIJ) 18 h after ovulation. After transfer on Day 2 of pregnancy, more than 50% of microspheres were retained in the ampulla, indicating that the AIJ changes again 34 h after ovulation. Treatment with oestradiol did not advance the passage of eggs or microspheres across the AIJ but caused accelerated transport through the isthmus as soon as the eggs or microspheres reached this segment. Dextran blue microspheres were seen to move back and forth in the isthmus of control anaesthetized rats at a frequency of 5-6 times/min. Between 7 and 20 h after treatment with oestradiol the frequency of these movements was significantly augmented, indicating that increased frequency of contractions of the smooth muscle of the isthmus precedes and accompanies accelerated transport of ova through this segment.     

Changes in quantities of high-mobility-group protein 1 in oviduct cellular fractions after oestrogen stimulation.

Antiserum against chick oviduct high-mobility-group protein 1 (HMG 1) has been induced in the rabbit. With this antiserum, immunobiochemical techniques have been used to probe the quantitative change of HMG 1 in the cellular fractions of chick oviduct before or after oestrogen stimulation. HMG 1 is detectable in the cytosol, microsomal and nuclear fraction of the chick oviduct cell. After administration of oestrogen to young chicks in vivo for 5 days, the quantity of HMG 1 is increased 4-fold in the cytosol, 3.5-fold in the microsomal fraction and 1.6-fold in the nuclear fraction. The finding of large amounts of HMG 1 in cytoplasm of oviduct cell is not likely due to its leakage from the nucleus. We anticipate that HMG 1 is synthesized in the cytoplasm and then transported into the nucleus. The synthesis and transportation of HMG proteins is probably regulated by oestrogen.     

Anti-glucocorticoid effects of progesterone in vivo on rat adipose tissue metabolism

Steroid hormones seem to be important for adipose tissue metabolism and accumulation. As progesterone has been suggested to modulate the glucocorticoid effects, the interactions between glucocortioid and progesterone on adipose tissue metabolism were investigated.Forty-eight male Wistar rats were adrenectomized and divided into four groups; controls (treated with vehicle only), dexamethasone treated (10 micro g per rat), progesterone treated (5mg per rat) and the last group received both dexamethasone and progesterone.The dexamethasone-treated group had a significant loss of body weight and smaller intra-abdominal fat depots compared to the control group in addition, dexamethasone increased LPL-activity and increased catecholamine stimulated lipolysis. When progesterone was given concomitantly the dexamethasone effects on adipose tissue mass, LPL-activity and lipolysis were blocked. When given alone progesterone had no influence on body weight, amount of adipose tissue, lipolysis or LPL-activity.These data indicate that progesterone acts as an anti-glucocorticoid in adipose tissue in vivo, thus attenuating the glucocorticoid effect on adipose tissue metabolism.     

Effects of danazol on incidence of progesterone and oestrogen receptors in benign breast disease.

Results from a continuing clinical trial in benign breast disease indicate that danazol may induce progesterone receptors and that this effect persists after treatment. In women given danazol the number of biopsy specimens that were positive for progesterone receptors rose from 14 out of 31 before treatment to 23 out of 30 at the end of six months'' treatment and remained raised at 21 out of 31 six months later. The number of biopsy specimens that were positive for oestrogen receptors rose transiently from eight out of 31 to 12 out of 30 and then fell to six out of 31. A satisfactory clinical response was achieved in 26 of the 31 patients but was maintained in only 11 six months or more after the end of treatment. It was only in this group that a significant and long-standing increase in progesterone receptors was observed. These findings suggest that in some women with benign breast disease who have been treated with danazol changes occur that may have long term benefit.