GST-Induced dimerization of DNA-binding domains alters characteristics of their interaction with DNA

The steroid hormone 20-hydroxyecdysone (20E) plays a key role in the induction and modulation of morphogenetic events throughout Drosophila melanogaster development. Two members of the nuclear receptor superfamily, the product of the EcR (EcR) and of the ultraspiracle genes (Usp), heterodimerize to form its functional receptor. To study the receptor-DNA interaction, critical for regulating 20E-dependent gene expression, it is necessary to produce large quantities of EcR and Usp DNA-binding domains. Toward this end DNA-binding domains of EcR and Usp (EcRDBD and UspDBD, respectively) were cloned and expressed in Escherichia coli as fusion proteins with glutathione S-transferase (GST). However, the results of DNA-binding studies obtained with purified GST-DBDs were found to be questionable because the fused proteins oligomerized in solution due to the presence of GST. Therefore DBDs were released from GST-chimeric proteins by thrombin cleavage and then purified by glutathione-Sepharose 4B chromatography and by gel filtration on Superdex 75 HR. The gel mobility-shift experiments showed that UspDBD exhibited higher affinity than EcRDBD toward a 20-hydroxyecdysone response element from the Drosophila hsp 27 gene (hsp 27pal). Furthermore, formation of the heterodimeric EcRDBD-UspDBD complex was observed to be synergistic when equimolar mixture of both DBDs was incubated with hsp 27pal. Surprisingly, GST-EcRDBD bound hsp 27pal with higher affinity than GST-UspDBD. This difference was accompanied by the impaired ability of the GST-DBDs to interact synergistically with hsp 27pal. This is the first report on expression and purification of the soluble DBDs of the functional ecdysteroid receptor with satisfying yields. Furthermore, our results add to the recent findings which indicate the need for caution in interpreting the activities of GST fusion proteins.     

Estrogen receptor accessory proteins augment receptor-DNA interaction and DNA bending

The human Cu/Zn superoxide dismutase (hSOD-1) gene, catalyses the dismutation of O2 to H2O2 and O2. It is located on chromosome 21 in q22.1 and is overexpressed in Down's syndrome (DS) patients. These patients present various abnormalities including mental retardation, congenital heart disease, immunological deficits and premature aging. In order to explore the potential role of SOD-1 overexpression in DS, we have generated two lineages of transgenic mice for the hSOD-1 gene and studied, at the ultrastructural level, the effect of hSOD-1 overexpression on the thymic microenvironment. Modification of the cellular architecture and morphology associated with a lipidic invasion, signs of a premature involution of the thymus, were observed in both lineages. A rupture of the filamentous network in the extracellular and probably also in the intracellular matrix was first observed. These results correlate the thymic alterations visualized in light microscopy, on the thymus from DS patients, and raise the question of the relationship between the SOD-1 overexpression and the different morphological alterations associated with the premature thymic involution observed in SOD-1 transgenic mice. They suggest that thymic and immunological impairments present in DS patients may be related to the SOD-1 gene dosage effect.     

Unliganded epidermal growth factor receptor dimerization induced by direct interaction of quinazolines with the ATP binding site

Receptor dimerization is critical for signaling by the epidermal growth factor receptor (EGFR) tyrosine kinase. This occurs after binding of the receptor's extracellular domain by ligand or bivalent antibodies. The role of other receptor domains in dimerization is less clear, and there are no examples of dimers induced by direct perturbation of the EGFR kinase domain. Submicromolar concentrations of AG-1478 and AG-1517, quinazolines specific for inhibition of the EGFR kinase, induced reversible receptor dimerization in vitro and in intact A431 cells. Consistent with the inhibitory effect of quinazolines on receptor kinase activity, the dimers formed lacked a detectable Tyr(P) signal. Quinazoline-induced EGFR dimerization was abrogated in vitro by ATP and the ATP analog adenyl-5'-yl imidodiphosphate. Receptors with a single-point mutation in the ATP binding site as well as wild-type EGFR with a covalent modification of the ATP site failed to dimerize in response to AG-1478 and AG-1517. These data suggest that EGFR dimerization can be induced by the interaction of quinazolines at the ATP site in the absence of receptor ligand binding. In SKBR-3 cells, the quinazolines induced the formation of inactive EGFR/ErbB-2 heterodimers, potentially sequestering ErbB-2 from interacting with other coreceptors of the ErbB family. Structural studies of the quinazoline interaction with the EGFR tyrosine kinase domain should allow for an analysis of receptor-specific chemical features required for binding to the ATP site and disruption of signaling, a strategy that can be perhaps applied to other tumor cell receptor systems.     

Identification of amino acids in the transmembrane and juxtamembrane domains of the platelet-derived growth factor receptor required for productive interaction with the bovine papillomavirus E5 protein

The bovine papillomavirus E5 protein forms a stable complex with the cellular platelet-derived growth factor (PDGF) beta receptor, resulting in receptor activation and cell transformation. Amino acids in both the putative transmembrane domain and extracytoplasmic carboxyl-terminal domain of the E5 protein appear important for PDGF receptor binding and activation. Previous analysis indicated that the transmembrane domain of the receptor was also required for complex formation and receptor activation. Here we analyzed receptor chimeras and point mutants to identify specific amino acids in the PDGF beta receptor required for productive interaction with the E5 protein. These receptor mutants were analyzed in murine Ba/F3 cells, which do not express endogenous receptor. Our results confirmed the importance of the transmembrane domain of the receptor for complex formation, receptor tyrosine phosphorylation, and mitogenic signaling in response to the E5 protein and established that the threonine residue in this domain is required for these activities. In addition, a positive charge in the extracellular juxtamembrane domain of the receptor was required for E5 interaction and signaling, whereas replacement of the wild-type lysine with either a neutral or acidic amino acid inhibited E5-induced receptor activation and transformation. All of the receptor mutants defective for activation by the E5 protein responded to acute treatment with PDGF and to stable expression of v-Sis, a form of PDGF. The required juxtamembrane lysine and transmembrane threonine are predicted to align precisely on the same face of an alpha helix packed in a left-handed coiled-coil geometry. These results establish that the E5 protein and v-Sis recognize distinct binding sites on the PDGF beta receptor and further clarify the nature of the interaction between the viral transforming protein and its cellular target.     

The role of T cell receptor dimerization for T cell antagonism and T cell specificity

Endemic nephropathy is a chronic renal disease with a high prevalence in a geographically limited area of Croatia. It has also been recorded in some parts of Bosnia, Serbia, Bulgaria and Romania. Despite numerous studies conducted to date, the etiology of this disease has not been clarified. Pathological studies of the kidney in the early stage of endemic nephropathy have shown renal tubules to be the primary sites of the pathologic process with an interstitial tissue reaction, whereas glomerular alterations are of a secondary character. Tubulointerstitial lesions can thus account for the symptoms of the disease, i.e. tubular proteinuria and reduced urine concentration capacity and urine acidification. Also, an increased incidence of malignant tumours of the urinary tract was found in the same geographic area.     

RING fingers and B-boxes: zinc-binding protein-protein interaction domains

The cysteine-rich zinc-binding motifs known as the RING and B-box are found in several unrelated proteins. Structural, biochemical, and biological studies of these motifs reveal that they mediate protein-protein interactions. Several RING-containing proteins are oncoproteins and recent data indicate that proapoptotic activities can be mediated through the RING. 1H NMR methods were used to determine the structures of RINGs and a B-box domain and to monitor the conformational changes these motifs undergo upon zinc ligation. This review discusses in detail the structural features of the RING and B-box domains. Further, possible structure function relationships for these motifs particularly in their role as protein interaction domains are discussed.     

Comparison of the sequence-selective DNA binding by peptide dimers with covalent and noncovalent dimerization domains

Sequence-specific DNA binding proteins generally consist of more than two DNA-contacting regions to ensure the selectivity of recognition. The multiple DNA binding modules are connected either through the covalent linker or through the noncovalent dimerization domain. We have compared the DNA binding of peptide dimers with covalent and noncovalent dimerization domains to explore the potential advantage of each linkage on the sequence-specific DNA binding. Three sets of head-to-tail peptide dimers were synthesized by using the same basic region peptide to target the same DNA sequence; one dimer was assembled with a bridged biphenyl derivative as a covalent dimerization domain, and two other dimers were assembled with the cyclodextrin guest noncovalent dimerization domains. One of the noncovalent dimers was a heterodimer that consisted of cyclodextrin and guest peptides, while the other was a homodimer that consisted of peptides bearing both cyclodextrin and the guest molecule within the same chain. Both noncovalent dimers formed the specific DNA complexes within narrower ranges of peptide concentrations and showed higher sequence selectivity than the covalent dimer did. Among the three dimers, the noncovalent homodimer that can form an intramolecular inclusion complex showed the highest sequence selectivity. Because the noncovalent homodimer with the higher stability of the circular intramolecular inclusion complex exhibited the higher sequence selectivity, it was concluded that an equilibrium involving a conformational transition of a monomeric peptide effectively reduced the stability of its nonspecific binding complex, hence increasing the efficacy of cooperative dimer formation at the specific DNA sequence.     

Estrogen receptor expression in bovine and rat retinas

PURPOSE: To investigate the expression and distribution of estrogen receptor protein and mRNA in bovine and rat retinas. METHODS: Western blot analysis with an antiestrogen receptor monoclonal antibody (mAb) was used to detect estrogen receptor protein in the bovine retina. Immunohistochemistry with an antiestrogen receptor mAb and in situ hybridization with an oligodeoxynucleotide sequence coding for estrogen receptor were applied to study the cellular distribution of estrogen receptor protein and its mRNA in male bovine retina and rat retina of both sexes. RESULTS: Estrogen receptor protein was detected in bovine retina by Western blot analysis. Immunohistochemical staining with the antiestrogen receptor mAb was widespread throughout the neural retina. Specific staining showed extensive distribution localizing in the nerve fiber layer, the ganglion cell layer, the inner nuclear layer, and the outer plexiform layer. Retinal pigment epithelium and choroid were also stained with the antiestrogen receptor mAb. By in situ hybridization, the expression of estrogen receptor mRNA was predominantly observed in ganglion cell layer, the inner nuclear layer, and outer portion of the outer nuclear layer. No significant difference was found between male and female rats in the immunostaining of retinas with the antiestrogen receptor mAb. CONCLUSIONS: This study provides the first evidence for the presence of estrogen receptor in bovine and rat retinas. Expression of estrogen receptor throughout the retina suggests that estrogen may have important functions in the retina.     

Thermodynamics of the interaction of human immunoglobulin E with its high-affinity receptor Fc epsilon RI

We have employed isothermal titration calorimetry (ITC) and circular dichroism (CD) spectroscopy to characterize the binding of soluble fragments of IgE (IgE-Fc and Fc epsilon 3-4) to a soluble fragment of the high-affinity receptor Fc epsilon RI alpha-chain (sFc epsilon RI alpha). The thermodynamic parameters for the interaction of IgE-Fc and Fc epsilon 3-4 with sFc epsilon RI alpha, determined using ITC, confirm the earlier conclusion that the C epsilon 2 domain is not involved in the interaction and that the stoichiometry of both complexes is 1:1. For both IgE-Fc and Fc epsilon 3-4, the value of Delta H degrees is -36.9 +/- 4.6 kcal mol-1 at 37.3 degreesC and Delta Cp degrees is -820 +/- 120 cal mol-1 K-1. The temperature at which DeltaS degrees is zero is 284 +/- 1 K, indicating that the entropy contribution to the thermodynamics of association is unfavorable at physiological temperature. Of particular interest is the large value of Delta Cp degrees. The large surface area of IgE and Fc epsilon RI alpha that is implicated in complex formation from previous mutagenesis studies on the two proteins may account in part for the magnitude of Delta Cp degrees. Additional contributions may arise from hydration within the binding site and changes in tertiary structure of the individual components of the complex. However, the CD spectra of IgE, IgE-Fc, and Fc epsilon 3-4 complexes with sFc epsilon RI alpha are merely the sum of the spectra of their individual components, indicating that the secondary structure of the immunoglobulin domain folds are preserved on complex formation. Thus, any change in tertiary structure must be limited to the relative disposition of the immunoglobulin domains C epsilon 3 and C epsilon 4 in IgE and the two immunoglobulin-like domains in the alpha-chain of Fc epsilon RI.     

Specificity of the SH2 domains of SHP-1 in the interaction with the immunoreceptor tyrosine-based inhibitory motif-bearing receptor gp49B

Inhibitory receptors on hemopoietic cells critically regulate cellular function. Despite their expression on a variety of cell types, these inhibitory receptors signal through a common mechanism involving tyrosine phosphorylation of the immunoreceptor tyrosine-based inhibitory motif (ITIM), which engages Src homology 2 (SH2) domain-containing cytoplasmic tyrosine or inositol phosphatases. In this study, we have investigated the proximal signal-transduction pathway of an ITIM-bearing receptor, gp49B, a member of a newly described family of murine NK and mast cell receptors. We demonstrate that the tyrosine residues within the ITIMs are phosphorylated and serve for the association and activation of the cytoplasmic tyrosine phosphatase SHP-1. Furthermore, we demonstrate a physiologic association between gp49B and SHP-1 by coimmunoprecipitation studies from NK cells. To address the mechanism of binding between gp49B and SHP-1, binding studies involving glutathione S-transferase SHP-1 mutants were performed. Utilizing the tandem SH2 domains of SHP-1, we show that either SH2 domain can interact with phosphorylated gp49B. Full-length SHP-1, with an inactivated amino SH2 domain, also retained gp49B binding. However, binding to gp49B was disrupted by inactivation of the carboxyl SH2 domain of full-length SHP-1, suggesting that in the presence of the phosphatase domain, the carboxyl SH2 domain is required for the recruitment of phosphorylated gp49B. Thus, gp49B signaling involves SHP-1, and this association is dependent on tyrosine phosphorylation of the gp49B ITIMs, and an intact SHP-1 carboxyl SH2 domain.     

ARS Presidential Address: Estrogen therapy: a causal role in endometrial cancer?

Publicity associated with recent reports of a greater frequency of adenocarcinoma of the endometrium among women who take exogenous estrogens has created confusion among physicians and fear among patients. An objective review of available data on estrogen effects and current epidemiologic evidence suggests that estrogen may play an indirect role in the development of endometrial carcinoma. Until definitive data are available, physicians should exercise caution in prescribing estrogens, monitoring the status of the endometrium at regular intervals by sampling and pathologic analysis.