Effects of the steroid antagonist RU486 on dimerization of the human progesterone receptor.

We previously reported, using a coimmunoprecipitation assay, that the B form (PR-B) of the human progesterone receptor from T47D human breast cancer cells dimerizes in solution with the A receptor (PR-A) and that the extent of dimerization correlates with receptor binding activity for specific DNA sequences [DeMarzo, A.M., Beck, C.A., O?ate, S.A., & Edwards, D.P. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 72-76]. This suggested that solution dimerization is an intermediate step in the receptor activation process. The present study has tested the effects of the progesterone antagonist RU486 on solution dimerization of progesterone receptors (PR). As determined by the coimmunoprecipitation assay, RU486 binding did not impair dimerization of receptors; rather, the antagonist promoted more efficient solution dimerization than the progestin agonist R5020. This enhanced receptor dimerization correlated with a higher DNA binding activity for transformed receptors bound with RU486. RU486 has been shown previously to produce two other alterations in the human PR when compared with R5020. PR-RU486 complexes in solution exhibit a faster sedimentation rate (6 S) on salt-containing sucrose density gradients than PR-R5020 complexes (4 S), and PR-DNA complexes have a faster electrophoretic mobility on gel-shift assays in the presence of RU486. We presently show that the 6 S PR-RU486 complex is a receptor monomer, not a dimer. The increased sedimentation rate and increased mobility on gel-shift assays promoted by RU486 were also observed with recombinant PR-A and PR-B separately expressed in insect cells from baculovirus vectors. These results suggest that RU486 induces a distinct conformational change both in PR monomers in solution and in dimers bound to DNA. We also examined whether conformational changes in PR induced by RU486 would prevent a PR polypeptide bound to RU486 from heterodimerization with another PR polypeptide bound to R5020. To evaluate this, PR-A and PR-B that were separately bound to R5020 or RU486 in whole cells were mixed in vitro. PR-A-RU486 was capable of dimerization with PR-B-R5020, and this was demonstrated for heterodimers both formed in solution and bound to specific DNA. The capability to form heterodimers in vitro raises the possibility that the antagonist action of RU486 in vivo could in part be imposed in a dominant negative fashion through heterodimerization between one receptor subunit bound to an agonist and another bound to RU486.     

Differential expression of uterine progesterone receptor forms A and B during the menstrual cycle

Recent studies suggest that the progesterone receptor isoforms (PR-A and PR-B) activate genes differentially and that PR-A may act as a repressor of PR-B function. Hence, the absolute and relative expression of the two isoforms will determine the response to progesterone. We have measured their relative expression in the uterus of cycling women who underwent endometrial biopsy. PR isoforms were identified on blots of SDS-PAGE gels by reaction with the AB-52 antibody after immunoprecipitation from endometrial extract. Both isoforms were highest in the peri-ovulatory phase, but levels of PR-A were always higher than those of PR-B. The ratio of PR-A to PR-B changed during the menstrual cycle. Between days 2 and 8, PR-B is almost undetectable and the A:B ratio is >10:1. From days 9 to 13, the ratio is about 5:1, and it is about 2:1 between days 14 and 16. Thereafter, PR-B dwindles rapidly and is virtually undetectable at the end of the cycle. In various hypoestrogenic environments, PR-B expression was reduced. However, exogenous estrogens in the follicular phase in the form of oral contraceptives, enhanced PR-B expression. These data support the possibility that progesterone acts through cycle-specific PR isoforms.     

Ontogenic variations in the content and distribution of progesterone receptor isoforms in the reproductive tract and brain of chicks

Progesterone participates in the regulation of several functions in chicks such as ovulation, gonadal differentiation, and sexual and nesting behaviors. Many progesterone actions are mediated by specific intracellular receptors (PR) which are ligand-induced transactivators. Two PR isoforms that are functionally distinct in their ability to activate genes and regulate distinct physiological processes have been described in chicks: a full length form PR-B and the N-terminally truncated one PR-A which lacks the amino-terminal 128 amino acids of PR-B. PR isoforms have been detected in several tissues of both the adult and the embryo chick such as brain, ovary and oviduct. PR isoforms expression ratio varies among progesterone target tissues and under different hormonal and environmental conditions such as those presented during avian sexual maturity and the seasons of the year. These data let us to conclude that progesterone actions in brain, ovary, and oviduct highly depend on PR isoforms expression pattern and regulation.     

Functional properties of the N-terminal region of progesterone receptors and their mechanistic relationship to structure

Progesterone receptors (PR) are present in two isoforms, PR-A and PR-B. The B-upstream segment (BUS) of PR-B is a 164 amino acid N-terminal extension that is missing in PR-A and is responsible for the functional differences reported between the two isoforms. BUS contains an activation function (AF3) which is defined by a core domain between residues 54–154 whose activity is dependent upon a single Trp residue and two LXXLL motifs. We have also identified sites both within and outside of BUS that repress the strong synergism between AF3 and AF1 in the N-terminal region and AF2 in the hormone binding domain. One of these repressor sites is a consensus binding motif for the small ubiquitin-like modifier protein, SUMO-1 (³⁸⁷IKEE). The DNA binding domain (DBD) structure is also important for function. When BUS is linked to the glucocorticoid receptor DBD, AF3 activity is substantially attenuated, suggesting that binding to a DNA response element results in allosteric communication between the DBD and N-terminal functional regions. Lastly, biochemical and biophysical analyses of highly purified PR-B and PR-A N-terminal regions reveal that they are unstructured unless the DBD is present. Thus, the DBD stabilizes N-terminal structure. We propose a model in which the DBD through DNA binding, and BUS through protein–protein interactions, stabilize active receptor conformers within an ensemble distribution of active and inactive conformational states. This would explain why PR-B are stronger transactivators than PR-A.     

Molecular size of nerve growth factor in dilute solution.

Nerve growth factor (NGF) is a protein composed of two identical chains of mass 13,259. An analysis of the sedimentation equilibrium, sedimentation velocity, and gel filtration behavior of dilute solutions of NGF indicates the existence of a rapidly reversible monomer in equilibrium dimer equilibrium and that the association constant K for the reaction at neutral pH is 9.4 X 10(6)M-1. Reaction mixtures consist of equal concentrations of monomer and dimer at a total protein concentration as high as 1.4 mug/ml, and at 1 ng/ml, monomer accounts for greater than 99% of the total. The latter concentration is 20 to 30 times that required for the biological activity of NGF. Several lines of evidence suggest that the dimerization reaction is highly stereospecific, although its biological significance is not known.     

Self-association mode of a flavoenzyme D-amino acid oxidase from hog kidney. II. Stoichiometry of holoenzyme association and energetics of subunit association

The self-association pattern of D-amino acid oxidase holoenzyme in 0.1 M sodium pyrophosphate, pH 8.3, at 25 degrees C was examined by the low-angle laser light-scattering method. As to the results of nonlinear least-squares analysis of the apparent weight-average molecular weight (Mwapp) versus protein concentration (c) data, the following three models fitted equally well the data over the concentration range of 0.03-11.4 mg/ml: 1) the model of isodesmic indefinite self-association of the monomer where the dimerization constant differs from the isodesmic association constant, 2) the model which involves the dimerization of the monomer and isodesmic indefinite self-association of the dimer, and 3) the model which involves the trimerization of the monomer and isodesmic indefinite self-association of the trimer. In a more limited concentration range (0.3-11.4 mg/ml), a model of isodesmic indefinite self-association of the stable dimer where the dimer does not dissociate into the monomers cannot be excluded from the above three models. Measurements with the concentration range lowered to 0.03 mg/ml enabled us to exclude unequivocally the model involving such a stable dimer and to extrapolate the Mwapp data to the Mr of the monomer at infinite dilution as in the case of the apoenzyme. The observed sedimentation boundary profiles were qualitatively consistent with the idealized boundary profiles calculated with the model which involves the dimerization of the monomer and isodesmic indefinite self-association of the dimer, so this model is the most probable of the models examined. These results provide the first evidence that the association mode of the holoenzyme is different from that of the apoenzyme, i.e. isodesmic indefinite self-association of the monomer (Tojo, H., Horiike, K., Shiga, K., Nishina, Y., Watari, H., and Yamano, T. (1985) J. Biol. Chem. 260, 12607-12614). The overall linkage scheme, between binding of coenzyme FAD and subunit association, was considered, and the overall free energy change in each process in the scheme was calculated. The total stabilization energies of the intersubunit interaction in the holoenzyme relative to the apoenzyme were found to be -2.2 kcal/mol at the dimerization step and -0.5 kcal/mol at the step of the addition of the dimer to any 2i-mer (i = 1,2, ...).     

Progesterone-independent effects of human progesterone receptors (PRs) in estrogen receptor-positive breast cancer: PR isoform-specific gene regulation and tumor biology

Progesterone receptors (PRs) are prognostic markers in breast cancers irrespective of the patient's progestational status. However, there are two PR isoforms, PR-A and PR-B, that are equimolar in the normal breast but dysregulated in advanced disease. Postmenopausal, tamoxifen-treated patients with estrogen receptor (ER)-positive, PR-A-rich tumors have much faster disease recurrence than patients with PR-B-rich tumors. To study the mechanisms we engineered ER+ breast cancer cells that express each PR isoform under control of an inducible promoter. We identified 79 genes regulated by progesterone (P), mainly by PR-B, and 51 genes regulated without progesterone, mainly by PR-A. Only nine genes were regulated with and without ligand, leading to definition of three classes: I) genes regulated only by liganded PR; II) genes regulated only by unliganded PR; III) genes regulated by both. Unliganded PR-A and PR-B differentially regulate genes that coordinate extracellular signaling pathways and influence tumor cell biology. Indeed, in the absence of P, compared with ER+/PR-B+ or PR- cells, ER+, PR-A+ cells exhibit an aggressive phenotype, are more adhesive to an extracellular matrix, and are more migratory. Additionally, unliganded PR-A and PR-B both inhibit cell growth and provoke resistance to Taxol-induced apoptosis. We propose that PR-A:PR-B ratios, even in the absence of P, influence the biology and treatment response of ER+ tumors, that PR-A isoforms are functionally dominant in P-deficient states, and that PR-A rich tumors are especially aggressive.     

The association−dissociation behavior of the ApoE proteins: kinetic and equilibrium studies

The apolipoprotein E family consists of three major protein isoforms: apolipoprotein E4 (ApoE4), ApoE3, and ApoE2. The isoforms, which contain 299 residues, differ only by single-amino acid changes, but of the three, only ApoE4 is a risk factor for Alzheimer’s disease. At micromolar concentrations, lipid-free ApoE exists predominantly as tetramers. In more dilute solutions, lower-molecular mass species predominate. Using fluorescence correlation spectroscopy (FCS), intermolecular fluorescence resonance energy transfer (FRET), and sedimentation methods, we found that the association?dissociation reaction of ApoE can be modeled with a monomer?dimer?tetramer process. Equilibrium constants have been determined from the sedimentation data, while the individual rate constants for association and dissociation were determined by measurement of the kinetics of dissociation of ApoE and are in agreement with the equilibrium constants. Dissociation kinetics as measured by intermolecular FRET show two phases reflecting the dissociation of tetramer to dimer and of dimer to monomer, with dissociation from tetramer to dimer being more rapid than the dissociation from dimer to monomer. The rate constants differ for the different ApoE isoforms, showing that the association?dissociation process is isoform specific. Strikingly, the association rate constants are almost 2 orders of magnitude slower than expected for a diffusion-controlled process. Dissociation kinetics were also monitored by tryptophan fluorescence in the presence of acrylamide and the data found to be consistent with the monomer?dimer?tetramer model. The approach combining multiple methods establishes the reaction scheme of ApoE self-association.     

Re-examining the oligomerization state of macrophage migration inhibitory factor (MIF) in solution

The state of oligomerization of macrophage migration inhibitory factor (MIF, also known as glycosylation inhibiting factor, GIF) in solution has been variously reported as monomer, dimer, trimer, or mixtures of all three. Several crystal structures show MIF to be a trimer. Sedimentation velocity shows a recombinant human MIF sample is quite homogeneous, with 98% as a species with s(20,w)=3.07 S and D(20,w)=8.29 x 10(-7) cm(2)/s. Using the partial specific volume calculated from the amino acid composition these values imply a mass of 33.56 kDa, well above that of dimer, but also 9% below the trimer mass of 37.035 kDa. Sedimentation equilibrium data at loading concentrations from 0.01 to 1 mg/ml show unequivocally that the self-association is extremely tight. However, the apparent mass is 33.53 kDa [95% confidence 33.25-33.82], again 9% below that expected for 100% trimer. To examine the possibility this protein has an unusual partial specific volume, sedimentation equilibrium was also done in H(2)O/D(2)O mixtures, giving 0.765+/-0.017 ml/g rather than the calculated 0.735 ml/g. With this revised partial specific volume, the equilibrium and velocity data each give M=37.9+/-2.8 kDa, fully consistent with a strongly-associated trimeric quaternary structure.     

Expression of progesterone receptor isoforms A and B is differentially regulated by estrogen in different breast cancer cell lines

Progesterone action in target tissues is mediated through two progesterone receptor (PR) isoforms, PR-A and PR-B, which display different regulatory functions in target cells. Relative expression ratio of these isoforms varies depending on cell and tissue types. Here, we studied the regulation of PR isoform expression by estradiol (E(2)), insulin, IGF-1 and cAMP in different breast cancer cell lines. Although, E(2) induced PR expression in all cell lines studied, the expression ratio of PR-A/PR-B induced by E(2) was dependent on the cell line. The differential regulation of the isoforms was also seen at the mRNA level suggesting that the PR-A and PR-B promoters are differentially regulated by E(2) in different breast cancer cells. Insulin, IGF-1 or cAMP previously reported to induce PR expression however failed to alter the PR expression in our study. This is the first report describing that in different breast cancer cell lines the expression of PR-A and PR-B is regulated by E(2) in a distinct way.     

Cooperative DNA binding by the B-isoform of human progesterone receptor: thermodynamic analysis reveals strongly favorable and unfavorable contributions to assembly

Progesterone receptors (PR) play critical roles in eukaryotic gene regulation, yet the mechanisms by which they assemble at multisite promoters are poorly understood. Here we present a thermodynamic analysis of the interactions of the PR B-isoform (PR-B) with promoters containing either one or two progesterone response elements (PREs). Utilizing quantitative footprinting, we have resolved the microscopic energetics of PR-B binding, including cooperativity terms. The results of this analysis challenge a number of assumptions found in traditional models of receptor function. First, PR-B interactions at a single PRE can be equally well described by mechanisms invoking either the receptor monomer or the dimer as the active DNA binding species. If, as is commonly accepted, PR-B interacts with response elements only as a preformed dimer, then its intrinsic binding affinity is not the typically observed nanomolar but is rather picomolar. This high affinity binding is opposed, however, by a large energetic penalty. The penalty presumably pays for costly structural rearrangements of the receptor dimer and/or response element that are needed to form the protein-DNA complex. If PR-B assembles at a single response element via successive monomer binding reactions, then this penalty minimizes cooperative interactions between adjacent monomers. When binding to two response elements, the receptor exhibits strong intersite cooperativity. Although this phenomenon has been observed before, the present work demonstrates that the energetics reach levels seen in highly cooperative systems such as lambda cI repressor. This first quantitative dissection of cooperative receptor-promoter interactions suggests that PR-B function is more complex than traditionally envisioned.