Kir4.1 channels were found to colocalize with the H+/K+-ATPase throughout the parietal cell (PC) acid secretory cycle. This study was undertaken to explore their functional role. Acid secretory rates, electrophysiological parameters, PC ultrastructure, and gene and protein expression were determined in gastric mucosae of 7–8-day-old Kir4.1-deficient mice and WT littermates. Kir4.1−/− mucosa secreted significantly more acid and initiated secretion significantly faster than WT mucosa. No change in PC number but a relative up-regulation of H+/K+-ATPase gene and protein expression (but not of other PC ion transporters) was observed. Electron microscopy revealed fully fused canalicular membranes and a lack of tubulovesicles in resting state Kir4.1−/− PCs, suggesting that Kir4.1 ablation may also interfere with tubulovesicle endocytosis. The role of this inward rectifier in the PC apical membrane may therefore be to balance between K+ loss via KCNQ1/KCNE2 and K+ reabsorption by the slow turnover of the H+/K+-ATPase, with consequences for K+ reabsorption, inhibition of acid secretion, and membrane recycling. Our results demonstrate that Kir4.1 channels are involved in the control of acid secretion and suggest that they may also affect secretory membrane recycling.
The molecular mechanisms underlying the endoplasmic reticulum (ER) export and cell surface transport of nascent G protein-coupled receptors (GPCRs) have just begun to be revealed and previous studies have shown that hydrophobic motifs in the putative amphipathic 8th α-helical region within the membrane-proximal C termini play an important role. In this study, we demonstrate that di-acidic motifs in the membrane-distal, nonstructural C-terminal portions are required for the exit from the ER and transport to the plasma membrane of angiotensin II receptors, but not adrenergic receptors. More interestingly, distinct di-acidic motifs dictate optimal export trafficking of different angiotensin II receptors and export ability of each acidic residue in the di-acidic motifs cannot be fully substituted by other acidic residue. Moreover, the function of the di-acidic motifs is likely mediated through facilitating the recruitment of the receptors onto the ER-derived COPII transport vesicles. Therefore, the di-acidic motifs located in the membrane-distal C termini may represent the first linear motifs which recruit selective GPCRs onto the COPII vesicles to control their export from the ER.
When deprived of anchorage to the extracellular matrix, fibroblasts arrest in G1 phase at least in part due to inactivation of G1 cyclin-dependent kinases. Despite great effort, how anchorage signals control the G1-S transition of fibroblasts remains highly elusive. We recently found that the mammalian target of rapamycin (mTOR) cascade might convey an anchorage signal that regulates S phase entry. Here, we show that Rho-associated kinase connects this signal to the TSC1/TSC2-RHEB-mTOR pathway. Expression of a constitutively active form of ROCK1 suppressed all of the anchorage deprivation effects suppressible by tsc2 mutation in rat embryonic fibroblasts. TSC2 contains one evolutionarily conserved ROCK target-like sequence, and an alanine substitution for Thr1203 in this sequence severely impaired the ability of ROCK1 to counteract the anchorage loss-imposed down-regulation of both G1 cell cycle factors and mTORC1 activity. Moreover, TSC2 Thr1203 underwent ROCK-dependent phosphorylation in vivo and could be phosphorylated by bacterially expressed active ROCK1 in vitro, providing biochemical evidence for a direct physical interaction between ROCK and TSC2.
The neuregulins (NRGs) play important roles in animal development and homeostasis, and their deregulation has been linked to diseases such as cancer and schizophrenia. The NRGs belong to the epidermal growth factor (EGF) family of transmembrane growth factors. Although NRGs may be synthesized as transmembrane proteins (the pro-NRGs), some of them lack an N-terminal signal sequence, raising the question of how these pro-NRGs are directed to the plasma membrane. Here we have explored the domains of pro-NRGs that are required for their membrane anchoring, cell surface exposure, and biological activity. We show that an internal hydrophobic region acts as a membrane-anchoring domain, but other regions of pro-NRG are required for proper sorting to the plasma membrane. Using mutants that are located in different subcellular compartments, we show that only plasma membrane-exposed pro-NRG is biologically active. At this location, the pro-NRGs may act as transautocrine molecules (i.e. as membrane factors able to activate receptors present in cells that are in physical contact with the pro-NRG-producing cells (in trans) or capable of activating receptors present in the pro-NRG-producing cells (in cis)).
The neural cell adhesion molecule (NCAM) was recently shown to be involved in the progression of various tumors with diverse effects. We previously demonstrated that NCAM potentiates the cellular invasion and metastasis of melanoma. Here we further report that the growth of melanoma is obviously retarded when the expression of NCAM is silenced. We found that the proliferation of murine B16F0 melanoma cells, their colony formation on soft agar, and growth of transplanted melanoma in vivo are clearly inhibited by the introduction of NCAM siRNA. Interestingly, change of NCAM expression level is shown to regulate the activity of Wnt signaling molecule, β-catenin, markedly. This novel machinery requires the function of FGF receptor and glycogen synthase kinase-3β but is independent of the Wnt receptors, MAPK-Erk and PI3K/Akt pathways. In addition, NCAM is found to form a functional complex with β-catenin, FGF receptor, and glycogen synthase kinase-3β. Moreover, up-regulation of NCAM140 and NCAM180 appears more potent than NCAM120 in activation of β-catenin, suggesting that the intracellular domain of NCAM is required for facilitating the β-catenin signaling. Furthermore, the melanoma cells also exhibit distinct differentiation phenotypes with the NCAM silencing. Our findings reveal a novel regulatory role of NCAM in the progression of melanoma that might serve as a new therapeutic target for the treatment of melanoma.
Activation of the intrinsic apoptotic pathway represents a major mechanism for breast cancer regression resulting from anti-estrogen therapy. The BH3-only protein BIK is inducible by estrogen-starvation and anti-estrogen treatment and plays an important role in anti-estrogen induced apoptosis of breast cancer cells. BIK is predominantly localized to the endoplasmic reticulum where it regulates BAX/BAK-dependent release of Ca2+ from the endoplasmic reticulum stores and cooperates with other BH3-only proteins such as NOXA to cause rapid release of cytochrome c from mitochondria and activate apoptosis. BIK is also known to inactivate BCL-2 through complex formation. Previously, we demonstrated that apoptosis triggered by BIK in estrogen-starved human breast cancer cells is suppressed by GRP78, a major endoplasmic reticulum chaperone. Here we described the isolation of a novel clonal human breast cancer cell line (MCF-7/BUS-10) resistant to long-term estrogen deprivation. These cells exhibit elevated level of GRP78, which protects them from estrogen starvation-induced apoptosis. Our studies revealed that overexpression of GRP78 suppresses apoptosis induced by BIK and NOXA, either alone or in combination. Surprisingly, the interaction of GRP78 with BIK does not require its BH3 domain...
The retinoic acid-related orphan nuclear receptor γt (RORγt)/RORγ2 is well known as a master regulator of interleukin 17 (IL-17)-producing helper T (Th17) cell development. To develop a therapeutic agent against Th17-mediated autoimmune diseases, we screened chemical compounds and successfully found that digoxin inhibited IL-17 production. Further studies revealed that digoxin bound to the ligand binding domain of RORγt and suppressed Th17 differentiation without affecting Th1 differentiation. To better understand the structural basis for the inhibitory activity of digoxin, we determined the crystal structure of the RORγt ligand-binding domain in complex with digoxin at 2.2 Å resolution. The structure reveals that digoxin binds to the ligand-binding pocket protruding between helices H3 and H11 from the pocket. In addition, digoxin disrupts the key interaction important for the agonistic activity, resulting in preventing the positioning of helix H12 in the active conformation, thus antagonizing coactivator interaction. Functional studies demonstrated that digoxin inhibited RORγt activity and decreased IL-17 production but not RORα activity. Digoxin inhibited IL-17 production in CD4+ T cells from experimental autoimmune encephalomyelitis mice. Our data indicates that RORγt is a promising therapeutic target for Th17-derived autoimmune diseases and our structural data will help to design novel RORγt antagonists.
p21Waf1/Cip1 protein levels respond to DNA damage; p21 is induced after ionizing radiation, but degraded after UV. p21 degradation after UV is necessary for optimal DNA repair, presumably because p21 inhibits nucleotide excision repair by blocking proliferating cell nuclear antigen (PCNA). Because p21 also inhibits DNA mismatch repair (MMR), we investigated how p21 levels respond to DNA alkylation by N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), which triggers the MMR system. We show that MNNG caused rapid degradation of p21, and this involved the ubiquitin ligase Cdt2 and the proteasome. p21 degradation further required MSH2 but not MLH1. p21 mutants that cannot bind PCNA or cannot be ubiquitinated were resistant to MNNG. MNNG induced the formation of PCNA complexes with MSH6 and Cdt2. Finally, when p21 degradation was blocked, MNNG treatment resulted in reduced recruitment of MMR proteins to chromatin. This study describes a novel pathway that removes p21 to allow cells to efficiently activate the MMR system.
We investigated the role of a Ca2+ channel and intracellular calcium concentration ([Ca2+]i) in osmotic stress-induced JNK activation and tight junction disruption in Caco-2 cell monolayers. Osmotic stress-induced tight junction disruption was attenuated by 1,2-bis(2-aminophenoxyl)ethane-N,N,N′,N′-tetraacetic acid (BAPTA)-mediated intracellular Ca2+ depletion. Depletion of extracellular Ca2+ at the apical surface, but not basolateral surface, also prevented tight junction disruption. Similarly, thapsigargin-mediated endoplasmic reticulum (ER) Ca2+ depletion attenuated tight junction disruption. Thapsigargin or extracellular Ca2+ depletion partially reduced osmotic stress-induced rise in [Ca2+]i, whereas thapsigargin and extracellular Ca2+ depletion together resulted in almost complete loss of rise in [Ca2+]i. L-type Ca2+ channel blockers (isradipine and diltiazem) or knockdown of the CaV1.3 channel abrogated [Ca2+]i rise and disruption of tight junction. Osmotic stress-induced JNK2 activation was abolished by BAPTA and isradipine, and partially reduced by extracellular Ca2+ depletion, thapsigargin, or CaV1.3 knockdown. Osmotic stress rapidly induced c-Src activation, which was significantly attenuated by BAPTA, isradipine, or extracellular Ca2+ depletion. Tight junction disruption by osmotic stress was blocked by tyrosine kinase inhibitors (genistein and PP2) or siRNA-mediated knockdown of c-Src. Osmotic stress induced a robust increase in tyrosine phosphorylation of occludin...