Mastermind-like domain-containing 1 (MAMLD1 or CXorf6) transactivates the Hes3 promoter, augments testosterone production, and contains the SF1 target sequence

Although chromosome X open reading frame 6 (CXorf6) has been shown to be a causative gene for hypospadias, its molecular function remains unknown. To clarify this, we first examined CXorf6 protein structure, identifying homology to mastermind-like 2 (MAML2) protein, which functions as a co-activator in canonical Notch signaling. Transactivation analysis for wild-type CXorf6 protein by luciferase assays showed that CXorf6 significantly transactivated the promoter of a noncanonical Notch target gene hairy/enhancer of split 3 (Hes3) without demonstrable DNA-binding capacity. Transactivation analysis was also performed for the previously described three apparently pathologic nonsense mutations, indicating that E124X and Q197X proteins had no transactivation function, whereas R653X protein retained a nearly normal transactivation function. Subcellular localization analysis revealed that wild-type and R653X proteins co-localized with MAML2 protein in nuclear bodies, whereas E124X and Q197X proteins were incapable of localizing to nuclear bodies. Thus, further studies were performed for R653X, revealing the occurrence of nonsense mediated mRNA decay in vivo. Next, transient knockdown of CXorf6 was performed using small interfering RNA, showing reduced testosterone production in mouse Leydig tumor cells. Furthermore, steroidogenic factor 1 (SF1) protein bound to a specific sequence in the upstream of the CXorf6 coding region and exerted a transactivation activity. These results suggest that CXorf6 transactivates the Hes3 promoter, augments testosterone production, and contains the SF1 target sequence, thereby providing the first clue to clarify the biological role of CXorf6. We designate CXorf6 as MAMLD1 (mastermind-like domain-containing 1) based on its characteristic structure.     

Rab5 association with the angiotensin II type 1A receptor promotes Rab5 GTP binding and vesicular fusion.

Previous studies have demonstrated that the internalization of the angiotensin II type 1A receptor (AT(1A)R) may be mediated by both beta-arrestin-sensitive and -insensitive mechanisms. Therefore, we have used the AT(1A)R carboxyl-terminal tail to screen a rat brain yeast two-hybrid expression library for novel AT(1A)R-interacting proteins that might contribute to the regulation of AT(1A)R internalization. We have identified Rab5a as an AT(1A)R-binding protein that selectively associates with the AT(1A)R and not with the beta2-adrenergic receptor. A Rab5a-S34N mutant defective in GTP binding does not prevent the internalization of the AT(1A)R but does prevent the trafficking of the AT(1A)R into larger hollow cored vesicular structures. Agonist activation of the AT(1A)R promotes both the formation of Rab5a.AT(1A)R protein complexes and Rab5a GTP binding. Rab5a interactions with the AT(1A)R are mediated in part by the last 10 amino acid residues of the AT(1A)R carboxyl-terminal tail, and although a mutant receptor lacking these residues internalizes normally, it does not redistribute into larger hollow vesicles. Our data suggest that AT(1A)R activation modulates Rab5a activity leading to the homotypic fusion of endocytic vesicles. These observations suggest that vesicular cargo proteins, such as the AT(1A)R, may control their targeting between intracellular compartments by directly regulating the activity of components of the intracellular trafficking machinery such as Rab5a.     

Visualization of comparative genomic analyses by BLAST score ratio

Background  

The first microbial genome sequence, Haemophilus influenzae, was published in 1995. Since then, more than 400 microbial genome sequences have been completed or commenced. This massive influx of data provides the opportunity to obtain biological insights through comparative genomics. However few tools are available for this scale of comparative analysis.     

GeneKeyDB: A lightweight,gene-centric,relational database to support data mining environments

Background  

The analysis of biological data is greatly enhanced by existing or emerging databases. Most existing databases, with few exceptions are not designed to easily support large scale computational analysis, but rather offer exclusively a web interface to the resource. We have recognized the growing need for a database which can be used successfully as a backend to computational analysis tools and pipelines. Such database should be sufficiently versatile to allow easy system integration.     

c-Src regulates clathrin adapter protein 2 interaction with beta-arrestin and the angiotensin II type 1 receptor during clathrin- mediated internalization

Beta-arrestins are multifunctional adapters involved in the internalization and signaling of G protein-coupled receptors (GPCRs). They target receptors to clathrin-coated pits (CCPs) through binding with clathrin and clathrin adapter 2 (AP-2) complex. They also act as transducers of signaling by recruiting c-Src kinase to certain GPCRs. Here we sought to determine whether c-Src regulates the recruitment of AP-2 to beta-arrestin and the angiotensin II (Ang II) type 1 receptor (AT1R) during internalization. We show that the agonist stimulation of native AT1R in vascular smooth muscle cells (VSMCs) induces the formation of an endogenous complex containing c-Src, beta-arrestins and AP-2. In vitro studies using coimmunoprecipitation experiments and a yeast three-hybrid assay reveal that c-Src stabilizes the agonist-independent association between beta-arrestin2 and the beta-subunit of AP-2 independently of the kinase activity of c-Src. However, although c-Src expression promoted the rapid dissociation of AP-2 from both beta-arrestin and AT1R after receptor stimulation, a kinase-inactive mutant of c-Src failed to induce the dissociation of AP-2 from the agonist-occupied receptor. Thus, the consequence of c-Src in regulating the dissociation of AP-2 from the receptor was also examined on the internalization of AT1R by depleting c-Src in human embryonic kidney (HEK) 293 cells using a small interfering RNA strategy. Experiments in c-Src depleted cells reveal that AT1R remained mostly colocalized with AP-2 at the plasma membrane after Ang II stimulation, consistent with the observed delay in receptor internalization. Moreover, coimmunoprecipitation experiments in c-Src depleted HEK 293 cells and VSMCs showed an increased association of AP-2 to the agonist-occupied AT1R and beta-arrestin, respectively. Together, our results support a role for c-Src in regulating the dissociation of AP-2 from agonist-occupied AT1R and beta-arrestin during the clathrin-mediated internalization of receptors and suggest a novel function for c-Src kinase in the internalization of AT1R.     

Dissociation of beta-arrestin from internalized bradykinin B2 receptor is necessary for receptor recycling and resensitization

Beta-arrestins are multifunctional adaptors that bind agonist-activated G protein-coupled receptors (GPCRs), mediate their desensitization and internalization, and control the rate at which receptors recycle back at the plasma membrane ready for subsequent stimulation. The activation of the bradykinin (BK) type 2 receptor (B2R) results in the rapid desensitization and internalization of the receptor. Little is known, however, about the role of beta-arrestin in regulating the intracellular trafficking and the resensitization of the B2R. Using confocal microscopy, we show that BK stimulation of COS-7 cells expressing B2R induces the colocalization of the agonist-activated receptor with beta-arrestin into endosomes. Fluorescent imaging and ligand binding experiments also reveal that upon agonist removal, beta-arrestin rapidly dissociates from B2R into endosomes, and that receptors return back to the plasma membrane, fully competent for reactivating B2R signaling as measured by NO production upon a second BK challenge. However, when the receptor is mutated in its C-terminal domain to increase its avidity for beta-arrestin, B2R remains associated with beta-arrestin into endosomes, and receptors fail to recycle to the plasma membrane postagonist wash. Similarly, the recycling of receptors is prevented when a beta-arrestin mutant exhibiting increased avidity for agonist-bound GPCRs is expressed with B2R. Stabilizing receptor/beta-arrestin complexes into endosomes results in the dampening of the BK-mediated NO production. These results provide evidence for the involvement of beta-arrestin in the intracellular trafficking of B2R, and highlight the importance of receptor recycling in reestablishing B2R signaling.     

Phosphoinositide 3-kinase regulates beta2-adrenergic receptor endocytosis by AP-2 recruitment to the receptor/beta-arrestin complex

Internalization of beta-adrenergic receptors (betaARs) occurs by the sequential binding of beta-arrestin, the clathrin adaptor AP-2, and clathrin. D-3 phosphoinositides, generated by the action of phosphoinositide 3-kinase (PI3K) may regulate the endocytic process; however, the precise molecular mechanism is unknown. Here we demonstrate that betaARKinase1 directly interacts with the PIK domain of PI3K to form a cytosolic complex. Overexpression of the PIK domain displaces endogenous PI3K from betaARK1 and prevents betaARK1-mediated translocation of PI3K to activated beta2ARs. Furthermore, disruption of the betaARK1/PI3K interaction inhibits agonist-stimulated AP-2 adaptor protein recruitment to the beta2AR and receptor endocytosis without affecting the internalization of other clathrin dependent processes such as internalization of the transferrin receptor. In contrast, AP-2 recruitment is enhanced in the presence of D-3 phospholipids, and receptor internalization is blocked in presence of the specific phosphatidylinositol-3,4,5-trisphosphate lipid phosphatase PTEN. These findings provide a molecular mechanism for the agonist-dependent recruitment of PI3K to betaARs, and support a role for the localized generation of D-3 phosphoinositides in regulating the recruitment of the receptor/cargo to clathrin-coated pits.     

Expression of myotubularin by an adenoviral vector demonstrates its function as a phosphatidylinositol 3-phosphate [PtdIns(3)P] phosphatase in muscle cell lines: involvement of PtdIns(3)P in insulin-stimulated glucose transport

X-linked myotubular myopathy is a muscle disorder caused by mutations on the myotubular myopathy-1 (MTM-1) gene, coding for myotubularin a 65-kDa polypeptide similar to protein phosphatases. Biochemical and in vivo studies define myotubularin as a phosphatidylinositol 3-phosphate [PtdIns(3)P] phosphatase. To efficiently express myotubularin in muscle cell lines and adipocytes, we used an adenoviral genome recombinogenic to pcDNA3, and to other widely used expression vectors, to produce adenoviruses expressing wild-type (wt), catalytically inactive C375S, and substrate trap D278A myotubularin.[32P]Orthophosphate labeling followed by phosphoinositide analysis of differentiated L6 and C2C12 cells expressing myotubularin demonstrated increased PtdIns(3)P levels upon expression of the C375S and D278A mutants. In keeping with its biochemical function, overexpression of wt myotubularin as an enhanced green fluorescent protein fusion disrupted the endosomal punctuated staining of the FYVE (Fab1p/YOTB Vac1p/EEA1)-domain-containing PtdIns(3)P binding protein early endosomal antigen 1 as well as of a gluathione-S-transferase-FYVE probe directed to PtdIns(3)P. Expression of wt myotubularin, although not affecting activation of proximal insulin signal transduction targets such as protein kinase B and MAPK, induced a decrease in insulin-induced glucose uptake, whereas basal glucose uptake was augmented by expression of D278A (DA) and C375S (CS) mutants. Moreover, overexpression of myotubularin in 3T3-L1 adipocytes impaired insulin-induced translocation at the plasma membrane of green fluorescent protein-tagged glucose transporter 4. These data indicate that PtdIns(3)P is required to direct glucose transporter 4 to insulin-responsive compartments and/or to allow the translocation of the latter at the plasma membrane.We conclude that myotubularin, by modulating the intracellular levels of PtdIns(3)P, plays a role in the control of vesicular traffic related to glucose transport, by counteracting the activities of the PtdIns(3)P-producing phosphatidylinositol 3-kinases.     

Production of phosphatidylinositol 5-phosphate by the phosphoinositide 3-phosphatase myotubularin in mammalian cells

MTM1, the gene encoding myotubularin (MTM1), is mutated in the X-linked myotubular myopathy (XLMTM), a severe genetic muscular disorder. MTM1 is a phosphoinositide phosphatase hydrolyzing phosphatidylinositol 3-phosphate (PtdIns(3)P) in yeast and in vitro. Because this lipid is implicated in the regulation of vesicular trafficking, we used established cell lines from XLMTM patients to evaluate whether the lack of endogenous MTM1 expression could affect PtdIns(3)P labeling patterns. Our results showed that the vesicular trafficking related to early endosomes was not significantly affected in the XLMTM cell lines compared with control cells. However, in addition to PtdIns(3)P, we found that MTM1 can hydrolyze phosphatidylinositol 3,5-bisphosphate both in vitro and in mammalian cells. Using a mass assay, we demonstrated that the product generated is phosphatidylinositol 5-phosphate (PtdIns(5)P), a recently discovered phosphoinositide, the function of which is still unknown. In L6 myotubes overexpressing MTM1, hyperosmotic shock induced an increase in the mass level of PtdIns(5)P that was reduced by 50% upon overexpression of the MTM1 inactive mutant D278A. These data demonstrate for the first time a role for MTM1 in the production of PtdIns(5)P in mammalian cells, suggesting that the lack of transformation of phosphatidylinositol 3,5-bisphosphate into PtdIns(5)P might be an important component in the etiology of myotubular myopathy.