Drosophila G␣ o . We identify Pins GoLoco domain 1 as necessary and sufficient for this unusual interaction with G␣ o -GTP. We further pinpoint a lysine residue located centrally in this domain as necessary for the interaction. Our studies thus identify Drosophila Pins as a target of G␣ o -mediated GPCR receptor signaling, e.g., in the context of the nervous system development, where G␣ o acts downstream from Frizzled and redundantly with G␣ i to control the asymmetry of cell divisions. INTRODUCTIONTrimeric G proteins transduce the signals from G proteincoupled receptors (GPCRs), the largest receptor family in the animal kingdom (Pierce et al., 2002). Signal specificity is mainly represented by the ␣-subunits of the trimeric G proteins; 16 genes for the ␣-subunits are present in the human genome, and six in Drosophila (Malbon, 2005). Both in flies and mammals, G␣ o is the predominant G␣-subunit in the nervous system (Sternweis and Robishaw, 1984;Wolfgang et al., 1990); up to 10% of the whole plasma membrane proteins of the neuronal growth cones is represented by the trimeric G o protein (Strittmatter et al., 1990). G␣ o is required for the proper brain functioning and development (Jiang et al., 1998;Ferris et al., 2006), e.g., controlling neurite outgrowth (Bromberg et al., 2008). Among the brain GPCRs activating G␣ o are the dopamine, serotonin, adenosine, cannabinoid, glutamate, and other receptors (Offermanns, 2003;Bromberg et al., 2008). Additional developmental functions of G␣ o are the transduction of the evolutionary conserved Frizzled receptors (Egger-Adam and Katanaev, 2008) and the regulation of the heart development and physiology (Valenzuela et al., 1997;Fremion et al., 1999).In the resting state the trimeric G proteins exist as complexes of the GDP-bound ␣-subunit and the ␤-and ␥-subunits. On ligand activation, GPCRs serve as guanine nucleotide exchange factors, catalyzing the substitution of GDP for GTP on the G␣-subunit. This leads to dissociation of the complex into the GTP-loaded G␣ and the ␤␥-heterodimer. Both components of the initial complex can interact with downstream effectors (Gilman, 1987).GoLoco domains (Willard et al., 2004) present in many different proteins across the animal kingdom can specifically bind ␣-subunits of the G i/o class of trimeric G protein (G␣ i , G␣ o , G␣ t , and G␣ z ) and thus might serve as a hallmark of a subclass of G␣ i/o target proteins. For example, interaction of G␣ i/o with the GoLoco-containing protein Rap1Gap (a negative regulator of a small G protein Rap1) has been proposed as a mechanism of GPCR-induced neurite outgrowth (Jordan et al., 1999;Jordan et al., 2005). However, in the majority of cases GoLoco domains bind to the GDP-, and not the GTP-loaded forms of free G␣-subunits (Willard et al., 2004); furthermore, some GoLoco motifs are able to dissociate the trimeric G protein complexes without nucleotide exchange (Takesono et al., 1999;Ghosh et al., 2003). These observations have led to proposition that GoLoco-containing proteins may serve not as targ...

Drosophila genome encodes six alpha-subunits of heterotrimeric G proteins. The Gαs alpha-subunit is involved in the post-eclosion wing maturation, which consists of the epithelial-mesenchymal transition and cell death, accompanied by unfolding of the pupal wing into the firm adult flight organ. Here we show that another alpha-subunit Gαo can specifically antagonize the Gαs activities by competing for the Gβ13F/Gγ1 subunits of the heterotrimeric Gs protein complex. Loss of Gβ13F, Gγ1, or Gαs, but not any other G protein subunit, results in prevention of post-eclosion cell death and failure of the wing expansion. However, cell death prevention alone is not sufficient to induce the expansion defect, suggesting that the failure of epithelial-mesenchymal transition is key to the folded wing phenotypes. Overactivation of Gαs with cholera toxin mimics expression of constitutively activated Gαs and promotes wing blistering due to precocious cell death. In contrast, co-overexpression of Gβ13F and Gγ1 does not produce wing blistering, revealing the passive role of the Gβγ in the Gαs-mediated activation of apoptosis, but hinting at the possible function of Gβγ in the epithelial-mesenchymal transition. Our results provide a comprehensive functional analysis of the heterotrimeric G protein proteome in the late stages of Drosophila wing development.

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Heterotrimeric G-proteins are molecular switches that regulate numerous aspects of cellular physiology by transducing the signals from G protein-coupled receptors (GPCRs). In the basal state, the Gα-subunits of the heterotrimeric G proteins are GDP-liganded (the inactive form) and bind to the βγ-complex. GPCRs can activate guanine nucleotide exchange on the Gα-subunits to produce the active, GTP-bound state. GoLoco domains present in many proteins play important roles in multiple heterotrimeric G protein-dependent activities, physically binding the Gα-subunits of the Gαi/o class. In most cases GoLoco binds exclusively to the GDP-loaded form of the Gα-subunits. Our biochemical and genetic experiments as well as structural modeling show that the poly-GoLoco protein Pins binds to both the GDP-and GTP-forms of Drosophila Gαo. We identify the Pins GoLoco domain 1 as necessary and sufficient for the unusual interaction with Gαo-GTP. We further pinpoint the central Lysine residue present in this domain as responsible for the interaction. Molecular modeling suggests that the side chain of this Lysine points directly into the guanine nucleotide-binding pocket of Gαo, stabilizing the extra negative charges of the γ-phosphate group of GTP. Such a positively charged amino acid is unique in the Drosophila GoLoco proteome, but is conserved in several GoLoco domains of other organisms. We conclude that Pins, through its GoLoco domain 1, is a target for Gαo-mediated GPCR signaling.

An earlier developed purified cell-free system was used to explore the potential of two RNA-directed RNA polymerases (RdRps), Q␤ phage replicase and the poliovirus 3Dpol protein, to promote RNA recombination through a primer extension mechanism. The substrates of recombination were fragments of complementary strands of a Q␤ phage-derived RNA, such that if aligned at complementary 3-termini and extended using one another as a template, they would produce replicable molecules detectable as RNA colonies grown in a Q␤ replicase-containing agarose. The results show that while 3Dpol efficiently extends the aligned fragments to produce the expected homologous recombinant sequences, only nonhomologous recombinants are generated by Q␤ replicase at a much lower yield and through a mechanism not involving the extension of RNA primers. It follows that the mechanisms of RNA recombination by poliovirus and Q␤ RdRps are quite different. The data favor an RNA transesterification reaction catalyzed by a conformation acquired by Q␤ replicase during RNA synthesis and provide a likely explanation for the very low frequency of homologous recombination in Q␤ phage.Recombinations (sequence exchange and rearrangements) between and within RNA molecules are rare but biologically important events contributing to the evolution and diversity of RNA viruses (1, 2) and generating defective interfering RNAs that attenuate viral infections (3). In contrast to splicing and other types of regular RNA rearrangements, recombinations occur without apparent sequence or structure specificity (1, 2). There are indications that recombination may occur between cellular RNAs (4 -6), eventually resulting, by means of reverse transcription and integration, in alterations in the chromosomal DNA. Spontaneous Mg 2ϩ -catalyzed rearrangements in RNA sequences (7) might have been a mechanism for evolution in the prebiotic RNA world and might have evolved into contemporary sequencespecific ribozyme-catalyzed reactions (8, 9).RNA recombination was discovered more than 40 years ago as an exchange of genetic markers between polioviruses (10, 11), and since then similar approaches were used to demonstrate that genomes of RNA viruses of animals, plants, and bacteria are all capable of recombination (2, 4, 12, 13). However, such in vivo experiments utilizing living cells, as well as in vitro studies that used crude cell lysates could not uncover the underlying molecular mechanisms or even definitely answer the question if recombining entities were RNA molecules themselves or their cDNA copies, because every living cell contained enzymes capable of reverse transcription and appropriate dNTP substrates. It became evident that further progress in this field depended on the availability of adequate in vitro systems whose composition and other parameters can be strictly controlled by the experimenter (2, 14).The first example of such a sort has been the cell-free system employing purified Q␤ replicase, RNA-directed RNA polymerase (RdRp) 1 of bacteriophage Q␤ (15). The system...

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Drug development requires high-tech, simple, and sensitive methods. AlphaLISA method was announced as a universal method that would fit the listed criteria. However, research of other works on the pharmacokinetics of drugs related to this method showed a small number of pharmacokinetic studies in clinical trials. In this review, we focused on not only the positive aspects of the Alpha method, but also its disadvantages.