Phosphorylation of the regulator of G protein signaling RGS9-1 by protein kinase A is a potential mechanism of light- and Ca2+-mediated regulation of G protein function in photoreceptors.

In vertebrate photoreceptors, photoexcited rhodopsin interacts with the G protein transducin, causing it to bind GTP and stimulate the enzyme cGMP phosphodiesterase. The rapid termination of the active state of this pathway is dependent upon a photoreceptor-specific regulator of G protein signaling RGS9-1 that serves as a GTPase activating protein (GAP) for transducin. Here, we show that, in preparations of photoreceptor outer segments (OS), RGS9-1 is readily phosphorylated by an endogenous Ser/Thr protein kinase. Protein kinase C and MAP kinase inhibitors reduced labeling by about 30%, while CDK5 and CaMK II inhibitors had no effect. cAMP-dependent protein kinase (PKA) inhibitor H89 reduced RGS9-1 labeling by more than 90%, while dibutyryl-cAMP stimulated it 3-fold, implicating PKA as the major kinase responsible for RGS9-1 phosphorylation in OS. RGS9-1 belongs to an RGS subfamily also including RGS6, RGS7, and RGS11, which exist as heterodimers with the G protein beta subunit Gbeta5. Phosphorylated RGS9-1 remains associated with Gbeta5L, a photoreceptor-specific splice form, which itself was not phosphorylated. RGS9-1 immunoprecipitated from OS was in vitro phosphorylated by exogenous PKA. The PKA catalytic subunit could also phosphorylate recombinant RGS9-1, and mutational analysis localized phosphorylation sites to Ser(427) and Ser(428). Substitution of these residues for Glu, to mimic phosphorylation, resulted in a reduction of the GAP activity of RGS9-1. In OS, RGS9-1 phosphorylation required the presence of free Ca(2+) ions and was inhibited by light, suggesting that RGS9-1 phosphorylation could be one of the mechanisms mediating a stronger photoresponse in dark-adapted cells.

Impact of metabolic genotypes on levels of biomarkers of genotoxic exposure.

Phase I and Phase II xenobiotic-metabolising enzyme families are involved in the metabolic activation and detoxification of various classes of environmental carcinogens. Particular genetic polymorphisms of these enzymes have been shown to influence individual cancer risk. A brief overview is presented about recent research of the relationship between metabolic genotypes and internal dose, biologically effective dose and cytogenetic effects of complex and specific genotoxic exposures of human study populations, and we report our new results from two molecular epidemiological studies. We investigated the effects of multiple interactions among CYP1A1 Ile462Val, CYP1A1 MspI, CYP1B1 Leu432Val, CYP2C9 Arg144Cys, CYP2C9 Ile359Leu, NQO1 Pro189Ser, GSTM1 gene deletion and GSTP1 Ile105Val genotypes on the levels of carcinogen-DNA adducts determined by (32)P-postlabelling and PAH-DNA immunoassay in peripheral blood lymphocytes from workers occupationally exposed to polycyclic aromatic hydrocarbons in aluminium plants, and in bronchial tissue from smoking lung patients. A statistically significant positive linear correlation was observed between white blood cell aromatic DNA adduct and urinary 1-hydroxypyrene (1-OHPY) levels from potroom workers with GSTM1 null genotype (P=0.011). Our results suggest interactions between GSTM1 and GSTP1 alleles in modulation of urinary 1-OHPY levels and white blood cell DNA adduct levels in the PAH-exposed workers. Interactions between GSTM1 and GSTP1 alleles, in association with particular genotype combinations of CYPs, were also recognised in bronchial aromatic DNA adduct levels of smoking lung patients. The impact of single metabolic genotypes and their combinations on biomarkers of exposure was usually weak, if any, in both our studies and reports of the literature. The effect of special metabolic gene interactions may be better recognised if the compared groups of individuals are stratified for multiple potential modulators of the observable biomarker end-point, and/or if chemical structure-specific biomarker methods are applied.

Complexes of the G protein subunit gbeta 5 with the regulators of G protein signaling RGS7 and RGS9. Characterization in native tissues and in transfected cells.

A novel protein class, termed regulators of G protein signaling (RGS), negatively regulates G protein pathways through a direct interaction with Galpha subunits and stimulation of GTP hydrolysis. An RGS subfamily including RGS6, -7, -9, and -11, which contain a characteristic Ggamma -like domain, also has the unique ability to interact with the G protein beta subunit Gbeta(5). Here, we examined the behavior of Gbeta(5), RGS7, RGS9, and Galpha in tissue extracts using immunoprecipitation and conventional chromatography. Native Gbeta(5) and RGS7 from brain, as well as photoreceptor-specific Gbeta(5)L and RGS9, always co-purified as tightly associated dimers, and neither RGS-free Gbeta(5) nor Gbeta(5)-free RGS could be detected. Co-expression in COS-7 cells of Gbeta(5) dramatically increased the protein level of RGS7 and vice versa, indicating that cells maintain Gbeta(5):RGS stoichiometry in a manner similar to Gbetagamma complexes. This mechanism is non-transcriptional and is based on increased protein stability upon dimerization. Thus, analysis of native Gbeta(5)-RGS and their coupled expression argue that in vivo, Gbeta(5) and Ggamma-like domain-containing RGSs only exist as heterodimers. Native Gbeta(5)-RGS7 did not co-precipitate or co-purify with Galpha(o) or Galpha(q); nor did Gbeta(5)L-RGS9 with Galpha(t). However, in transfected cells, RGS7 and Gbeta(5)-RGS7 inhibited Galpha(q)-mediated Ca(2+) response to muscarinic M3 receptor activation. Thus, Gbeta(5)-RGS dimers differ from other RGS proteins in that they do not bind to Galpha with high affinity, but they can still inhibit G protein signaling.

Gbeta5 prevents the RGS7-Galphao interaction through binding to a distinct Ggamma-like domain found in RGS7 and other RGS proteins.

The G protein beta subunit Gbeta5 deviates significantly from the other four members of Gbeta-subunit family in amino acid sequence and subcellular localization. To detect the protein targets of Gbeta5 in vivo, we have isolated a native Gbeta5 protein complex from the retinal cytosolic fraction and identified the protein tightly associated with Gbeta5 as the regulator of G protein signaling (RGS) protein, RGS7. Here we show that complexes of Gbeta5 with RGS proteins can be formed in vitro from the recombinant proteins. The reconstituted Gbeta5-RGS dimers are similar to the native retinal complex in their behavior on gel-filtration and cation-exchange chromatographies and can be immunoprecipitated with either anti-Gbeta5 or anti-RGS7 antibodies. The specific Gbeta5-RGS7 interaction is determined by a distinct domain in RGS that has a striking homology to Ggamma subunits. Deletion of this domain prevents the RGS7-Gbeta5 binding, although the interaction with Galpha is retained. Substitution of the Ggamma-like domain of RGS7 with a portion of Ggamma1 changes its binding specificity from Gbeta5 to Gbeta1. The interaction of Gbeta5 with RGS7 blocked the binding of RGS7 to the Galpha subunit Galphao, indicating that Gbeta5 is a specific RGS inhibitor.

Localization of the sites for Ca2+-binding proteins on G protein-coupled receptor kinases.

Inhibition of G protein-coupled receptor kinases (GRKs) by Ca2+-binding proteins has recently emerged as a general mechanism of GRK regulation. While GRK1 (rhodopsin kinase) is inhibited by the photoreceptor-specific Ca2+-binding protein recoverin, other GRKs can be inhibited by Ca2+-calmodulin. To dissect the mechanism of this inhibition at the molecular level, we localized the GRK domains involved in Ca2+-binding protein interaction using a series of GST-GRK fusion proteins. GRK1, GRK2, and GRK5, which represent the three known GRK subclasses, were each found to possess two distinct calmodulin-binding sites. These sites were localized to the N- and C-terminal regulatory regions within domains rich in positively charged and hydrophobic residues. In contrast, the unique N-terminally localized GRK1 site for recoverin had no clearly defined structural characteristics. Interestingly, while the recoverin and calmodulin-binding sites in GRK1 do not overlap, recoverin-GRK1 interaction is inhibited by calmodulin, most likely via an allosteric mechanism. Further analysis of the individual calmodulin sites in GRK5 suggests that the C-terminal site plays the major role in GRK5-calmodulin interaction. While specific mutation within the N-terminal site had no effect on calmodulin-mediated inhibition of GRK5 activity, deletion of the C-terminal site attenuated the effect of calmodulin on GRK5, and the simultaneous mutation of both sites rendered the enzyme calmodulin-insensitive. These studies provide new insight into the mechanism of Ca2+-dependent regulation of GRKs.

A potyvirus polymerase interacts with the viral coat protein and VPg in yeast cells.

The two-hybrid system was used to test for pairwise interactions between the tobacco vein mottling virus (TVMV)-encoded RNA-dependent RNA polymerase (or NIb protein) and two other TVMV-encoded proteins: the NIa protein, which consists of genome-linked protein (VPg) and proteinase domains, and the viral coat protein (CP). Using this approach, we find that the NIb protein interacts with both the NIa protein and the CP in yeast cells. Moreover, we find that a mutation in the conserved GDD domain of the NIb protein diminishes the NIb-CP interaction but not the NIb-NIa interaction. Likewise, mutations in the vicinity of the NIa protein to which the genomic RNA is covalently attached eliminate the NIb-NIa interaction. We conclude that the NIb protein interacts with the VPg domain of the NIa protein and that this interaction requires a functional RNA attachment site. This interaction may be important for the initiation of viral RNA synthesis in infected cells. We also conclude that the CP interacts with the NIb in a manner that is sensitive in changes in the highly conserved GDD motif. The role of this interaction in the functioning of the NIb protein or the CP is unclear, but may involve regulation of viral RNA synthesis in infected cells.

Nucleotide sequence of shallot virus X RNA reveals a 5'-proximal cistron closely related to those of potexviruses and a unique arrangement of the 3'-proximal cistrons.

The 8890 nucleotide RNA sequence of shallot virus X (ShVX), a new virus isolated from shallot, has been determined. The sequence contains six open reading frames (ORFs) which encode putative proteins (in the 5' to 3' direction) of M(r) 194528 (ORF1), 26333 (ORF2), 11245 (ORF3), 42209 (ORF4), 28486 (ORF5) and 14741 (ORF6). The ORF1 protein was found to be highly homologous to the putative potexvirus RNA replicases; ORF2, -3, -5 and -6 proteins also have analogues among the potex- and/or carlavirus-encoded proteins. ORF3 is followed by an AUG-lacking frame coding for an amino acid sequence homologous to that of the 7K to 8K proteins of the triple gene block of the above-mentioned viruses. The putative ORF4 protein has no reliable homology with proteins in the database. The results obtained testify that, except for the unique 42K protein gene, the ShVX genome combines a number of elements typical of both carla- and potexviruses.

Nucleotide sequence and gene organization of the 3'-terminal region of chrysanthemum virus B genomic RNA.

DNA clones complementary to the 3'-terminal 3426 nucleotides of the genomic RNA of the carlavirus chrysanthemum virus B (CVB) have been sequenced. The sequence contains six open reading frames (ORFs) which encode putative proteins (in the 5'----3' direction) of Mr 25,749 (ORF2), Mr 11,435 (ORF3), Mr 6984 (ORF4), the triple gene block proteins, a protein Mr 34,638 (ORF5); the coat protein, and a protein of Mr 12,609 (ORF6). The latter protein is basic and contains a putative zinc finger motif. The 5'-proximal ORF1 encodes a product with substantial homology to the C-terminal portions of the putative RNA replicases of two other carlaviruses, potato viruses M and S. The analysis of the minus-sense sequence shows one ORF which encodes a polypeptide of Mr 16,817. The sequenced portion of the CVB genomic RNA contains three short internal non-coding regions, two of which are typical for carlaviruses (those between ORFs 1 and 2, and between ORFs 4 and 5), and one (between ORFs 5 and 6) is unusual. There is significant similarity in the amino acid sequences of the CVB RNA-encoded proteins and the corresponding proteins of other carlaviruses.

The genome organization of potato virus M RNA.

The 8534 nucleotide sequence of the genome of the carlavirus, potato virus M (PVM), has been determined. The sequence contains six large open reading frames (ORFs) and non-coding regions consisting of 75 nucleotides at the 5' end, 70 nucleotides followed by a poly(A) tail at the 3' end and 38 and 21 nucleotides between three large blocks of coding sequences. The ORF beginning at the first initiation codon at nucleotide 76 encodes a polypeptide of 223K which, according to its primary sequence analysis, seems to be a virus RNA replicase. The next coding block consists of three ORFs encoding polypeptides of 25K, 12K and 7K. The third block consists of two ORFs encoding polypeptides of 34K (PVM coat protein) and 11K. The 11K polypeptide contains a pattern resembling the consensus for a metal-binding nucleic acid-binding 'finger'.

The putative RNA replicase of potato virus M: obvious sequence similarity with potex- and tymoviruses.

On the basis of comparison of the protein sequences of the putative virus-specific replicases, carlaviruses can be placed in the "Sindbis-like" supergroup of plus-stranded RNA viruses. Among these, the amino acid sequences of the replication proteins of potex- and tymoviruses showed the highest similarity to potato virus M. The possible functions of conserved domains are suggested to be methyltransferase, nucleotide-binding domain, and RNA polymerase.

32P-postlabelling analysis of DNA adducts of benzo[a]pyrene formed in complex metabolic activation systems in vitro.

The influence of cytochrome P-450-linked monooxygenase, epoxide hydrolase, UDP-glucuronyltransferase and glutathione S-transferases on the metabolic activation of benzo[a]pyrene (BP) was studied by incubating BP with preparations of rat-liver microsomal and cytosolic fractions in the presence of exogenous DNA. 32P-Postlabelling analysis of the DNA revealed the presence of covalently bound adducts formed by BP, which were visualised as radioactive spots on autoradiographs of thin-layer chromatograms. The effects on the adduct profile of adding different combinations of 1,2-epoxy-3,3,3-trichloropropane (TCPO), UDP-glucoronic acid (UDPGA) and glutathione (GSH) to the incubation mixture were determined. As many as 14 different DNA adducts were resolved and quantitated on the chromatograms, the numbers and quantities of which varied within a large range depending on the incubation conditions. The influence of the enzyme inhibitor and cofactors on the adduct patterns reflects the complex effects of simultaneous enzyme interactions on the metabolic activation of BP.