Novel GABAA receptor alpha subunit is expressed only in cerebellar granule cells

GABA (gamma-aminobutyric acid), the major inhibitory neurotransmitter in the vertebrate brain, mediates neuronal inhibition by opening a chloride channel integral to the GABAA receptor. This action is potentiated by both benzodiazepine and barbiturate drugs. Since the isolation of cDNAs encoding GABAA receptor alpha 1 and beta 1 subunits, a further eight subunits have been identified. These subunits show GABAA receptor heterogeneity, unpredicted from classical pharmacological studies. I now report the isolation of a mouse cDNA clone encoding a novel GABAA receptor alpha subunit. The striking feature of this subunit is its regional distribution in the mouse brain. Northern hybridization and in situ hybridization experiments demonstrate that the subunit mRNA is expressed only in cerebellar granule cells. This is the first demonstration of the exclusive presence of a neuroreceptor subtype in a single neuronal cell type.     

Ubiquitylation-dependent negative regulation of WASp is essential for actin cytoskeleton dynamics

The Wiskott-Aldrich syndrome protein (WASp) is a key regulator of actin dynamics during cell motility and adhesion, and mutations in its gene are responsible for Wiskott-Aldrich syndrome (WAS). Here, we demonstrate that WASp is ubiquitylated following T-cell antigen receptor (TCR) activation. WASp phosphorylation at tyrosine 291 results in recruitment of the E3 ligase Cbl-b, which, together with c-Cbl, carries out WASp ubiquitylation. Lysine residues 76 and 81, located at the WASp WH1 domain, which contains the vast majority of WASp gene mutations, serve as the ubiquitylation sites. Disruption of WASp ubiquitylation causes WASp accumulation and alters actin dynamics and the formation of actin-dependent structures. Our data suggest that regulated degradation of activated WASp might be an efficient strategy by which the duration and localization of actin rearrangement and the intensity of T-cell activation are controlled.     

Integrins on eggs: the betaC subunit is essential for formation of the cortical actin cytoskeleton in sea urchin eggs

Eggs of several metazoans have been demonstrated to express integrins; however, their function is unclear. Previous studies have shown that the betaC integrin subunit is expressed on unfertilized sea urchin eggs and proteolytically removed at fertilization. Here we report that the betaC subunit is reexpressed on the egg surface immediately after fertilization. Using morpholino antisense oligonucleotides to block translation, we show that without betaC expression, eggs undergo cleavage resulting in loosely adherent cells that fail to develop beyond a blastula. Without betaC containing integrins, the cortical actin network of the egg does not form, yet contractile rings appear. Coinjection of RNA encoding the betaC or chicken beta1 subunit, but lacking the morpholino target sequence, rescues the cortical actin network and normal embryos result. Coinjection of RNA encoding the betaC subunit lacking the cytoplasmic domain fails to rescue. These studies demonstrate that the cortical actin cytoskeleton is anchored by betaC integrins and contractile ring actin is not. We suggest that one important function of egg integrins is to organize the actin cortex.     

Developmental expression of the GABAA receptor alpha 1 subunit mRNA in the rat brain

Recent studies have suggested that the GABAA, receptor complex, the site of action of the inhibitory neurotransmitter gamma amino-butyric acid (GABAA) and the anxiolytic benzodiazepines, is heterogeneous. Moreover, its composition may change during development. To better understand the molecular basis of receptor heterogeneity, the levels and distribution of the mRNA encoding the alpha 1 receptor subunit were examined in the developing and adult rat brain with quantitative in situ hybridization histochemistry. Our studies demonstrate that alpha 1 subunit mRNA expression changes during ontogeny. At late embryonic stages and in the first postnatal week, low levels of the mRNA were detected in the cortex, inferior colliculus, and hippocampus. The mRNA levels in these regions increased during the second and third postnatal weeks. Furthermore, a dramatic change in the distribution of the alpha 1 subunit mRNA was seen in the second postnatal week when the message first became detectable in the cerebellar cortex. During subsequent development and in the mature brain, the alpha 1 subunit mRNA was most abundant in the cerebellum, olfactory bulb, and inferior colliculus, although the absolute levels of mRNA varied by as much as sixfold in selected brain regions. The mature distribution of alpha 1 subunit mRNA, along with its temporal appearance in the cerebellum, suggests that this subunit is a constituent of the Type 1 benzodiazepine site of the GABAA receptor complex. Furthermore, the onset of alpha 1 subunit mRNA expression in the cerebellar cortex coincides with a period of extensive synapse formation, raising the possibility that synaptic interactions modulate the appearance of this GABAA receptor subunit in the cerebellum.     

Binding of Par-4 to the actin cytoskeleton is essential for Par-4/Dlk-mediated apoptosis

Prostate apoptosis response-4 (Par-4) is a 38-kDa protein originally identified as a gene product upregulated in prostate cancer cells undergoing apoptosis. Cell death mediated by Par-4 and its interaction partner DAP like kinase (Dlk) is characterized by dramatic changes of the cytoskeleton. To uncover the role of the cytoskeleton in Par-4/Dlk-mediated apoptosis, we analyzed Par-4 for a direct association with cytoskeletal structures. Confocal fluorescence microscopy revealed that endogenous Par-4 is specifically associated with stress fibers in rat fibroblasts. In vitro cosedimentation analyses and in vivo FRET analyses showed that Par-4 directly binds to F-actin. Actin binding is mediated by the N-terminal 266 amino acids, but does not require the C-terminal region of Par-4 containing the leucine zipper and the death domain. Furthermore, the interaction of Par-4 with actin filaments leads to the formation of actin bundles in vitro and in vivo. In rat fibroblasts, this microfilament association is essential for the pro-apoptotic function of Par-4, since both disruption of the actin cytoskeleton by cytochalasin D treatment and overexpression of Par-4 constructs impaired in actin binding result in a significant decrease of apoptosis induction by Par-4 and Dlk. We propose a model, in which Par-4 recruits Dlk to stress fibers, leading to enhanced phosphorylation of the regulatory light chain of myosin II (MLC) and to the induction of apoptosis.     

Translation elongation factor 1A is essential for regulation of the actin cytoskeleton and cell morphology

The binding of eukaryotic translation elongation factor 1A (eEF1A) to actin is a noncanonical function that may link two distinct cellular processes, cytoskeleton organization and gene expression. Using the yeast Saccharomyces cerevisiae, we have established an in vivo assay that directly identifies specific regions and residues of eEF1A responsible for actin interactions and bundling. Using a unique genetic screen, we isolated a series of eEF1A mutants with reduced actin bundling activity. These mutations alter actin cytoskeleton organization but not translation, indicating that these are separate functions of eEF1A. This demonstrates for the first time a direct consequence of eEF1A on cytoskeletal organization in vivo and the physiological significance of this interaction.     

The Drosophila acetylcholine receptor subunit D alpha5 is part of an alpha-bungarotoxin binding acetylcholine receptor

The central nervous system of Drosophila melanogaster contains an alpha-bungarotoxin-binding protein with the properties expected of a nicotinic acetylcholine receptor. This protein was purified 5800-fold from membranes prepared from Drosophila heads. The protein was solubilized with 1% Triton X-100 and 0.5 M sodium chloride and then purified using an alpha-cobratoxin column followed by a lentil lectin affinity column. The purified protein had a specific activity of 3.9 micromol of 125I-alpha-bungarotoxin binding sites/g of protein. The subunit composition of the purified receptor was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. This subunit profile was identical with that revealed by in situ labeling of the membrane-bound protein using the photolyzable methyl-4-azidobenzoimidate derivative of 125I-alpha-bungarotoxin. The purified receptor reveals two different protein bands with molecular masses of 42 and 57 kDa. From sedimentation analysis of the purified protein complex in H2O and D2O and gel filtration, a mass of 270 kDa was calculated. The receptor has a s(20,w) of 9.4 and a Stoke's radius of 7.4 nm. The frictional coefficient was calculated to be 1.7 indicating a highly asymmetric protein complex compatible with a transmembrane protein forming an ion channel. The sequence of a peptide obtained after tryptic digestion of the 42-kDa protein allowed the specific identification of the Drosophila D alpha5 subunit by sequence comparison. A peptide-specific antibody raised against the D alpha5 subunit provides further evidence that this subunit is a component of an alpha-bungarotoxin binding nicotinic acetylcholine receptor from the central nervous system of Drosophila.     

The GABAA receptor alpha 1 subunit Pro174-Asp191 segment is involved in GABA binding and channel gating

The GABA-binding site undergoes structural rearrangements during the transition from agonist binding to channel opening. To define possible roles of the GABA(A) receptor alpha(1) subunit Pro(174)-Asp(191) segment in these processes, we used the substituted cysteine accessibility method to characterize this region. Each residue was individually mutated to cysteine, expressed with wild-type beta(2) subunits in Xenopus laevis oocytes, and examined using two-electrode voltage clamp. Most mutations did not alter GABA EC(50) values. The D183C mutation produced a 7-fold reduction in GABA sensitivity. There were no significant changes in the K(I) values for the competitive antagonist, SR-95531. N-Biotinylaminoethyl methanethiosulfonate modified P174C-, R176C-, S177C-, V178C-, V180C-, A181C-, D183C-, R186C- and N188C-containing receptors. The pattern of accessibility suggests that this protein segment is aqueous-exposed and adopts a random coil conformation. Both GABA and SR-95531 slowed covalent modification of V178C, V180C, and D183C, indicating that these residues may line the GABA-binding site. Further, pentobarbital-induced channel activation accelerated modification of V180C and A181C and slowed the modification of R186C, suggesting that this region of the alpha(1) subunit may act as a dynamic element during channel-gating transitions.     

Methamphetamine induces long-term changes in GABAA receptor alpha2 subunit and GAD67 expression

The present study investigated whether GABA(A) receptor alpha2 subunit and GAD(67) are involved in chronic high dose methamphetamine (METH)-induced sensitization and neurotoxicity. The METH sensitization was established in rats by 7-day pump infusion plus daily injection (25mg/kg/day) and a subsequent 28-day withdrawal period. Behavioral sensitization was assessed by behavioral ratings after challenge with METH (0.5mg/kg). The neurotoxicity was evaluated by the expression of glial fibrillary acidic protein (GFAP). Western blot assay showed that METH sensitization decreases GABA(A) alpha2 subunit and GAD(67) protein levels in the nucleus accumbens (NAc) core and shell, and conversely, these proteins were increased in the caudate. An upregulation of GFAP expression was observed in the caudate, but not in the NAc core and shell. These data suggest that inhibition of GABA transmission in the NAc is related to METH behavioral sensitization, whereas activation of GABA transmission in the caudate is associated with METH-induced neurotoxicity.     

C-terminal and heparin-binding domains of collagenic tail subunit are both essential for anchoring acetylcholinesterase at the synapse

The collagen-tailed form of acetylcholinesterase (A(12)-AChE) appears to be localized at the neuromuscular junction in association with the transmembrane dystroglycan complex through binding of its collagenic tail (ColQ) to the proteoglycan perlecan. The heparan sulfate binding domains (HSBD) of ColQ are thought to be involved in anchoring ColQ to the synaptic basal lamina. The C-terminal domain (CTD) of ColQ is also likely involved, but there has been no direct evidence. Mutations in COLQ cause endplate AChE deficiency in humans. Nine previously reported and three novel mutations are in CTD of ColQ, and most CTD mutations do not abrogate formation of A(12)-AChE in transfected COS cells. Patient endplates, however, are devoid of AChE, suggesting that CTD mutations affect anchoring of ColQ to the synaptic basal lamina. Based on our observations that purified AChE can be transplanted to the heterologous frog neuromuscular junction, we tested insertion competence of nine naturally occurring CTD mutants and two artificial HSBD mutants. Wild-type human A(12)-AChE inserted into the frog neuromuscular junction, whereas six CTD mutants and two HSBD mutants did not. Our studies establish that the CTD mutations indeed compromise anchoring of ColQ and that both HSBD and CTD are essential for anchoring ColQ to the synaptic basal lamina.     

The GK domain of the voltage-dependent calcium channel beta subunit is essential for binding to the alpha subunit

The β subunits of voltage-dependent calcium channels bind the pore-forming α₁ subunit and play an important role in the regulation of calcium channel function. Recently, we have identified a new splice variant of the β₄ subunit, which we have termed the β_4d subunit. The β_4d subunit is a truncated splice variant of the β_4b subunit and lacks parts of the guanylate kinase (GK) domain and the C-terminus. The calcium current in BHK cells expressing α_1C and α₂δ with the β_4d subunit was as small as that without the β_4d subunit. Western blot analysis revealed that β_4d protein was expressed to a lesser extent that the β_4b protein. In addition, a GST pull down assay showed that the β_4d subunit could not interact with the α₁ subunit of the calcium channel. Collectively, our results suggest that the GK domain of the β subunit is essential for the expression of the functional calcium channel.     

Laminin alpha5 is essential for the formation of the zebrafish fins

The vertebrate fin fold, the presumptive evolutionary antecedent of the paired fins, consists of two layers of epidermal cells extending dorsally and ventrally over the trunk and tail of the embryo, facilitating swimming during the embryonic and larval stages. Development of the fin fold requires dramatic changes in cell shape and adhesion during early development, but the proteins involved in this process are completely unknown. In a screen of mutants defective in fin fold morphogenesis, we identified a mutant with a severe fin fold defect, which also displays malformed pectoral fins. We find that the cause of the defect is a non-sense mutation in the zebrafish lama5 gene that truncates laminin α5 before the C-terminal laminin LG domains, thereby preventing laminin α5 from interacting with its cell surface receptors. Laminin is mislocalized in this mutant, as are the membrane-associated proteins, actin and β-catenin, that normally form foci within the fin fold. Ultrastructural analysis revealed severe morphological abnormalities and defects in cell-cell adhesion within the epidermis of the developing fin fold at 36 hpf, resulting in an epidermal sheet that can not extend away from the body. Examining the pectoral fins, we find that the lama5 mutant is the first zebrafish mutant identified in which the pectoral fins fail to make the transition from an apical epidermal ridge to an apical fold, a transformation that is essential for pectoral fin morphogenesis. We propose that laminin α5, which is concentrated at the distal ends of the fins, organizes the distal cells of the fin fold and pectoral fins in order to promote the morphogenesis of the epidermis. The lama5 mutant provides novel insight into the role of laminins in the zebrafish epidermis, and the molecular mechanisms driving fin formation in vertebrates.     

Cyclase-associated protein is essential for the functioning of the endo-lysosomal system and provides a link to the actin cytoskeleton

Data from mutant analysis in yeast and Dictyostelium indicate a role for the cyclase-associated protein (CAP) in endocytosis and vesicle transport. We have used genetic and biochemical approaches to identify novel interacting partners of Dictyostelium CAP to help explain its molecular interactions in these processes. Cyclase-associated protein associates and interacts with subunits of the highly conserved vacuolar H(+)-ATPase (V-ATPase) and co-localizes to some extent with the V-ATPase. Furthermore, CAP is essential for maintaining the structural organization, integrity and functioning of the endo-lysosomal system, as distribution and morphology of V-ATPase- and Nramp1-decorated membranes were disturbed in a CAP mutant (CAP bsr) accompanied by an increased endosomal pH. Moreover, concanamycin A (CMA), a specific inhibitor of the V-ATPase, had a more severe effect on CAP bsr than on wild-type cells, and the mutant did not show adaptation to the drug. Also, the distribution of green fluorescent protein-CAP was affected upon CMA treatment in the wildtype and recovered after adaptation. Distribution of the V-ATPase in CAP bsr was drastically altered upon hypo-osmotic shock, and growth was slower and reached lower saturation densities in the mutant under hyper-osmotic conditions. Taken together, our data unravel a link of CAP with the actin cytoskeleton and endocytosis and suggest that CAP is an essential component of the endo-lysosomal system in Dictyostelium.     

Cloning, pharmacological characteristics and expression pattern of the rat GABAA receptor alpha 4 subunit.

A cDNA of rat brain encoding the GABAA receptor alpha 4 subunit has been cloned. Recombinant receptors composed of alpha 4, beta 2 and gamma 2 subunit bind with high affinity the GABA agonist [3H]muscimol and the benzodiazepine 'alcohol antagonist' [3H]Ro 15-4513, but fail to bind benzodiazepine agonists. The alpha 4 subunit is expressed mainly in the thalamus, as assessed by in situ hybridization histochemistry, and may participate in a major population of thalamic GABAA receptors. The alpha 4 mRNA is found at lower levels in cortex and caudate putamen, and is rare in cerebellum.     

Dynamics of ligand-induced, Rac1-dependent anchoring of cadherins to the actin cytoskeleton

Cadherin receptors are key morphoregulatory molecules during development. To dissect their mode of action, we developed an approach based on the use of myogenic C2 cells and beads coated with an Ncad-Fc ligand, allowing us to mimic cadherin-mediated adhesion. We used optical tweezers and video microscopy to investigate the dynamics of N-cadherin anchoring within the very first seconds of bead-cell contact. The analysis of the bead movement by single-particle tracking indicated that N-cadherin molecules were freely diffusive in the first few seconds after bead binding. The beads rapidly became diffusion-restricted and underwent an oriented rearward movement as a result of N-cadherin anchoring to the actin cytoskeleton. The kinetics of anchoring were dependent on ligand density, suggesting that it was an inducible process triggered by active cadherin recruitment. This anchoring was inhibited by the dominant negative form of Rac1, but not that of Cdc42. The Rac1 mutant had no effect on cell contact formation or cadherin-catenin complex recruitment, but did inhibit actin recruitment. Our results suggest that cadherin anchoring to the actin cytoskeleton is an adhesion-triggered, Rac1-regulated process enabling the transduction of mechanical forces across the cell membrane; they uncover novel aspects of the action of cadherins in cell sorting, cell migration, and growth cone navigation.     

p116Rip targets myosin phosphatase to the actin cytoskeleton and is essential for RhoA/ROCK-regulated neuritogenesis

Activation of the RhoA-Rho kinase (ROCK) pathway stimulates actomyosin-driven contractility in many cell systems, largely through ROCK-mediated inhibition of myosin II light chain phosphatase. In neuronal cells, the RhoA-ROCK-actomyosin pathway signals cell rounding, growth cone collapse, and neurite retraction; conversely, inhibition of RhoA/ROCK promotes cell spreading and neurite outgrowth. The actin-binding protein p116(Rip), whose N-terminal region bundles F-actin in vitro, has been implicated in Rho-dependent neurite remodeling; however, its function is largely unknown. Here, we show that p116(Rip), through its C-terminal coiled-coil domain, interacts directly with the C-terminal leucine zipper of the regulatory myosin-binding subunits of myosin II phosphatase, MBS85 and MBS130. RNA interference-induced knockdown of p116(Rip) inhibits cell spreading and neurite outgrowth in response to extracellular cues, without interfering with the regulation of myosin light chain phosphorylation. We conclude that p116(Rip) is essential for neurite outgrowth and may act as a scaffold to target the myosin phosphatase complex to the actin cytoskeleton.     

The cell-surface-expressed nucleolin is associated with the actin cytoskeleton

Nucleolin is a RNA- and protein-binding multifunctional protein. Mainly characterized as a nucleolar protein, nucleolin is continuously expressed on the surface of different types of cells along with its intracellular pool within the nucleus and cytoplasm. By confocal and electron microscopy using specific antibodies against nucleolin, we show that cytoplasmic nucleolin is found in small vesicles that appear to translocate nucleolin to the cell surface. Translocation of nucleolin is markedly reduced at low temperature or in serum-free medium, whereas conventional inhibitors of intracellular glycoprotein transport have no effect. Thus, translocation of nucleolin is the consequence of an active transport by a pathway which is independent of the endoplasmic reticulum-Golgi complex. The cell-surface-expressed nucleolin becomes clustered at the external side of the plasma membrane when cross-linked by the nucleolin-specific monoclonal antibody mAb D3. This clustering, occurring at 20 degrees C and in a well-organized pattern, is dependent on the existence of an intact actin cytoskeleton. At 37 degrees C, mAb D3 becomes internalized, thus illustrating that surface nucleolin can mediate intracellular import of specific ligands. Our results point out that nucleolin should also be considered a component of the cell surface where it could be functional as a cell surface receptor for various ligands reported before.