β-Neuregulin-1 is required for the in vivo development of functional Ca2+-activated K+ channels in parasympathetic neurons

The development of functional Ca2+-activated K+ channels (KCa) in chick ciliary ganglion (CG) neurons requires interactions with afferent preganglionic nerve terminals. Here we show that the essential preganglionic differentiation factor is an isoform of β-neuregulin-1. β-Neuregulin-1 transcripts are expressed in the midbrain preganglionic Edinger–Westphal nucleus at developmental stages that coincide with or precede the normal onset of macroscopic KCa in CG neurons. Injection of β-neuregulin-1 peptide into the brains of developing embryos evoked a robust stimulation of functional KCa channels at stages before the normal appearance of these channels in CG neurons developing in vivo. Conversely, injection of a neutralizing antiserum specific for β-neuregulin-1 inhibited the development of KCa channels in CG neurons. Low concentrations of β-neuregulin-1 evoked a robust increase in whole-cell KCa in CG neurons cocultured with iris target tissues. By contrast, culturing CG neurons with iris cells or low concentrations of β-neuregulin-1 by themselves was insufficient to stimulate KCa. These data suggest that the preganglionic factor required for the development of KCa in ciliary ganglion neurons is an isoform of β-neuregulin-1, and that this factor acts in concert with target-derived trophic molecules to regulate the differentiation of excitability.

Cyclic AMP Increases Cell Surface Expression of Functional Na,K-ATPase Units in Mammalian Cortical Collecting Duct Principal Cells

Cyclic AMP (cAMP) stimulates the transport of Na+ and Na,K-ATPase activity in the renal cortical collecting duct (CCD). The aim of this study was to investigate the mechanism whereby cAMP stimulates the Na,K-ATPase activity in microdissected rat CCDs and cultured mouse mpkCCDc14 collecting duct cells. db-cAMP (10−3 M) stimulated by 2-fold the activity of Na,K-ATPase from rat CCDs as well as the ouabain-sensitive component of 86Rb+ uptake by rat CCDs (1.7-fold) and cultured mouse CCD cells (1.5-fold). Pretreatment of rat CCDs with saponin increased the total Na,K-ATPase activity without further stimulation by db-cAMP. Western blotting performed after a biotinylation procedure revealed that db-cAMP increased the amount of Na,K-ATPase at the cell surface in both intact rat CCDs (1.7-fold) and cultured cells (1.3-fold), and that this increase was not related to changes in Na,K-ATPase internalization. Brefeldin A and low temperature (20°C) prevented both the db-cAMP-dependent increase in cell surface expression and activity of Na,K-ATPase in both intact rat CCDs and cultured cells. Pretreatment with the intracellular Ca2+ chelator bis-(o-aminophenoxy)-N,N,N′,N′-tetraacetic acid also blunted the increment in cell surface expression and activity of Na,K-ATPase caused by db-cAMP. In conclusion, these results strongly suggest that the cAMP-dependent stimulation of Na,K-ATPase activity in CCD results from the translocation of active pump units from an intracellular compartment to the plasma membrane.

Functional transitions in myosin: Formation of a critical salt-bridge and transmission of effect to the sensitive tryptophan

For analyzing the mechanism of energy transduction in the “motor” protein, myosin, it is opportune both to model the structural change in the hydrolytic transition, ATP (myosin-bound) + H2O → ADP⋅Pi (myosin-bound) and to check the plausibility of the model by appropriate site-directed mutations in the functional system. Here, we made a series of mutations to investigate the role of the salt-bridge between Glu-470 and Arg-247 (of chicken smooth muscle myosin) that has been inferred from crystallography to be a central feature of the transition [Fisher, A. J., Smith, C. A., Thoden, J. B., Smith, R., Sutoh, K., Holden, H. M., & Rayment, I. (1995) Biochemistry 34, 8960–8972]. Our results suggest that whether in the normal, or in the inverted, direction an intact salt-bridge is necessary for ATP hydrolysis, but when the salt-bridge is in the inverted direction it does not support actin activation. Normally, fluorescence changes result from adding nucleotides to myosin; these signals are reported by Trp-512 (of chicken smooth muscle myosin). Our results also suggest that structural impairments in the 470–247 region interfere with the transmission of these signals to the responsive Trp.

Two-domain reconstitution of a functional protein histidine kinase

In prokaryotes, in the absence of protein serine/threonine/tyrosine kinases, protein histidine kinases play a major role in signal transduction involved in cellular adaptation to various environmental changes and stresses. Histidine kinases phosphorylate their cognate response regulators at a specific aspartic acid residue with ATP in response to particular environmental signals. In this His-Asp phosphorelay signal transduction system, it is still unknown how the histidine kinase exerts its enzymatic function. Here we demonstrate that the cytoplasmic kinase domain of EnvZ, a transmembrane osmosensor of Escherichia coli can be further divided into two distinct functional subdomains: subdomain A [EnvZ(C)⋅(223–289); 67 residues] and subdomain B [EnvZ(C)⋅(290–450); 161 residues]. Subdomain A, with a high helical content, contains the autophosphorylation site, H–243, and forms a stable dimer having the recognition site for OmpR, the cognate response regulator of EnvZ. Subdomain B, an α/β-protein, exists as a monomer. When mixed, the two subdomains reconstitute the kinase function to phosphorylate subdomain A at His-243 in the presence of ATP. Subsequently, the phosphorylated subdomain A is able to transfer its phosphate group to OmpR. The two-domain structure of this histidine kinase provides an insight into the structural arrangement of the enzyme and its transphosphorylation mechanism.

Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma

Inactivation of the genes involved in DNA mismatch repair is associated with microsatellite instability (MSI) in colorectal cancer. We report that hypermethylation of the 5′ CpG island of hMLH1 is found in the majority of sporadic primary colorectal cancers with MSI, and that this methylation was often, but not invariably, associated with loss of hMLH1 protein expression. Such methylation also occurred, but was less common, in MSI− tumors, as well as in MSI+ tumors with known mutations of a mismatch repair gene (MMR). No hypermethylation of hMSH2 was found. Hypermethylation of colorectal cancer cell lines with MSI also was frequently observed, and in such cases, reversal of the methylation with 5-aza-2′-deoxycytidine not only resulted in reexpression of hMLH1 protein, but also in restoration of the MMR capacity in MMR-deficient cell lines. Our results suggest that microsatellite instability in sporadic colorectal cancer often results from epigenetic inactivation of hMLH1 in association with DNA methylation.

Purification and characterization of a CENP-B homologue protein that binds to the centromeric K-type repeat DNA of Schizosaccharomyces pombe

We have purified and characterized a novel 60-kDa protein that binds to centromeric K-type repeat DNA from Schizosaccharomyces pombe. This protein was initially purified by its ability to bind to the autonomously replicating sequence 3002 DNA. Cloning of the gene encoding this protein revealed that it possesses significant homology to the mammalian centromere DNA-binding protein CENP-B and S. pombe Abp1, and this gene was designated as cbh+ (CENP-B homologue). Cbh protein specifically interacts in vitro with the K-type repeat DNA, which is essential for centromere function. The Cbh-binding consensus sequence was determined by DNase I footprinting assays as PyPuATATPyPuTA, featuring an inverted repeat of the first four nucleotides. Based on its binding activity to centromeric DNA and homology to centromere proteins, we suggest that this protein may be a functional homologue of the mammalian CENP-B in S. pombe.

Molecular and functional characterization of a novel low-affinity cation transporter (LCT1) in higher plants

The transport of cations across membranes in higher plants plays an essential role in many physiological processes including mineral nutrition, cell expansion, and the transduction of environmental signals. In higher plants the coordinated expression of transport mechanisms is essential for specialized cellular processes and for adaptation to variable environmental conditions. To understand the molecular basis of cation transport in plant roots, a Triticum aestivum cDNA library was used to complement a yeast mutant deficient in potassium (K+) uptake. Two genes were cloned that complemented the mutant: HKT1 and a novel cDNA described in this report encoding a cation transporter, LCT1 (low-affinity cation transporter). Analysis of the secondary structure of LCT1 suggests that the protein contains 8–10 transmembrane helices and a hydrophilic amino terminus containing sequences enriched in Pro, Ser, Thr, and Glu (PEST). The transporter activity was assayed using radioactive isotopes in yeast cells expressing the cDNA. LCT1 mediated low-affinity uptake of the cations Rb+ and Na+, and possibly allowed Ca2+ but not Zn2+ uptake. LCT1 is expressed in low abundance in wheat roots and leaves. The precise functional role of this cation transporter is not known, although the competitive inhibition of cation uptake by Ca2+ has parallels to whole plant and molecular studies that have shown the important role of Ca2+ in reducing Na+ uptake and ameliorating Na+ toxicity. The structure of this higher plant ion transport protein is unique and contains PEST sequences.

An ancient family of human endogenous retroviruses encodes a functional homolog of the HIV-1 Rev protein

The human endogenous retrovirus K (HERV-K) family of endogenous retroviruses consists of ≈50 proviral copies per haploid human genome. Herein, the HERV-Ks are shown to encode a sequence-specific nuclear RNA export factor, termed K-Rev, that is functionally analogous to the HIV-1 Rev protein. Like HIV-1 Rev, K-Rev binds to both the Crm1 nuclear export factor and to a cis-acting viral RNA target to activate nuclear export of unspliced RNAs. Surprisingly, this HERV-K RNA sequence, which is encoded within the HERV-K long terminal repeat, is also recognized by HIV-1 Rev. These data provide surprising evidence for an evolutionary link between HIV-1 and a group of endogenous retroviruses that first entered the human genome ≈30 million years ago.

Two functionally distinct subsites for the binding of internal blockers to the pore of voltage-activated K+ channels

Many blockers of Na+ and K+ channels act by blocking the pore from the intracellular side. For Shaker K+ channels, such intracellular blockers vary in their functional effect on slow (C-type) inactivation: Some blockers interfere with C-type inactivation, whereas others do not. These functional differences can be explained by supposing that there are two overlapping “subsites” for blocker binding, only one of which inhibits C-type inactivation through an allosteric effect. We find that the ability to bind to these subsites depends on specific structural characteristics of the blockers, and correlates with the effect of mutations in two distinct regions of the channel protein. These interactions are important because they affect the ability of blockers to produce use-dependent inhibition.

Correlation of the structural and functional domains in the membrane protein Vpu from HIV-1

Vpu is an 81-residue membrane protein encoded by the HIV-1 genome. NMR experiments show that the protein folds into two distinct domains, a transmembrane hydrophobic helix and a cytoplasmic domain with two in-plane amphipathic α-helices separated by a linker region. Resonances in one-dimensional solid-state NMR spectra of uniformly 15N labeled Vpu are clearly segregated into two bands at chemical shift frequencies associated with NH bonds in a transmembrane α-helix, perpendicular to the membrane surface, and with NH bonds in the cytoplasmic helices parallel to the membrane surface. Solid-state NMR spectra of truncated Vpu2–51 (residues 2–51), which contains the transmembrane α-helix and the first amphipathic helix of the cytoplasmic domain, and of a construct Vpu28–81 (residues 28–81), which contains only the cytoplasmic domain, support this structural model of Vpu in the membrane. Full-length Vpu (residues 2–81) forms discrete ion-conducting channels of heterogeneous conductance in lipid bilayers. The most frequent conductances were 22 ± 3 pS and 12 ± 3 pS in 0.5 M KCl and 29 ± 3 pS and 12 ± 3 pS in 0.5 M NaCl. In agreement with the structural model, truncated Vpu2–51, which has the transmembrane helix, forms discrete channels in lipid bilayers, whereas the cytoplasmic domain Vpu28–81, which lacks the transmembrane helix, does not. This finding shows that the channel activity is associated with the transmembrane helical domain. The pattern of channel activity is characteristic of the self-assembly of conductive oligomers in the membrane and is compatible with the structural and functional findings.

Human γ-aminobutyric acid type B receptors are differentially expressed and regulate inwardly rectifying K+ channels

γ-Aminobutyric acid type B receptors (GABABRs) are involved in the fine tuning of inhibitory synaptic transmission. Presynaptic GABABRs inhibit neurotransmitter release by down-regulating high-voltage activated Ca2+ channels, whereas postsynaptic GABABRs decrease neuronal excitability by activating a prominent inwardly rectifying K+ (Kir) conductance that underlies the late inhibitory postsynaptic potentials. Here we report the cloning and functional characterization of two human GABABRs, hGABABR1a (hR1a) and hGABABR1b (hR1b). These receptors closely match the pharmacological properties and molecular weights of the most abundant native GABABRs. We show that in transfected mammalian cells hR1a and hR1b can modulate heteromeric Kir3.1/3.2 and Kir3.1/3.4 channels. Heterologous expression therefore supports the notion that Kir3 channels are the postsynaptic effectors of GABABRs. Our data further demonstrate that in principle either of the cloned receptors could mediate inhibitory postsynaptic potentials. We find that in the cerebellum hR1a and hR1b transcripts are largely confined to granule and Purkinje cells, respectively. This finding supports a selective association of hR1b, and not hR1a, with postsynaptic Kir3 channels. The mapping of the GABABR1 gene to human chromosome 6p21.3, in the vicinity of a susceptibility locus (EJM1) for idiopathic generalized epilepsies, identifies a candidate gene for inherited forms of epilepsy.

Growth suppression of glioma cells by PTEN requires a functional phosphatase catalytic domain

Deletions of all or part of chromosome 10 are the most common genetic alterations in high-grade gliomas. The PTEN gene (also called MMAC1 and TEP1) maps to chromosome region 10q23 and has been implicated as a target of alteration in gliomas and also in other cancers such as those of the breast, prostate, and kidney. Here we sought to provide a functional test of its candidacy as a growth suppressor in glioma cells. We used a combination of Northern blot analysis, protein truncation assays, and sequence analysis to determine the types and frequency of PTEN mutations in glioma cell lines so that we could define appropriate recipients to assess the growth suppressive function of PTEN by gene transfer. Introduction of wild-type PTEN into glioma cells containing endogenous mutant alleles caused growth suppression, but was without effect in cells containing endogenous wild-type PTEN. The ectopic expression of PTEN alleles, which carried mutations found in primary tumors and have been shown or are expected to inactivate its phosphatase activity, caused little growth suppression. These data strongly suggest that PTEN is a protein phosphatase that exhibits functional and specific growth-suppressing activity.

The restriction–modification genes of Escherichia coli K-12 may not be selfish: They do not resist loss and are readily replaced by alleles conferring different specificities

Type II restriction and modification (R-M) genes have been described as selfish because they have been shown to impose selection for the maintenance of the plasmid that encodes them. In our experiments, the type I R-M system EcoKI does not behave in the same way. The genes specifying EcoKI are, however, normally residents of the chromosome and therefore our analyses were extended to monitor the deletion of chromosomal genes rather than loss of plasmid vector. If EcoKI were to behave in the same way as the plasmid-encoded type II R-M systems, the loss of the relevant chromosomal genes by mutation or recombination should lead to cell death because the cell would become deficient in modification enzyme and the bacterial chromosome would be vulnerable to the restriction endonuclease. Our data contradict this prediction; they reveal that functional type I R-M genes in the chromosome are readily replaced by mutant alleles and by alleles encoding a type I R-M system of different specificity. The acquisition of allelic genes conferring a new sequence specificity, but not the loss of the resident genes, is dependent on the product of an unlinked gene, one predicted [Prakash-Cheng, A., Chung, S. S. & Ryu, J. (1993) Mol. Gen. Genet. 241, 491–496] to be relevant to control of expression of the genes that encode EcoKI. Our evidence suggests that not all R-M systems are evolving as “selfish” units; rather, the diversity and distribution of the family of type I enzymes we have investigated require an alternative selective pressure.

A system for functional analysis of Ebola virus glycoprotein

Ebola virus causes hemorrhagic fever in humans and nonhuman primates, resulting in mortality rates of up to 90%. Studies of this virus have been hampered by its extraordinary pathogenicity, which requires biosafety level 4 containment. To circumvent this problem, we developed a novel complementation system for functional analysis of Ebola virus glycoproteins. It relies on a recombinant vesicular stomatitis virus (VSV) that contains the green fluorescent protein gene instead of the receptor-binding G protein gene (VSVΔG*). Herein we show that Ebola Reston virus glycoprotein (ResGP) is efficiently incorporated into VSV particles. This recombinant VSV with integrated ResGP (VSVΔG*-ResGP) infected primate cells more efficiently than any of the other mammalian or avian cells examined, in a manner consistent with the host range tropism of Ebola virus, whereas VSVΔG* complemented with VSV G protein (VSVΔG*-G) efficiently infected the majority of the cells tested. We also tested the utility of this system for investigating the cellular receptors for Ebola virus. Chemical modification of cells to alter their surface proteins markedly reduced their susceptibility to VSVΔG*-ResGP but not to VSVΔG*-G. These findings suggest that cell surface glycoproteins with N-linked oligosaccharide chains contribute to the entry of Ebola viruses, presumably acting as a specific receptor and/or cofactor for virus entry. Thus, our VSV system should be useful for investigating the functions of glycoproteins from highly pathogenic viruses or those incapable of being cultured in vitro.

Mutations in Drosophila Enabled and Rescue by Human Vasodilator-stimulated Phosphoprotein (VASP) Indicate Important Functional Roles for Ena/VASP Homology Domain 1 (EVH1) and EVH2 Domains

Drosophila Enabled (Ena) was initially identified as a dominant genetic suppressor of mutations in the Abelson tyrosine kinase and, more recently, as a member of the Ena/human vasodilator-stimulated phosphoprotein (VASP) family of proteins. We have used genetic, biochemical, and cell biological approaches to demonstrate the functional relationship between Ena and human VASP. In addition, we have defined the roles of Ena domains identified as essential for its activity in vivo. We have demonstrated that VASP rescues the embryonic lethality associated with loss of Ena function in Drosophila and have shown that Ena, like VASP, is associated with actin filaments and focal adhesions when expressed in cultured cells. To define sequences that are central to Ena function, we have characterized the molecular lesions present in two lethal ena mutant alleles that affected the Ena/VASP homology domain 1 (EVH1) and EVH2. A missense mutation that resulted in an amino acid substitution in the EVH1 domain eliminated in vitro binding of Ena to the cytoskeletal protein zyxin, a previously reported binding partner of VASP. A nonsense mutation that resulted in a C-terminally truncated Ena protein lacking the EVH2 domain failed to form multimeric complexes and exhibited reduced binding to zyxin and the Abelson Src homology 3 domain. Our analysis demonstrates that Ena and VASP are functionally homologous and defines the conserved EVH1 and EVH2 domains as central to the physiological activity of Ena.