The Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) carries 1 molecule of Na(+) and K(+) along with 2 molecules of Cl(-) across the cell membrane. It is expressed in a broad spectrum of tissues and has been implicated in cell volume regulation and in ion transport by secretory epithelial tissue. However, the specific contribution of NKCC1 to the physiology of the various organ systems is largely undefined. We have generated mouse lines carrying either of 2 mutant alleles of the Slc12a2 gene, which encodes this cotransporter: a null allele and a mutation that results in deletion of 72 amino acids of the cytoplasmic domain. Both NKCC1-deficient mouse lines show behavioral abnormalities characteristic of mice with inner ear defects. Male NKCC1-deficient mice are infertile because of defective spermatogenesis, as shown by the absence of spermatozoa in histological sections of their epididymides and the small number of spermatids in their testes. Consistent with this observation, we show that Slc12a2 is expressed in Sertoli cells, pachytene spermatocytes, and round spermatids isolated from wild-type animals. Our results indicate a critical role for NKCC1-mediated ion transport in spermatogenesis and suggest that the cytoplasmic domain of NKCC1 is essential in the normal functioning of this protein.
BRCA1 is a tumor suppressor gene implicated in transcriptional regulation. We have generated cell lines with inducible expression of BRCA1 as a tool to identify downstream targets that may be important mediators of BRCA1 function. Oligonucleotide array-based expression profiling identified 11 previously described interferon regulated genes that were up-regulated following inducible expression of BRCA1. Northern blot analysis revealed that a subset of the identified targets including IRF-7, MxA, and ISG-54 were synergistically up-regulated by BRCA1 in the presence of interferon ␥ (IFN-␥) but not interferons ␣ or . Importantly, IFN-␥-mediated induction of IRF-7 and MxA was attenuated in the BRCA1 mutant cell line HCC1937, an effect that was rescued following reconstitution of exogenous wild type BRCA1 in these cells. Furthermore, reconstituted BRCA1 sensitized HCC1937 cells to IFN-␥-induced apoptotic cell death. This study identifies BRCA1 as a component of the IFN-␥-regulated signaling pathway and suggests that BRCA1 may play a role in the regulation of IFN-␥-mediated apoptosis.BRCA1 encodes a tumor suppressor gene that is mutated in the germline of women with a genetic predisposition to breast and ovarian cancer (1). Germline mutations of BRCA1 are found in half of breast-ovarian cancer pedigrees and in ϳ10% of women with early onset breast cancer, irrespective of family history (2). BRCA1 has been implicated in a number of important cellular functions including DNA damage repair, transcriptional regulation, cell cycle control, and, more recently, ubiquitination (3). The exact mechanism through which BRCA1 inactivation may lead to malignant transformation, however, remains to be defined.A large body of evidence has accumulated over the last few years demonstrating a role for BRCA1 in DNA damage repair and in particular repair of double-strand breaks. It was initially observed that BRCA1 co-localized in nuclear foci during the S phase with RAD51 (4), the mammalian homologue of bacterial recA, which is involved in homologous recombination and the repair of double-strand breaks in DNA following ionizing radiation. Subsequently, it has been shown that BRCA1is also a component of the RAD50-MRE11-NBS1 complex implicated in homologous recombination and nonhomologous end joining in response to DNA damage (5). Consistent with a potential role in the repair of double-strand breaks, treatment of cultured cells with ionizing radiation leads to BRCA1 hyperphosphorylation, an effect that is mediated in part by the ATM (6) and CHIT2 kinases (7). Genetic studies support a role for BRCA1 in the repair of double-strand breaks. Significantly, embryonic stem (ES) cells from BRCA1 knockout mice exhibit a defect in the repair of double-strand breaks by homologous recombination (8). Recently it has been reported that BRCA1 can bind to single-strand DNA in a non-sequence-specific manner with a high affinity for branched DNA structures (9). This has lead to the suggestion that BRCA1 is actively recruited to the sites of stalled replica...
BRCA1 gene is a tumor suppressor for breast and ovarian cancers with the putative role in DNA repair and transcription. To characterize the role of BRCA1 in transcriptional regulation, we analyzed gene expression profiles of mouse embryonic stem cells deficient in BRCA1 using microarray technology. We found that loss of BRCA1 correlated with decreased expression of several groups of genes including stress response genes, cytoskeleton genes, and genes involved in protein synthesis and degradation. Previous study showed that BRCA1 is a transcriptional co-activator of p53 protein; however the majority of p53 target genes remained at the same expression levels in BRCA1 knockout cells as in the wild type cells. The only p53 target gene downregulated with the loss of BRCA1 was 14-3-3, a major G 2 /M checkpoint control gene. Similar to cells with decreased 14-3-3 activity, BRCA1-deficient cells were unable to sustain G 2 /M growth arrest after exposure to ionizing radiation. We find that BRCA1 induction of 14-3-3 requires the presence of wild type p53 and can be regulated by a minimal p53 response element.
A genetic contribution to asthma susceptibility is well recognized, and linkage studies have identified a large number of genes associated with asthma pathogenesis. Recently, a locus encoding a seven-transmembrane protein was shown to be associated with asthma in founder populations. The expression of the protein GPRA (G protein-coupled receptor for asthma susceptibility) in human airway epithelia and smooth muscle, and its increased expression in a mouse model of asthma, suggested that a gain-of-function mutation in this gene increased the disease risk. However, we report here that the development of allergic lung disease in GPRA-deficient mice is unaltered. A possible explanation for this finding became apparent upon reexamination of the expression of this gene. In contrast to initial studies, our analyses failed to detect expression of GPRA in human lung tissue or in mice with allergic lung disease. We identify a single parameter that distinguishes GPRA-deficient and wild-type mice. Whereas the change in airway resistance in response to methacholine was identical in control and GPRA-deficient mice, the mutant animals showed an attenuated response to thromboxane, a cholinergic receptor-dependent bronchoconstricting agent. Together, our studies fail to support a direct contribution of GPRA to asthma pathogenesis. However, our data suggest that GPRA may contribute to the asthmatic phenotype by altering the activity of other pathways, such as neurally mediated mechanisms, that contribute to disease. This interpretation is supported by high levels of GPRA expression in the brain and its recent identification as the neuropeptide S receptor.
Calcium-sensing receptors are present in membranes as dimers that can be reduced to monomers with sufhydryl reagents. All studies were carried out on the human calcium-sensing receptor tagged at the carboxyl terminus with green fluorescent protein (hCaR-GFP) to permit identification and localization of expressed proteins. Truncations containing either the extracellular agonist binding domain plus transmembrane helix 1 (ECD/TMH1-GFP) or the transmembrane domain plus the intracellular carboxyl terminus (TMD/carboxyl terminus-GFP) were used to identify the dimerization domain. ECD/TMH1-GFP was a dimer in the absence of reducing reagents, whereas TMD/carboxyl-terminal GFP was a monomer in the absence or presence of reducing agents, suggesting that dimerization occurs via the ECD. To identify the residue(s) involved in dimerization within the ECD, cysteine 3 serine point mutations were made in residues that are conserved between hCaR and metabotropic glutamate receptors. Mutations at positions 60 and 131 were expressed at levels comparable to wild type in HEK 293 cells, had minimal effects on hCaR function, and did not eliminate dimerization, whereas mutations at positions 101 and 236 greatly decreased receptor expression and resulted in significant amounts of monomer in the absence of reducing agents. The double point mutant hCaR(C101S/C236S)-GFP was expressed more robustly than either C101S or C236S and covalent dimerization was eliminated. hCaR(C101S/ C236S)-GFP had a decreased affinity for extracellular Ca 2؉ and slower response kinetics upon increases or decreases in agonist concentration. These results suggest that covalent, disulfide bond-mediated dimerization of the calcium-sensing receptor contributes to stabilization of the ECD and to acceleration of the transitions between inactive and active receptor conformations.
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