No abstract

Human genomic DNA fragments containing catechol 0-methyltransferase (COMT) sequences were isolated and the exon-intron structure analysed by sequencing, PCR and comparing to the human COMT cDNA sequences. The gene contains six exons, of which exons 1 and 2 are noncoding. MB-ATG and S-ATG codons, responsible for the initiation of translation of the membranebound (MB) and soluble (S) forms of the enzyme, are located in exon 3. Two distinct COMTspecific transcripts, 1.3 kb and 1.5 kb, were detected in various human tissues and cell lines. Different quantities of the shorter COMT-specific mRNA in the tissues studied suggest a tissue-specific regulation of the COMT gene at transcriptional level. Mapping of the 5' ends of the COMT mRNAs showed that transcription initiates at multiple sites in two separate DNA regions, which are preceded by functional promoter sequences. The proximal promoter (Pl), located between the two translation initiation codons and extending approximately 200 bp upstream of the MB-ATG initiation codon, apparently gives rise to the 1.3-kb S-COMT mRNA (S-mRNA). The distal promoter (P2) is located in a DNA fragment in front of and partly overlapping the transcription-start region of the 1.5-kb transcript, suggesting that it controls the expression of this MB-mRNA. Similarities between the rat and human COMT gene promoters are analyzed.

Cyclase-associated proteins (CAPs) are highly conserved actin monomer binding proteins present in all eukaryotes. However, the mechanism by which CAPs contribute to actin dynamics has been elusive. In mammals, the situation is further complicated by the presence of two CAP isoforms whose differences have not been characterized. Here, we show that CAP1 is widely expressed in mouse nonmuscle cells, whereas CAP2 is the predominant isoform in developing striated muscles. In cultured NIH3T3 and B16F1 cells, CAP1 is a highly abundant protein that colocalizes with cofilin-1 to dynamic regions of the cortical actin cytoskeleton. Analysis of CAP1 knockdown cells demonstrated that this protein promotes rapid actin filament depolymerization and is important for cell morphology, migration, and endocytosis. Interestingly, depletion of CAP1 leads to an accumulation of cofilin-1 into abnormal cytoplasmic aggregates and to similar cytoskeletal defects to those seen in cofilin-1 knockdown cells, demonstrating that CAP1 is required for proper subcellular localization and function of ADF/cofilin. Together, these data provide the first direct in vivo evidence that CAP promotes rapid actin dynamics in conjunction with ADF/cofilin and is required for several central cellular processes in mammals.

Midbrain GABAergic neurons control several aspects of behavior, but regulation of their development and diversity is poorly understood. Here, we further refine the midbrain regions active in GABAergic neurogenesis and show their correlation with the expression of the transcription factor Gata2. Using tissue-specific inactivation and ectopic expression, we show that Gata2 regulates GABAergic neuron development in the mouse midbrain, but not in rhombomere 1, where it is needed in the serotonergic lineage. Without Gata2, all the precursors in the embryonic midbrain fail to activate GABAergic neuron-specific gene expression and instead switch to a glutamatergic phenotype. Surprisingly, this fate switch is also observed throughout the neonatal midbrain, except for the GABAergic neurons located in the ventral dopaminergic nuclei, suggesting a distinct developmental pathway for these neurons. These studies identify Gata2 as an essential post-mitotic selector gene of the GABAergic neurotransmitter identity and demonstrate developmental heterogeneity of GABAergic neurons in the midbrain.

The WH2 (WASP homology domain-2) is a small actin monomer-binding motif and is found in many proteins that regulate the actin cytoskeleton, including the ␤-thymosins, ciboulot, WASP, and verprolin/WIP (WASPinteracting protein). In sequence database searches we identified a novel mouse protein containing a WH2 domain in its C-terminal region. This mouse gene also shows strong sequence homology to human MIM (Missing in Metastasis), a cDNA fragment that is present in non-metastatic but absent in metastatic bladder cancer cell lines. Northern blot and in situ hybridizations show that MIM is strongly expressed in the developing neurons and skeletal and cardiac muscles in mouse embryos. In adult mice, the strongest expression of MIM mRNA is in liver, outer layers of the kidney, and in the Purkinje cells of the brain. Recombinant MIM protein interacts with actin monomers and inhibits actin filament nucleation in vitro. However, the MIM/ATP-G-actin complex can participate in actin filament assembly at the barbed end. MIM binds ATP-G-actin with a higher affinity (K D ‫؍‬ 0.06 M) than ADP-G-actin (K D ‫؍‬ 0.3 M) and inhibits the nucleotide exchange on actin monomers. Site-directed mutagenesis demonstrates that the actin monomer-binding site resides in the C-terminal WH2 domain of MIM. Overexpression of mouse MIM in NIH 3T3 cells results in the disappearance of actin stress fibers and appearance of abnormal actin filament structures. These data show that MIM is an ATP-G-actin binding protein that regulates cytoskeletal dynamics in specialized mammalian cell-types.The actin cytoskeleton is central in a number of cellular processes such as motility, morphogenesis, cytokinesis, and endocytosis. The structure and dynamics of the actin cytoskeleton are spatially and temporally regulated by a large number of actin-binding proteins, whose own activities and localities are precisely regulated by various signaling pathways (1). Sequence and structural data on actin-binding proteins has revealed that many of these proteins interact with actin through a relatively small number of protein motifs. These include the calponin homology domain, the gelsolin homology domain, the actin-depolymerizing-factor homology domain, and the WASP homology 2 (WH2) 1 domain (for reviews see 2-5). The WH2 domain is a small (ϳ35 residue) protein motif that interacts only with monomeric actin (5, 6). WH2 domains are found in many regulators of actin dynamics, including ␤-thymosins and ciboulot, which bind actin monomers and regulate filament assembly. ␤-thymosins are actin monomer-sequestering proteins, whereas ciboulot promotes actin assembly at the barbed end of the filaments (7,8). WH2 domains are also present in more complex proteins such as WASP/Scar, verprolin/WIP, and Srv2/CAP. These are multifunctional regulators of actin dynamics that link intracellular signaling pathways to actin dynamics (5). For example, WASP and Scar mediate signals from PIP 2 and the small GTPases Cdc42 and Rac to the actin cytoskeleton by inducing actin assembly through acti...