Interactions of phosducin with the subunits of G-proteins. Binding to the alpha as well as the betagamma subunits.

The high affinity interactions of phosducin with G-proteins involve binding of phosducin to the G-protein betagamma subunits. Here we have investigated whether phosducin interacts also with G-protein alpha subunits. Interactions of phosducin with the individual subunits of Go were measured by retaining phosducin-G-protein subunit complexes on columns containing immobilized anti-phosducin antibodies. Both the alpha and the beta subunits of trimeric Go were specifically retained by the antibodies in the presence of phosducin. This binding was almost completely abolished for both subunits following protein kinase A-mediated phosphorylation of phosducin and was reduced, more for alpha than for beta subunits, by the stable GTP analog guanosine 5'-(3-O-thio)triphosphate. Isolated alphao was also retained on the columns in the presence of phosducin but not in the presence of protein kinase A-phosphorylated phosducin. Likewise, purified G-protein betagamma subunit complexes as well as purified alpha subunits of Go and Gt were precipitated together with His6-tagged phosducin with nickel-agarose; this co-precipitation occurred concentration-dependently, with apparent affinities for phosducin of 55 nM (Gbetagamma), 110 nM (alphao), and 200 nM (alphat). In functional experiments, the steady state GTPase activity of isolated alphao was inhibited by phosducin by approximately 60% with an IC50 value of approximately 300 nM, whereas the GTPase activity of trimeric Go was inhibited by approximately 90% with an IC50 value of approximately 10 nM. Phosducin did not inhibit the GTP-hydrolytic activity of isolated alphao as measured by single-turnover assays, but it inhibited the release of GDP from alphao; the rate constant of GDP release was decreased approximately 40% by 500 nM phosducin, and the inhibition occurred with an IC50 value for phosducin of approximately 100 nM. These data suggest that phosducin binds with high affinity to G-protein betagamma subunits and with lower affinity to G-protein alpha subunits. We propose that the alpha subunit-mediated effects of phosducin might increase both the extent and the rapidity of its inhibitory effects compared with an action via the betagamma subunit complex alone.

Comparative pharmacology of human adenosine receptor subtypes - characterization of stably transfected receptors in CHO cells.

Four adenosine receptor subtypes of the family of G protein-coupled receptors, designated A1, A2A, A2B and A3 are currently known. In this study all human subtypes were stably transfected into Chinese hamster ovary (CHO) cells in order to be able to study their pharmacological profile in an identical cellular background utilizing radioligand binding studies (A1, A2A, A3) or adenylyl cyclase activity assays (A2B). The A1 subtype showed the typical pharmacological profile with 2-chloro-N6-cyclopentyladenosine (CCPA) as the agonist with the highest affinity and a marked stereoselectivity for the N6-phenylisopropyladenosine (PIA) diastereomers. In competition with antagonist radioligand biphasic curves were observed for agonists. In the presence of GTP all receptors were converted to a single low affinity state indicating functional coupling to endogenous G proteins. For A2A adenosine receptors CGS 21680 (2-[p-(2-carboxyethyl)phenylethylamino]-5'-N-ethylcarboxamidoadeno sine) and N-ethylcarboxamidoadenosine (NECA) were found to be the most potent agonists followed by R- and S-PIA with minor stereoselectivity. The relative potencies of agonists for the A2B adenosine receptor could only be tested by measurement of receptor-stimulated adenylyl cyclase activity. NECA was the most potent agonist with an EC50-value of 2.3 microM whereas all other compounds tested were active at concentrations in the high micromolar range. Inhibition of NECA-stimulated adenylyl cyclase identified xanthine amino congener (XAC; 8-[4-[[[[(2-aminoethyl)amino]-carbonyl]methyl]oxy]phenyl]-1,3-dipropylxa nthine) as the most potent antagonist at this receptor subtype. The A3 receptor was characterized utilizing the nonselective agonist [3H]NECA. The N6-benzyl substituted derivatives of adenosine-5'-N-methyluronamide (MECA) turned out to be the most potent agonists. The notion of xanthine-insensitivity of the A3 receptor should be dropped at least for the human receptor as xanthines with submicromolar affinity were found. Overall, the pharmacological characteristics of the human receptors are similar to other species with some species-specific characteristics. In this study we present for the first time the comparative pharmacology of all known human adenosine receptor subtypes. The CHO cells with stably transfected adenosine receptors provide an identical cellular background for such a pharmacological characterization. These cells are valuable systems for further characterization of specific receptor subtypes and for the development of new ligands.

A dileucine motif in the C terminus of the beta2-adrenergic receptor is involved in receptor internalization.

The cytoplasmic C terminus of the beta2-adrenergic receptor and many other G protein-coupled receptors contains a dileucine sequence that has been implicated in endosome/lysosome targeting of diverse proteins. In the present study, we provide evidence for an essential role of this motif in the agonist-induced internalization of the beta2-adrenergic receptor. Mutation of Leu-339 and/or Leu-340 to Ala caused little changes in surface expression, ligand binding, G protein coupling, and signaling to adenylyl cyclase, when these receptors were transiently or stably expressed in CHO or HEK-293 cells. However, agonist-induced receptor internalization was markedly impaired in the L339,340A double mutant and reduced in the two single mutants. This impairment in receptor internalization was seen by using various approaches to determine internalization: binding of hydrophobic vs. hydrophilic ligands, loss of surface beta2-adrenergic receptor immunoreactivity, and immunofluorescence microscopy. The selective effects of these mutations suggest that the C-terminal dileucine motif is involved in agonist-induced internalization of the beta2-adrenergic receptor.

Quantification of oxidative DNA modifications in mitochondria.

Specific repair endonucleases were used to quantify oxidative modifications in mitochondrial DNA (mtDNA) from rat liver and from porcine liver and kidney by means of a relaxation assay. In rat liver mitochondria the number of modifications sensitive to formamidopyrimidine--DNA glycosylase (FPG protein), which include 8-hydroxyguanine (8-oxo-7,8-dihydroguanine) residues, was only 0.8 +/- 0.2 per 10(5) base pairs (bp). Even lower values were observed in porcine kidney (0.5 +/- 0.3 per 10(5) bp) and liver (0.4 +/- 0.2 per 10(5) bp). The numbers of sites of base loss (AP sites) sensitive to T4 endonuclease V and of 5,6-dihydropyrimidines sensitive to endonuclease III were less than 0.2 per 10(5) bp in all cases. The data provide evidence that the steady-state levels of oxidative mtDNA modifications are low under physiological conditions, either because reactive oxygen species generated in the mitochondria are instantly inactivated or because of efficient DNA repair processes inside mitochondria.

Use of repair endonucleases to characterize DNA damage induced by reactive oxygen species in cellular and cell-free systems.

A number of repair endonuclease, viz. endonuclease III, formamidopyrimidine-DNA glycosylase (FPG protein), endonuclease IV, exonuclease III and UV endonuclease, is used to simultaneously quantify various types of DNA modifications, which were induced by agents that generate reactive oxygen species. Under cell-free conditions, two types of DNA damage profiles are obtained. The profiles induced by chemically generated singlet oxygen and by various photosensitizers (acridine orange, methylene blue, riboflavin, hematoporphyrin) plus light are dominated by base modifications sensitive to FPG protein, while 5,6-dihydropyrimidines (recognized by endonuclease III), sites of base loss (AP sites, recognized by endonuclease IV and exonuclease III) and strand breaks are minor lesions. In contrast, the DNA damage profile induced by hydroxyl radicals (gamma-rays) consists of approx. equal levels of base modifications. AP sites and strand breaks. The damage profiles induced by Fe(III)-EDTA in the presence of superoxide and by Fe(III)-nitrilotriacetate in the presence of H2O2 do not differ from that by hydroxyl radicals. The damage profile induced by Cu(II)-phenanthroline deviates by high levels of AP sites that are recognized by endonuclease IV and exonuclease III-but not by those AP endonucleases which cleave at the 3' site-and probably represent AP sites oxidized at C-1'. The damage induced by Fe(III)-bleomycin plus H2O2 deviates by an increased level of double strand breaks and the absence of endonuclease-sensitive base modifications. Cellular DNA damage profiles are obtained from bacteria, cultured mammalian cells and mammalian mitochondria after exposure to acridine orange plus visible light. A comparison with the cell-free profiles reveals that the damage in all three systems is not induced indirectly by hydroxyl radicals or an activation of cellular nucleases, but by the same mechanism that is responsible for the cell-free DNA damage.

Identification of ultimate DNA damaging oxygen species.

DNA damage induced by various reactive oxygen species can be characterized using a set of repair endonucleases with defined substrate specificities. DNA damage profiles thus obtained in a cell-free system can be compared with those observed in cellular DNA. Using this approach, we have demonstrated that an illumination of Salmonella typhimurium cells with visible light in the presence of methylene blue gives rise to a DNA damage profile very similar to that of singlet oxygen in a cell-free system. Therefore, the genotoxicity observed under these conditions most probably is attributable to the direct action of this species. The damage consists mainly of base modifications that are subject to repair by uvrABC-independent pathways. Revertant frequencies observed in parallel in the strains TA100 and TA2638 indicate a pronounced mutagenicity of the lesions induced. Exposure of Salmonella typhimurium to tert-butylhydroperoxide gives rise to another form of damage profile that is also different from that produced by hydroxyl radicals in a cell-free system. However, the latter dissimilarity does not exclude hydroxyl radicals as ultimate reactive species, as a very rapid repair of the induced base modifications is observed, which might have distorted the damage profile despite immediate work up.

Singlet oxygen as an ultimately reactive species in Salmonella typhimurium DNA damage induced by methylene blue/visible light.

The specific recognition of various DNA modifications by repair enzymes is exploited for the analysis of DNA damage induced by visible light in the presence of methylene blue in Salmonella typhimurium. The relative frequencies of various endonuclease-sensitive sites and strand breaks are determined in the plasmid pAQ1 of the treated bacteria and are compared with those observed after exposure of isolated DNA to various conditions. This comparison of damage profiles indicates that the cellular DNA damage by illumination in the presence of methylene blue is caused predominantly by the direct action of singlet oxygen. Indirect mechanisms, e.g. involving a generation of superoxide and hydroxyl radicals or the activation of cellular nucleases, do not contribute very much. The damage is dominated by base modifications. These are subject to an efficient repair that is not mediated by uvrABC proteins and therefore most probably involves recognition by specific glycosylases. Revertant frequencies observed under these conditions in the strains TA1535, TA100, TA2638 and TA104 indicate a pronounced mutagenicity of the lesions induced. On the other hand, the DNA damage does not contribute significantly to the cytotoxicity caused by the treatment as an excision repair deficiency (uvrB) has no influence on cell killing.

Site-specific covalent binding of stilbene-type and steroidal estrogens to tubulin following metabolic activation in vitro.

Both the steroidal estrogen, 2-hydroxy estradiol, and the stilbene-type estrogen, diethylstilbestrol, bind covalently and selectively to the C-terminal domain of beta-tubulin after peroxidative activation in vitro. The binding probably has to be attributed to quinonoid metabolites, as estrogens such as estradiol and hexestrol, which are unable to form quinones under these conditions, fail to bind. Albumin is not simultaneously modified, demonstrating the selectivity of the binding. The observed protein binding is discussed with respect to estrogen-induced aneuploidy and neoplastic cell transformation.