Differential DNA binding by calf uterine estrogen and progesterone receptors results from differences in oligomeric states

The studies presented here provided evidence that the calf uterine estrogen and progesterone receptors exhibit different DNA-binding properties in vitro as a result of having different dimerization constants. The affinity of the estrogen and progesterone receptors for DNA was measured by using isocratic elution from DNA-Sepharose. The hormone-free estrogen receptor had a 10-fold higher affinity for DNA than did the hormone-free progesterone receptor when measured at receptor concentrations of 6-12 nM and 180 mM KCl. No effect on DNA binding by binding progesterone to its receptor was detected. This contrasts with the increased affinity for DNA and increased number of ions released upon DNA binding exhibited by the hormone-bound estrogen receptor. Between 2 and 3 ions were released when the progesterone receptor and the diluted estrogen receptor bound DNA. These observations suggested the progesterone receptor was in the monomeric state, whereas the estrogen receptor was in the dimeric state at receptor concentrations of 6-12 nM. When the dimerization constant of the progesterone receptor was measured, the value of approximately 7 nM obtained was 20-fold higher than the value of 0.3 nM reported for the estrogen receptor. This makes it likely the two receptors exist in different forms at the same concentration in vitro. It is also suggested the predominant form of the estrogen and progesterone receptors in vivo could differ.     

Mutations of tyrosine 537 in the human estrogen receptor-alpha selectively alter the receptor's affinity for estradiol and the kinetics of the interaction

Mutation of tyrosine 537 (Y537) of the human estrogen receptor-alpha (hERalpha) produces receptors having a range of constitutive activity, which suggests that this residue modulates the conformational changes of the receptor. We investigated the effect of several mutations at this position, to phenylalanine (Y537F), to serine (Y537S), and to glutamic acid (Y537E), on the hormone-binding properties of the receptor. The affinities of the wt, the Y537F mutant, and the Y537S mutant for estradiol were similar: K(a) = 2.2 +/- 0.2, 3.9 +/- 0.5, and 2.8 +/- 0.4 nM(-1), respectively. By contrast, the affinity of the Y537E mutant for estradiol was reduced 10-fold, K(a) = 0.2 +/- 0.1 nM(-1). All proteins bound [(3)H]estradiol with a positive cooperative mechanism (n(H) = 1.7-1.9), indicating they can form dimers. The wt receptor and the Y537S and Y537E mutants exhibited biphasic dissociation kinetics, which is also indicative of dimerization. Surprisingly, the half-lives of the slow component of the wt and the Y537E mutant were indistinguishable, 118 +/- 3.4 and 122 +/- 4.5 min, respectively, even though the affinity of the Y537E mutant for hormone was reduced 10-fold. The half-life of the slow component of the Y537S mutant was reduced to 96.5 +/- 3.8 min. Molecular models were constructed and compared to identify changes in the structure that correlate with the observed effects on hormone binding. Local alterations in hydrogen bonding, the position of side chains, and the position of the peptide backbone were observed. Taken together, these results show that mutations at Y537 selectively alter the affinity and kinetics of hormone binding to the receptor, and are consistent with the idea that the estradiol-estrogen receptor interaction can follow more than one pathway.     

Hydrocortisone modulates the effect of estradiol on endothelial nitric oxide synthase expression in human endothelial cells.

The interaction between hydrocortisone and estradiol on the regulation of endothelial nitric oxide synthase (eNOS) expression was investigated in human umbilical vein endothelial cells (HUVECs). Following incubation in medium containing dextran-coated-charcoal-stripped serum (DCC-stripped medium) for 4 days, incubation of HUVECs with 0.1 nM estradiol for 24 hr in the absence of hydrocortisone increased levels of eNOS mRNA measured by ribonuclease protection assay above control (0 nM estradiol). 2 microM hydrocortisone applied for 24 hr preceding and during estradiol application inhibited the estradiol-elicited increase in eNOS mRNA levels, reducing mRNA levels from 134% +/- 14% of control to 85% +/- 5% of control. Significant (ANOVA, p<0.01) reductions of estradiol-mediated increases of mRNA levels occurred over a range of hydrocortisone concentrations (10 nM, p<0.05; 2 microM, p<0.05; n=3-12). In the presence of 2 microM hydrocortisone, 10 nM estradiol significantly reduced eNOS mRNA levels to 59% +/- 3% of control. The ability of hydrocortisone to block or reverse the estradiol-mediated increase in eNOS mRNA levels may provide a link between elevated hydrocortisone levels and decreased NO production, potentially contributing to the development of hypertension and cardiovascular disease in vivo and antagonizing cardioprotective effects of estrogens.     

Numerous transposed sequences of mitochondrial cytochrome oxidase I-II in aphids of the genus Sitobion (Hemiptera: Aphididae)

Polymerase chain reaction (PCR) products corresponding to 803 bp of the cytochrome oxidase subunits I and II region of mitochondrial DNA (mtDNA COI-II) were deduced to consist of multiple haplotypes in three Sitobion species. We investigated the molecular basis of these observations. PCR products were cloned, and six clones from one individual per species were sequenced. In each individual, one sequence was found commonly, but also two or three divergent sequences were seen. The divergent sequences were shown to be nonmitochondrial by sequencing from purified mtDNA and Southern blotting experiments. All seven nonmitochondrial clones sequenced to completion were unique. Nonmitochondrial sequences have a high proportion of unique sites, and very few characters are shared between nonmitochondrial clones to the exclusion of mtDNA. From these data, we infer that fragments of mtDNA have been transposed separately (probably into aphid chromosomes), at a frequency only known to be equalled in humans. The transposition phenomenon appears to occur infrequently or not at all in closely related genera and other aphids investigated. Patterns of nucleotide substitution in mtDNA inferred over a parsimony tree are very different from those in transposed sequences. Compared with mtDNA, nonmitochondrial sequences have less codon position bias, more even exchanges between A, G, C and T, and a higher proportion of nonsynonymous replacements. Although these data are consistent with the transposed sequences being under less constraint than mtDNA, changes in the nonmitochondrial sequences are not random: there remains significant position bias, and probable excesses of synonymous replacements and of conservative inferred amino acid replacements. We conclude that a proportion of the inferred change in the nonmitochondrial sequences occurred before transposition. We believe that Sitobion aphids (and other species exhibiting mtDNA transposition) may be important for studying the molecular evolution of mtDNA and pseudogenes. However, our data highlight the need to establish the true evolutionary relationships between sequences in comparative investigations.