Stimulation of premature retinoic acid synthesis in Xenopus embryos following premature expression of aldehyde dehydrogenase ALDH1. (49/8290)

In order for nuclear retinoic acid receptors to mediate retinoid signaling, the ligand retinoic acid must first be produced from its vitamin A precursor retinal. Biochemical studies have shown that retinal can be metabolized in vitro to retinoic acid by members of the aldehyde dehydrogenase enzyme family, including ALDH1. Here we describe the first direct evidence that ALDH1 plays a physiological role in retinoic acid synthesis by analysis of retinoid signaling in Xenopus embryos, which have plentiful stores of maternally derived retinal. The Xenopus ALDH1 gene was cloned and shown to be highly conserved with chick and mammalian homologs. Xenopus ALDH1 was not expressed at blastula and gastrula stages, but was expressed at the neurula stage. We used a retinoic acid bioassay to demonstrate that retinoic acid is normally undetectable in embryos from fertilization to the initial gastrula stage, but that a tremendous increase in retinoic acid occurs during neurulation when ALDH1 is first expressed. Overexpression of ALDH1 by injection of Xenopus embryos with mRNAs encoding the mouse, chick or Xenopus ALDH1 homologs induced high levels of retinoic acid detection during the blastula stage. Thus, premature expression of ALDH1 stimulates premature synthesis of retinoic acid. These findings reveal an important conserved role for ALDH1 in retinoic acid synthesis in vivo, and demonstrate that conversion of retinoids from the aldehyde form to the carboxylic acid form is a crucial regulatory step in retinoid signaling.  (+info)

Regulation of beta-catenin signaling by the B56 subunit of protein phosphatase 2A. (50/8290)

Dysregulation of Wnt-beta-catenin signaling disrupts axis formation in vertebrate embryos and underlies multiple human malignancies. The adenomatous polyposis coli (APC) protein, axin, and glycogen synthase kinase 3beta form a Wnt-regulated signaling complex that mediates the phosphorylation-dependent degradation of beta-catenin. A protein phosphatase 2A (PP2A) regulatory subunit, B56, interacted with APC in the yeast two-hybrid system. Expression of B56 reduced the abundance of beta-catenin and inhibited transcription of beta-catenin target genes in mammalian cells and Xenopus embryo explants. The B56-dependent decrease in beta-catenin was blocked by oncogenic mutations in beta-catenin or APC, and by proteasome inhibitors. B56 may direct PP2A to dephosphorylate specific components of the APC-dependent signaling complex and thereby inhibit Wnt signaling.  (+info)

A heptad motif of leucine residues found in membrane proteins can drive self-assembly of artificial transmembrane segments. (51/8290)

Specific interactions between alpha-helical transmembrane segments are important for folding and/or oligomerization of membrane proteins. Previously, we have shown that most transmembrane helix-helix interfaces of a set of crystallized membrane proteins are structurally equivalent to soluble leucine zipper interaction domains. To establish a simplified model of these membrane-spanning leucine zippers, we studied the homophilic interactions of artificial transmembrane segments using different experimental approaches. Importantly, an oligoleucine, but not an oligoalanine, se- quence efficiently self-assembled in membranes as well as in detergent solution. Self-assembly was maintained when a leucine zipper type of heptad motif consisting of leucine residues was grafted onto an alanine host sequence. Analysis of point mutants or of a random sequence confirmed that the heptad motif of leucines mediates self-recognition of our artificial transmembrane segments. Further, a data base search identified degenerate versions of this leucine motif within transmembrane segments of a variety of functionally different proteins. For several of these natural transmembrane segments, self-interaction was experimentally verified. These results support various lines of previously reported evidence where these transmembrane segments were implicated in the oligomeric assembly of the corresponding proteins.  (+info)

A region in IVS5 of the human cardiac L-type calcium channel is required for the use-dependent block by phenylalkylamines and benzothiazepines. (52/8290)

Mutations in motif IVS5 and IVS6 of the human cardiac calcium channel were made using homologous residues from the rat brain sodium channel 2a. [3H]PN200-110 and allosteric binding assays revealed that the dihydropyridine and benzothiazepine receptor sites maintained normal coupling in the chimeric mutant channels. Whole cell voltage clamp recording from Xenopus oocytes showed a dramatically slowed inactivation and a complete loss of use-dependent block for mutations in the cytoplasmic connecting link to IVS5 (HHT-5371) and in IVS5 transmembrane segment (HHT-5411) with both diltiazem and verapamil. However, the use-dependent block by isradipine was retained by these two mutants. For mutants HHT-5411 and HHT-5371, the residual current appeared associated with a loss of voltage dependence in the rate of inactivation indicating a destabilization of the inactivated state. Furthermore, both HHT-5371 and -5411 recovered from inactivation significantly faster after drug block than that of the wild type channel. Our data demonstrate that accelerated recovery of HHT-5371 and HHT-5411 decreased accumulation of these channels in inactivation during pulse trains and suggest a close link between inactivation gating of the channel and use-dependent block by phenylalkylamines and benzothiazepines and provide evidence of a role for the transmembrane and cytoplasmic regions of IVS5 in the use-dependent block by diltiazem and verapamil.  (+info)

Identification of two amino acids in activin A that are important for biological activity and binding to the activin type II receptors. (53/8290)

Activins are members of the transforming growth factor-beta family of growth and differentiation factors. In this paper, we report the results of a structure-function analysis of activin A. The primary targets for directed mutagenesis were charged, individual amino acids located in accessible domains of the protein, concentrating on those that differ from transforming growth factor-beta2, the x-ray crystal structure of which is known. Based on the activities of the recombinant activin mutants in two bioassays, 4 out of 39 mutant proteins (D27K, K102A, K102E, and K102R) produced in a vaccinia virus system were selected for further investigation. After production in insect cells and purification of these four mutants to homogeneity, they were studied in bioassays and in cross-linking experiments involving transfected receptor combinations. Mutant D27K has a 2-fold higher specific bio-activity and binding affinity to an ActRIIA/ALK-4 activin receptor complex than wild type activin, whereas mutant K102E had no detectable biological activity and did not bind to any of the activin receptors. Mutant K102R and wild type activin bound to all the activin receptor combinations tested and were equipotent in bioassays. Our results with the Lys-102 mutants indicate that the positive charge of amino acid 102 is important for biological activity and type II receptor binding of activins.  (+info)

Cloning of a novel four repeat protein related to voltage-gated sodium and calcium channels. (54/8290)

Cloning has led to the discovery of more ion channels than predicted by functional studies, yet there remain channels that have not been cloned. We report the cloning of a novel protein that contains the four domain structure found in voltage-gated Ca2+ and Na+ channels. Phylogenetic relationships suggested that the protein might have diverged from an ancestral four repeat channel before the divergence of Ca2+ and Na+ channels. Northern blot analysis showed that mRNA transcripts encoding the protein are expressed predominantly in the brain, moderately in the heart, and weakly in the pancreas. Despite extensive expression attempts, currents from the putative channel were not detected. Based on its sequence, we propose that the novel protein might be a voltage-activated cation channel with unique gating properties.  (+info)

Restoration of lectin activity to an inactive abrin B chain by substitution and mutation of the 2 gamma subdomain. (55/8290)

Abrin is a heterodimeric plant protein that occurs in several isoforms (abrin-a, abrin-b, abrin-c and abrin-d), whose B chains are believed to either have (abrin-a and abrin-d) or lack (abrin-b and abrin-c) the ability to bind galactose. The 5' signal sequence and toxin B chain (ATB)-coding region were excised from a preproabrin cDNA [K. A. Wood, J. M. Lord, E. J. Wawrzynczak, and M. Piatak (1991) Eur. J. Biochem. 198, 723-732], tentatively identified as abrin-c, which was predicted to lack lectin activity, and fused in-frame to generate pre-ATB cDNA. Transcripts, synthesized in vitro from pre-ATB cloned into the transcription vector pSP64T, were expressed after microinjection into Xenopus oocytes. The recombinant ATB was shown, using a qualitative sugar-binding assay, to be devoid of lectin activity. Lectin activity could not be restored to this nonbinding ATB by replacing the 2 gamma subdomain with the corresponding galactose-binding 2 gamma subdomain from ricin B chain, but it was restored by replacement with the active galactose-binding 2 gamma subdomain from a different abrin isoform (abrin-a). The putative galactose-binding pocket of the nonbinding ATB 2 gamma subdomain contained a His residue at the position occupied by a residue with an aromatic side chain (Tyr or Trp) in functional 2 gamma subdomains. Mutationally converting this His to either Tyr or Trp restored lectin activity to the nonbinding ATB, emphasizing the contribution of an aromatic side chain in a functional 2 gamma subdomain galactose-binding site for members of this lectin family.  (+info)

A Meis family protein caudalizes neural cell fates in Xenopus. (56/8290)

A homologue of the Drosophila homothorax (hth) gene, Xenopus Meis3 (XMeis3), was cloned from Xenopus laevis. XMeis3 is expressed in a single stripe of cells in the early neural plate stage. By late neurula, the gene is expressed predominantly in rhombomeres two, three and four, and in the anterior spinal cord. Ectopic expression of RNA encoding XMeis3 protein causes anterior neural truncations with a concomitant expansion of hindbrain and spinal cord. Ectopic XMeis3 expression inhibits anterior neural induction in neuralized animal cap ectoderm explants without perturbing induction of pan-neural markers. In naive animal cap ectoderm, ectopic XMeis3 expression activates transcription of the posteriorly expressed neural markers, but not pan-neural markers. These results suggest that caudalizing proteins, such as XMeis3, can alter A-P patterning in the nervous system in the absence of neural induction. Regionally expressed proteins like XMeis3 could be required to overcome anterior signals and to specify posterior cell fates along the A-P axis.  (+info)