Drosophila sine oculis and eyes absent genes synergize in compound-eye formation. The murine homologues of these genes, Six and Eya, respectively, show overlapping expression patterns during development. We hypothesized that Six and Eya proteins cooperate to regulate their target genes. Cotransfection assays were performed with various combinations of Six and Eya to assess their effects on a potential natural target, myogenin promoter, and on a synthetic promoter, the thymidine kinase gene promoter fused to multimerized Six4 binding sites. A clear synergistic activation of these promoters was observed in certain combinations of Six and Eya. To investigate the molecular basis for the cooperation, we first examined the intracellular distribution of Six and Eya proteins in transfected COS7 cells. Coexpression of Six2, Six4, or Six5 induced nuclear translocation of Eya1, Eya2, and Eya3, which were otherwise distributed in the cytoplasm. In contrast, coexpression of Six3 did not result in nuclear localization of any Eya proteins. Six and Eya proteins were coimmunoprecipitated from nuclear extracts prepared from cotransfected COS7 cells and from rat liver. Six domain and homeodomain, two evolutionarily conserved domains among various Six proteins, were necessary and sufficient for the nuclear translocation of Eya. In contrast, the Eya domain, a conserved domain among Eya proteins, was not sufficient for the translocation. A specific interaction between the Six domain and homeodomain of Six4 and Eya2 was observed by yeast two-hybrid analysis. Our results suggest that transcription regulation of certain target genes by Six proteins requires cooperative interaction with Eya proteins: complex formation through direct interaction and nuclear translocation of Eya proteins. This implies that the synergistic action of Six and Eya is conserved in the mouse and is mediated through cooperative activation of their target genes.Six genes are mouse homologues of the Drosophila sine oculis (so) gene, which is essential for compound-eye formation (9, 31). Six members of the Six family of genes have so far been identified in the mouse (17,18,27,28,35). Each Six gene shows a specific expression pattern during development of the mouse embryo. Six1 and Six2 show expression in mesenchymal cells around E8.5 to E10.5 and in muscles and limb tendons in later stages (28). Six3 is expressed in the rostral forebrain in earlier stages and is confined to the prospective eye region (27). Six4 proteins are distributed in the peripheral region of the mantle layer of the developing brain and spinal cord and in various ganglia between E9.5 and E14.5 (25). Six5 mRNA is expressed as early as E7 and is abundantly expressed in neonatal heart and skeletal muscles (24). Human SIX5 resides downstream of a CTG repeat, whose expansion leads to myotonic dystrophy (DM) (7). Since SIX5 is expressed in several tissues affected by DM and the transcription of SIX5 is repressed by the causative a CTG repeat expansion, it has been proposed that SIX5 is involved in som...

Heterotrimeric GTP-binding proteins (G proteins) transmit extracellular stimuli perceived by G protein-coupled receptors (GPCRs) to intracellular signaling cascades. Hundreds of GPCRs exist in humans and are the targets of a large percentage of the pharmaceutical drugs used today. Because G proteins are regulated by GPCRs, small molecules that directly modulate G proteins have the potential to become therapeutic agents. However, strategies to develop modulators have been hampered by a lack of structural knowledge of targeting sites for specific modulator binding. Here we present the mechanism of action of the cyclic depsipeptide YM-254890, which is a recently discovered G q -selective inhibitor. YM-254890 specifically inhibits the GDP/GTP exchange reaction of α subunit of G q protein (Gα q ) by inhibiting the GDP release from Gα q . X-ray crystal structure analysis of the Gα q βγ–YM-254890 complex shows that YM-254890 binds the hydrophobic cleft between two interdomain linkers connecting the GTPase and helical domains of the Gα q . The binding stabilizes an inactive GDP-bound form through direct interactions with switch I and impairs the linker flexibility. Our studies provide a novel targeting site for the development of small molecules that selectively inhibit each Gα subunit and an insight into the molecular mechanism of G protein activation.

Previous studies have reported that ST2 is preferentially expressed on Th2 cells and plays a critical part in controlling airway inflammation in murine models of asthma. However, the clinical role of ST2 in patients with bronchial asthma remains unclear. In our study, we examined 56 patients with atopic asthma in a nonattack phase and 200 nonatopic normal volunteers for healthy control, and analyzed the relationship of their serum ST2 levels to asthma severity, pulmonary function, and laboratory data. Of the 56 patients with atopic asthma, 30 exhibited asthmatic exacerbation, and their serum ST2 levels were also analyzed. The serum ST2 levels were low, but a statistical difference was found between patients with nonattack asthma and the healthy control group (p < 0.05). We also found a differential rise of serum ST2 level that correlates well with the severity of asthma exacerbation. Furthermore, the serum ST2 levels during asthma exacerbation statistically correlated with the percentage of predicted peak expiratory flow (r = -0.634, p = 0.004) and Pa(CO(2)) (r = 0.516, p = 0.003). These results suggest that soluble human ST2 protein in sera may be related to Th2-mediated allergic inflammation inducing acute exacerbation in patients with atopic asthma.

In the developing forebrain, the migration and positioning of neural progenitor cells (NPCs) are regulated coordinately by various molecules. Mutation of these molecules, therefore, causes cortical malformation. GPR56 has been reported as a cortical malformation-related gene that is mutated in patients with bilateral frontoparietal polymicrogyria. GPR56 encodes an orphan G protein-coupled receptor, and its mutations reduce the cell surface expression. It has also been reported that the expression level of GPR56 is involved in cancer cell adhesion and metastasis. However, it remains to be clarified how GPR56 functions in brain development and which signaling pathways are activated by GPR56. In this study, we showed that GPR56 is highly expressed in NPCs and has the ability to inhibit NPC migration. We found that GPR56 coupled with G␣ 12/13 and induced Rho-dependent activation of the transcription mediated through a serum-responsive element and NF-B-responsive element and actin fiber reorganization. The transcriptional activation and actin reorganization were inhibited by an RGS domain of the p115 Rho-specific guanine nucleotide exchange factor (p115 RhoGEF RGS) and dominant negative form of Rho. Moreover, we have demonstrated that a functional anti-GPR56 antibody, which has an agonistic activity, inhibited NPC migration. This inhibition was attenuated by p115 RhoGEF RGS, C3 exoenzyme, and GPR56 knockdown. These results indicate that GPR56 participates in the regulation of NPC movement through the G␣ 12/13 and Rho signaling pathway, suggesting its important role in the development of the central nervous system.

The ST2 gene encodes receptor-like molecules that are very similar to the type I interleukin-1 receptor. Two distinct types of the ST2 gene products, ST2 (a soluble secreted form) and ST2L (a transmembrane form) are produced by alternative splicing. Here we demonstrate that the human ST2 gene has two alternative promoters followed by distinct noncoding first exons, which are located more than 8 kb apart and are spliced to the common exon 2 containing the translation initiation site. Within 1001 bp upstream of the transcription initiation site of the cloned distal promoter, there are four GATA-1. The main promoter used for the expression of the ST2 gene in UT-7, a human leukaemic cell line, is distinct from that in TM12, a human fibroblastic cell line. Although UT-7 cells use both distal and proximal promoters, the distal promoter is used dominantly for expression of both ST2 and ST2L mRNA. On the other hand, almost all transcription in TM12 cells starts from the proximal promoter. These results contrast with those of former studies on the rat system, in which ST2 and ST2L mRNA were generated by use of the proximal and distal promoters, respectively. Furthermore, UT-7 cells use multiple transcription initiation sites in both the proximal and distal promoters, whereas the transcription of the ST2 gene in TM12 cells starts at a unique site. Intriguingly, these results suggest that ST2 and ST2L proteins have distinct functions in different cells within different biological systems, such as those of growth control, differentiation and immunological responses.Keywords: immunoglobulin superfamily; interleukin-1 receptor-related protein; orphan receptor; promoter usage; ST2 gene.The ST2 gene, also designated as T1, Fit-1 or DER4, was cloned as one of the primary response genes in the G 0 /G 1 transitional state of BALB/c-3T3 cells [1], a H-ras oncogeneresponsive gene [2], a Fos-responsive gene [3], and a delayed early serum response gene [4]. Subsequently, ST2L cDNA, encoding a membrane-bound protein the extracellular domain of which is almost identical to the ST2 protein, was cloned [5]. The mRNAs of ST2 and ST2L were produced by alternative 3 H splicing of the primary transcript of the ST2 gene [6]. Based on this discovery of the ST2 gene and its expression in fibroblastic cell lines, our previous studies focused on the function of the ST2 gene in growth control.On the other hand, structural analysis of the ST2 cDNA revealed that the ST2 protein was remarkably similar to the members of the immunoglobulin superfamily, especially to the extracellular portion of the mouse interleukin-1 receptor (IL-1R) [1], and the ST2L protein showed a striking overall similarity to the mouse type I interleukin (IL)-1 receptor (IL-1RI) [5]. Furthermore, the genes encoding ST2 and the two IL-1 receptors, IL-1RI and IL-1RII, were tightly linked on mouse chromosome 1 [7] and human chromosome 2 [8]. The human ST2 gene was assigned to chromosome 2q11.2 [9]. However, IL-1a, b, and receptor antagonist did not bind to the ST2L protein, suggesti...