NotI Sites Research Articles

Epigenetics: differential DNA methylation in mammalian somatic tissues

Epigenetics refers to heritable phenotypic alterations in the absence of DNA sequence changes, and DNA methylation is one of the extensively studied epigenetic alterations. DNA methylation is an evolutionally conserved mechanism to regulate gene expression in mammals. Because DNA methylation is preserved during DNA replication it can be inherited. Thus, DNA methylation could be a major mechanism by which to produce semi-stable changes in gene expression in somatic tissues. Although it remains controversial whether germ-line DNA methylation in mammalian genomes is stably heritable, frequent tissue-specific and disease-specific de novo methylation events are observed during somatic cell development/differentiation. In this minireview, we discuss the use of restriction landmark genomic scanning, together with in silico analysis, to identify differentially methylated regions in the mammalian genome. We then present a rough overview of quantitative DNA methylation patterns at 4600 NotI sites and more than 150 differentially methylated regions in several C57BL/6J mouse tissues. Comparative analysis between mice and humans suggests that some, but not all, tissue-specific differentially methylated regions are conserved. A deeper understanding of cell-type-specific differences in DNA methylation might lead to a better illustration of the mechanisms behind tissue-specific differentiation in mammals.

Congenital Chloride-losing Diarrhea Causing Mutations in the STAS Domain Result in Misfolding and Mistrafficking of SLC26A3

Congenital chloride-losing diarrhea (CLD) is a genetic disorder causing watery stool and dehydration. Mutations in SLC26A3 (solute carrier 26 family member 3), which functions as a coupled Cl(-)/HCO(3)(-) exchanger, cause CLD. SLC26A3 is a membrane protein predicted to contain 12 transmembrane-spanning alpha-helices and a C-terminal STAS (sulfate transporters and anti-sigma-factor) domain homologous to the bacterial anti-sigma-factor antagonists. The STAS domain is required for SLC26A3 Cl(-)/HCO(3)(-) exchange function and for the activation of cystic fibrosis transmembrane conductance regulator by SLC26A3. Here we investigate the molecular mechanism(s) by which four CLD-causing mutations (DeltaY526/7, I544N, I675/6ins, and G702Tins) in the STAS domain lead to disease. In a heterologous mammalian expression system biochemical, immunohistochemical, and ion transport experiments suggest that the four CLD mutations cause SLC26A3 transporter misfolding and/or mistrafficking. Expression studies with the isolated STAS domain suggest that the I675/6ins and G702Tins mutations disrupt the STAS domain directly, whereas limited proteolysis experiments suggest that the DeltaY526/7 and I544N mutations affect a later step in the folding and/or trafficking pathway. The data suggest that these CLD-causing mutations cause disease by at least two distinct molecular mechanisms, both ultimately leading to loss of functional protein at the plasma membrane.

Inhibition of p97-dependent Protein Degradation by Eeyarestatin I

Elimination of misfolded proteins from the endoplasmic reticulum (ER) by ER-associated degradation involves substrate retrotranslocation from the ER lumen into the cytosol for degradation by the proteasome. For many substrates, retrotranslocation requires the action of ubiquitinating enzymes, which polyubiquitinate substrates emerging from the ER lumen, and of the p97-Ufd1-Npl4 ATPase complex, which hydrolyzes ATP to dislocate polyubiquitinated substrates into the cytosol. Polypeptides extracted by p97 are eventually transferred to the proteasome for destruction. In mammalian cells, ERAD can be blocked by a chemical inhibitor termed Eeyarestatin I, but the mechanism of EerI action is unclear. Here we report that EerI can associate with a p97 complex to inhibit ERAD. The interaction of EerI with the p97 complex appears to negatively influence a deubiquitinating process that is mediated by p97-associated deubiquitinating enzymes. We further show that ataxin-3, a p97-associated deubiquitinating enzyme previously implicated in ER-associated degradation, is among those affected. Interestingly, p97-associated deubiquitination is also involved in degradation of a soluble substrate. Our analyses establish a role for a novel deubiquitinating process in proteasome-dependent protein turnover.

A Hybrid Vector System Expands Adeno-associated Viral Vector Packaging Capacity in a Transgene-independent Manner

The trans-splicing (ts) and overlapping (ov) vectors expand the packaging capacity of adeno-associated virus (AAV). But their application depends on the inherent properties of the target gene. The ts vectors require an optimal gene-splitting site and the ov vectors require a highly recombinogenic domain. In order to overcome these limitations, we developed a hybrid dual (hd) vector system. In the hd vectors, we inserted a highly recombinogenic alkaline phosphatase (AP) sequence in the ts vectors to allow for transgene-independent reconstitution through homologous recombination of the AP sequences. We first tested the hybrid system with the LacZ gene. Both in the cell line (in vitro) and in the mouse muscle (in vivo), the hd vectors significantly outperformed the ts and ov vectors. In muscle, the transduction efficiency of the hybrid vectors reached 80% of that from the single intact vector. Southern blot confirmed AP sequence-mediated transgene reconstitution. In order to validate the hybrid system, we split the 6 kilobase (kb) mini-dystrophin gene at the exon 55/56 junction, a predicted poor site for the ts approach. In dystrophic mdx mouse muscle, the hd vectors yielded 5.6-fold higher transduction than the ts vectors did. Taken together, these data suggest that the hybrid system efficiently expresses large therapeutic genes that are poor candidates for the ts and ov approaches.

Sequential changes in genome-wide DNA methylation status during adipocyte differentiation

DNA methylation is an epigenetic mark on the mammalian genome. There are numerous tissue-dependent and differentially methylated regions (T-DMRs) in the unique sequences distributed throughout the genome. To determine the epigenetic changes during adipocyte differentiation, we investigated the sequential changes in DNA methylation status of 3T3-L1 cells at the growing, confluent, postconfluent and mature adipocyte cell stages. Treatment of 3T3-L1 cells with 5-aza-2′-deoxycytidine inhibited differentiation in a stage-dependent manner, supporting the idea that formation of accurate DNA methylation profile, consisting of methylated and unmethylated T-DMRs, may be involved in differentiation. Analysis by methylation-sensitive quantitative real-time PCR of the 65 known T-DMRs which contain NotI sites detected 8 methylations that changed during differentiation, and the changes in the patterns of these methylations were diverse, confirming that the differentiation process involves epigenetic alteration at the T-DMRs. Intriguingly, the dynamics of the methylation change vary depending on the T-DMRs and differentiation stages. Restriction landmark genomic scanning detected 32 novel T-DMRs, demonstrating that differentiation of 3T3-L1 cells involves genome-wide epigenetic changes by temporal methylation/demethylation, in addition to maintenance of a static methylated/demethylated state, and both depend on differentiation stage.

Responses of Nontransformed Human Hepatocytes to Conditional Expression of Full-Length Hepatitis C Virus Open Reading Frame

Hepatitis C virus (HCV) is a major cause of chronic hepatitis that can lead to cirrhosis and hepatocellular carcinoma. To study the effects of HCV protein expression on host cells, we established conditional expression of the full-length open reading frame (ORF) of an infectious cDNA clone of HCV (genotype 1a, H77 strain) in the nontransformed human hepatocyte line cell HH4 using the ecdysone receptor regulatory system. Treatment with the ecdysone analog ponasterone-A induced tightly regulated and dose-dependent full-length HCV ORF expression and properly processed HCV proteins. HCV Core, NS3, and NS5A colocalized in perinuclear regions and associated with the early endosomal protein EEA1. HCV ORF expression caused marked growth inhibition, increased intracellular reactive oxygen species, up-regulation of glutamate-l-cysteine ligase activity, increased glutathione level, and activation of nuclear factor kappaB. Although it was not directly cytotoxic, HCV ORF expression sensitized HH4 cells to Fas at certain concentrations but not to tumor necrosis factor-related apoptosis-inducing ligand. HCV ORF expression in HH4 cells up-regulated genes involved in innate immune response/inflammation and oxidative stress responses and down-regulated cell growth-related genes. Expression of HCV ORF in host cells may contribute to HCV pathogenesis by producing oxidative stress and increasing the expression of genes related to the innate immune response and inflammation.

The Role of the N Terminus and Transmembrane Domain of TRPM8 in Channel Localization and Tetramerization

Transient receptor potential (TRP) channels are a family of cation channels involved in diverse cellular functions. They are composed of a transmembrane domain of six putative transmembrane segments flanked by large N- and C-terminal cytoplasmic domains. The melastatin subfamily (TRPM) channels have N-terminal domains of approximately 700 amino acids with four regions of shared homology and C-terminal domains containing the conserved TRP domain followed by a coiled-coil region. Here we investigated the effects of N- and C-terminal deletions on the cold and menthol receptor, TRPM8, expressed heterologously in Sf21 insect cells. Patch-clamp electrophysiology was used to study channel activity and revealed that only deletion of the first 39 amino acids was tolerated by the channel. Further N-terminal truncation or any C-terminal deletions prevented proper TRPM8 function. Confocal microscopy with immunofluorescence revealed that amino acids 40-86 are required for localization to the plasma membrane. Furthermore, analysis of deletion mutant oligomerization shows that the transmembrane domain is sufficient for TPRM8 assembly into tetramers. TRPM8 channels with C-terminal deletions tetramerize and localize properly but are inactive, indicating that although not essential for tetramerization and localization, the C terminus is critical for proper function of the channel sensor and/or gate.

Evidence for 26 distinct acyl-coenzyme A synthetase genes in the human genome

Acyl-coenzyme A synthetases (ACSs) catalyze the fundamental, initial reaction in fatty acid metabolism. "Activation" of fatty acids by thioesterification to CoA allows their participation in both anabolic and catabolic pathways. The availability of the sequenced human genome has facilitated the investigation of the number of ACS genes present. Using two conserved amino acid sequence motifs to probe human DNA databases, 26 ACS family genes/proteins were identified. ACS activity in either humans or rodents was demonstrated previously for 20 proteins, but 6 remain candidate ACSs. For two candidates, cDNA was cloned, protein was expressed in COS-1 cells, and ACS activity was detected. Amino acid sequence similarities were used to assign enzymes into subfamilies, and subfamily assignments were consistent with acyl chain length preference. Four of the 26 proteins did not fit into a subfamily, and bootstrap analysis of phylograms was consistent with evolutionary divergence. Three additional conserved amino acid sequence motifs were identified that likely have functional or structural roles. The existence of many ACSs suggests that each plays a unique role, directing the acyl-CoA product to a specific metabolic fate. Knowing the full complement of ACS genes in the human genome will facilitate future studies to characterize their specific biological functions.

A new class of tissue‐specifically methylated regions involving entire CpG islands in the mouse

CpG islands, which have higher GC content and CpG frequencies compared to the genome as a whole, are generally believed to be unmethylated in tissues except at promoters of genes undergoing X chromosome inactivation or genomic imprinting. Recent studies, however, have shown that CpG islands at promoters of a number of genes contain tissue-dependent, differentially methylated regions (T-DMRs). In general, the tissue-specific methylation is restricted to a part of the promoter CpG island, with hypomethylation of the remaining sequence. In the current study, using comparison between Restriction Landmark Genomic Scanning (RLGS) and in silico RLGS, we identified ten sperm-specific unmethylated NotI sites, T-DMRs located in CpG islands that were hypomethylated in sperm but near-completely methylated in the kidney and brain. Unusually, these T-DMRs involve the whole CpG island at each of these loci. We characterized one of these genes, adenine nucleotide translocator 4 (Ant4), which is expressed in germ cells. Using a promoter assay, we demonstrated that expression of Ant4 gene is controlled by DNA methylation at the CpG island sequences within the promoter region. Ant4 and other sperm-specific hypomethylated loci represent a new class of CpG islands that become completely methylated in different cell lineages. T-DMRs at CpG islands are functionally important gene regulatory elements that may now be categorized into two classes: T-DMRs involving a subregion of the CpG island and those that occupy the whole CpG island.

LPT1 Encodes a Membrane-bound O-Acyltransferase Involved in the Acylation of Lysophospholipids in the Yeast Saccharomyces cerevisiae

Phospholipids are major components of cellular membranes that participate in a range of cellular processes. Phosphatidic acid (PA) is a key molecule in the phospholipid biosynthetic pathway. In Saccharomyces cerevisiae, SLC1 has been identified as the gene encoding lysophosphatidic acid acyltransferase, which catalyzes PA synthesis. However, despite the importance of PA, disruption of SLC1 does not affect cell viability (Nagiec, M. M., Wells, G. B., Lester, R. L., and Dickson, R. C. (1993) J. Biol. Chem. 268, 22156-22163). We originally aimed to identify the acetyl-CoA:lyso platelet-activating factor acetyltransferase (lysoPAF AT) gene in yeast. Screening of a complete set of yeast deletion clones (4741 homozygous diploid clones) revealed a single mutant strain, YOR175c, with a defect in lysoPAF AT activity. YOR175c has been predicted to be a member of the membrane-bound O-acyltransferase superfamily, and we designated the gene LPT1. An Lpt1-green fluorescent protein fusion protein localized at the endoplasmic reticulum. Other than lysoPAF AT activity, Lpt1 catalyzed acyltransferase activity with a wide variety of lysophospholipids as acceptors, including lysophosphatidic acid, lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylglycerol, lysophosphatidylinositol, and lysophosphatidylserine. A liquid chromatography-mass spectrometry analysis indicated that lysophosphatidylcholine and lysophosphatidylethanolamine accumulated in the Deltalpt1 mutant strain. Although the Deltalpt1 mutant strain did not show other detectable defects, the Deltalpt1 Deltaslc1 double mutant strain had a synthetic lethal phenotype. These results indicate that, in concert with Slc1, Lpt1 plays a central role in PA biosynthesis, which is essential for cell viability.

Rational design of a chimeric endonuclease targeted to NotI recognition site

A cleavage-deficient variant of NotI restriction endonuclease (GCGGCCGC) was isolated by random mutagenesis of the notIR gene. The NotI variant D160N was shown to bind DNA and protect plasmid DNA from EagI (CGGCCG) and NotI digestions. The EDTA-resistant BmrI restriction endonuclease cleaves DNA sequence ACTGGG N5/N4. The N-terminal cleavage domain of BmrI (residues 1-198) with non-specific nuclease activity was fused to the NotI variant D160N with a short linker. The engineered chimeric endonuclease (CH-endonuclease) recognizes NotI sites specifically in the presence of high salt (100-150 mM NaCl) and divalent cations Mg++ or Ca++. In contrast to wild-type NotI, which cuts within its recognition sequence, BmrI198-NotI (D160N) cleaves DNA outside of NotI sites, resulting in deletion of the NotI site and the adjacent sequences. The fusion of the BmrI cleavage domain to cleavage-deficient variants of Type II restriction enzymes to generate novel cleavage sites will provide useful tools for DNA manipulation.

Cloning and Characterization of Two Human VIP1-like Inositol Hexakisphosphate and Diphosphoinositol Pentakisphosphate Kinases

Eukaryotes possess numerous inositol phosphate (IP) and diphosphoinositol phosphate (PP-IPs or inositol pyrophosphates) species that act as chemical codes important for intracellular signaling pathways. Production of IP and PP-IP molecules occurs through several classes of evolutionarily conserved inositol phosphate kinases. Here we report the characterization of a human inositol hexakisphosphate (IP6) and diphosphoinositol pentakisphosphate (PP-IP5 or IP7) kinase with similarity to the yeast enzyme Vip1, a recently identified IP6/IP7 kinase (Mulugu, S., Bai, W., Fridy, P. C., Bastidas, R. J., Otto, J. C., Dollins, D. E., Haystead, T. A., Ribeiro, A. A., and York, J. D. (2007) Science 316, 106-109). Recombinant human VIP1 exhibits in vitro IP6 and IP7 kinase activities and restores IP7 synthesis when expressed in mutant yeast. Expression of human VIP1 in HEK293T cells engineered to produce high levels of IP7 results in dramatic increases in bisdiphosphoinositol tetrakisphosphate (PP2-IP4 or IP8). Northern blot analysis indicates that human VIP1 is expressed in a variety of tissues and is enriched in skeletal muscle, heart, and brain. The subcellular distribution of tagged human VIP1 is indicative of a cytoplasmic non-membrane localization pattern. We also characterized human and mouse VIP2, an additional gene product with nearly 90% similarity to VIP1 in the kinase domain, and observed both IP6 and IP7 kinase activities. Our data demonstrate that human VIP1 and VIP2 function as IP6 and IP7 kinases that act along with the IP6K/Kcs1-class of kinases to convert IP6 to IP8 in mammalian cells, a process that has been found to occur in response to various stimuli and signaling events.

Myoferlin Regulates Vascular Endothelial Growth Factor Receptor-2 Stability and Function

Myoferlin and dysferlin are members of the ferlin family of membrane proteins. Recent studies have shown that mutation or genetic disruption of myoferlin or dysferlin promotes muscular dystrophy-related phenotypes in mice, which are the result of impaired plasma membrane integrity. However, no biological functions have been ascribed to myoferlin in non-muscle tissues. Herein, using a proteomic analysis of endothelial cell (EC) caveolae/lipid raft microdomains we identified myoferlin in these domains and show that myoferlin is highly expressed in ECs and vascular tissues. The loss of myoferlin results in lack of proliferation, migration, and nitric oxide (NO) release in response to vascular endothelial growth factor (VEGF). Western blotting and surface biotinylation experiments show that loss of myoferlin reduces the expression level and autophosphorylation of VEGF receptor-2 (VEGFR-2) in native ECs. In a reconstituted cell system, transfection of myoferlin increases VEGFR-2 membrane expression and autophosphorylation in response to VEGF. In vivo, VEGFR-2 levels and VEGF-induced permeability are impaired in myoferlin-deficient mice. Mechanistically, myoferlin forms a complex with dynamin-2 and VEGFR-2, which prevents CBL-dependent VEGFR-2 polyubiquitination and proteasomal degradation. These data are the first to report novel biological activities for myoferlin and reveal the role of membrane integrity to VEGF signaling.

Cell type‐specific methylation profiles occurring disproportionately in CpG‐less regions that delineate developmental similarity

Our previous studies using restriction landmark genomic scanning (RLGS) defined tissue- or cell-specific DNA methylation profiles. It remains to be determined whether the DNA sequence compositions in the genomic contexts of the NotI loci tested by RLGS influence their tendency to change with differentiation. We carried out 3834 methylation measurements consisting of 213 NotI loci in the mouse genome in 18 different tissues and cell types, using quantitative real-time PCR based on a Virtual image rlgs database. Loci were categorized as CpG islands or other, and as unique or repetitive sequences, each category being associated with a variety of methylation categories. Strikingly, the tissue-dependently and differentially methylated regions (T-DMRs) were disproportionately distributed in the non-CpG island loci. These loci were located not only in 5'-upstream regions of genes but also in intronic and non-genic regions. Hierarchical clustering of the methylation profiles could be used to define developmental similarity and cellular phenotypes. The results show that distinctive tissue- and cell type-specific methylation profiles by RLGS occur mostly at NotI sites located at non-CpG island sequences, which delineate developmental similarity of different cell types. The finding indicates the power of NotI methylation profiles in evaluating the relatedness of different cell types.

Enhanced Lysosomal Pathology Caused by β-Synuclein Mutants Linked to Dementia with Lewy Bodies

Two missense mutations (P123H and V70M) of beta-synuclein (beta-syn), the homologue of alpha-syn, have been recently identified in dementia with Lewy bodies. However, the mechanism through which these mutations influence the pathogenesis of dementia with Lewy bodies is unclear. To investigate the role of the beta-syn mutations in neurodegeneration, each mutant was stably transfected into B103 neuroblastoma cells. Cells overexpressing mutated beta-syn had eosinophilic cytoplasmic inclusion bodies immunopositive for mutant beta-syn, and electron microscopy revealed that these cells were abundant in various cytoplasmic membranous inclusions resembling the histopathology of lysosomal storage disease. Consistent with these findings, the inclusion bodies were immunopositive for lysosomal markers, including cathepsin B, LAMP-2, GM2 ganglioside, and ATP13A2, which has recently been linked to PARK9. Notably, formation of these lysosomal inclusions was greatly stimulated by co-expression of alpha-syn, was dependent on the phosphorylation of alpha-syn at Ser-129, and was more efficient with the A53T familial mutant of alpha-syn compared with wild type. Furthermore, the inclusion formation in cells overexpressing mutant beta-syn and transfected with alpha-syn was significantly suppressed by treatment with autophagy-lysosomal inhibitors, which were associated with impaired clearance of syn proteins and enhanced apoptosis, indicating that formation of lysosomal inclusions might be protective. Collectively, the results demonstrated unambiguously that overexpression of beta-syn mutants (P123H and V70M) in neuroblastoma cells results in an enhanced lysosomal pathology. We suggest that these missense mutations of beta-syn might play a causative role in stimulating neurodegeneration.

Dimerization and Actin-bundling Properties of Villin and Its Role in the Assembly of Epithelial Cell Brush Borders

Villin is a major actin-bundling protein in the brush border of epithelial cells. In this study we demonstrate for the first time that villin can bundle actin filaments using a single F-actin binding site, because it has the ability to self-associate. Using fluorescence resonance energy transfer, we demonstrate villin self-association in living cells in microvilli and in growth factor-stimulated cells in membrane ruffles and lamellipodia. Using sucrose density gradient, size-exclusion chromatography, and matrix-assisted laser desorption ionization time-of-flight, the majority of villin was identified as a monomer or dimer. Villin dimers were also identified in Caco-2 cells, which endogenously express villin and Madin-Darby canine kidney cells that ectopically express villin. Using truncation mutants of villin, site-directed mutagenesis, and fluorescence resonance energy transfer, an amino-terminal dimerization site was identified that regulated villin self-association in parallel conformation as well as actin bundling by villin. This detailed analysis describes for the first time microvillus assembly by villin, redefines the actin-bundling function of villin, and provides a molecular mechanism for actin bundling by villin, which could have wider implications for other actin cross-linking proteins that share a villin-like headpiece domain. Our study also provides a molecular basis to separate the morphologically distinct actin-severing and actin-bundling properties of villin.

A Ferritin-responsive Internal Ribosome Entry Site Regulates Folate Metabolism

Cytoplasmic serine hydroxymethyltransferase (cSHMT) enzyme levels are elevated by the expression of the heavy chain ferritin (H ferritin) cDNA in cultured cells without corresponding changes in mRNA levels, resulting in enhanced folate-dependent de novo thymidylate biosynthesis and impaired homocysteine remethylation. In this study, the mechanism whereby H ferritin regulates cSHMT expression was determined. cSHMT translation is shown to be regulated by an H ferritin-responsive internal ribosome entry site (IRES) located within the cSHMT mRNA 5'-untranslated region (5'-UTR). The cSHMT 5'-UTR exhibited IRES activity during in vitro translation of bicistronic mRNA templates, and in MCF-7 and HeLa cells transfected with bicistronic mRNAs. IRES activity was depressed in H ferritin-deficient mouse embryonic fibroblasts and elevated in cells expressing the H ferritin cDNA. H ferritin was shown to interact with the mRNA-binding protein CUGBP1, a protein known to interact with the alpha and beta subunits of eukaryotic initiation factor eIF2. Small interference RNA-mediated depletion of CUGBP1 decreased IRES activity from bicistronic templates that included the cSHMT 3'-UTR in the bicistronic construct. The identification of this H ferritin-responsive IRES represents a mechanism that accounts for previous observations that H ferritin regulates folate metabolism.

Lateral Diffusion of the GABAB Receptor Is Regulated by the GABAB2 C Terminus

GABAB (gamma-aminobutyric acid, type B) is a heterodimeric G-protein-coupled receptor. The GABAB1 subunit, which contains an endoplasmic reticulum retention sequence, is only transported to the cell surface when it is associated with the GABAB2 subunit. Fluorescence recovery after photobleaching studies in transfected COS-7 cells and hippocampal neurons revealed that GABAB2 diffuses slowly within the plasma membrane whether expressed alone or with the GABAB1 subunit. Treatment of cells with brefeldin A revealed that GABAB2 moves freely within the endoplasmic reticulum, suggesting that slow movement of GABAB2 is a result of its plasma membrane insertion. Disruption of the cytoskeleton did not affect the mobility of GABAB2, indicating that its restricted diffusion is not due to direct interactions with actin or tubulin. To determine whether the C terminus of GABAB2 regulates its diffusion, this region of the subunit was attached to the lymphocyte membrane protein, CD2, which then exhibited a slower rate of lateral diffusion. Furthermore, co-expression of a cytoplasmically expressed soluble form of the GABAB2 C terminus increased movement of the GABAB2 subunit. We constructed forms of GABAB2 with various C-terminal truncations. Truncation of GABAB2 after residue 862, but not residue 886, caused a dramatic increase in its mobility, suggesting that the region between these two residues is critical for restricting GABAB2 diffusion. Finally, we investigated whether activation of GABAB might modulate its movement. Treatment of COS-7 cells with the GABAB receptor agonist baclofen significantly increased its mobile fraction. These data show that the restricted movement of GABAB at the cell surface is regulated by a region within its C terminus.

Essential, Completely Conserved Glycine Residue in the Domain III S2–S3 Linker of Voltage-gated Calcium Channel α1 Subunits in Yeast and Mammals

Voltage-gated Ca2+ channels (VGCCs) mediate the influx of Ca2+ that regulates many cellular events, and mutations in VGCC genes cause serious hereditary diseases in mammals. The yeast Saccharomyces cerevisiae has only one gene encoding the putative pore-forming alpha1 subunit of VGCC, CCH1. Here, we identify a cch1 allele producing a completely nonfunctional Cch1 protein with a Gly1265 to Glu substitution present in the domain III S2-S3 cytoplasmic linker. Comparison of amino acid sequences of this linker among 58 VGCC alpha1 subunits from 17 species reveals that a Gly residue whose position corresponds to that of the Cch1 Gly1265 is completely conserved from yeasts to humans. Systematic amino acid substitution analysis using 10 amino acids with different chemical and structural properties indicates that the Gly1265 is essential for Cch1 function because of the smallest residue volume. Replacement of the Gly959 residue of a rat brain Cav1.2 alpha1 subunit (rbCII), positionally corresponding to the yeast Cch1 Gly1265, with Glu, Ser, Lys, or Ala results in the loss of Ba2+ currents, as revealed by the patch clamp method. These results suggest that the Gly residue in the domain III S2-S3 linker is functionally indispensable from yeasts to mammals. Because the Gly residue has never been studied in any VGCC, these findings provide new insights into the structure-function relationships of VGCCs.

Role of Laminin Terminal Globular Domains in Basement Membrane Assembly

Laminins contribute to basement membrane assembly through interactions of their N- and C-terminal globular domains. To further analyze this process, recombinant laminin-111 heterotrimers with deletions and point mutations were generated by recombinant expression and evaluated for their ability to self-assemble, interact with nidogen-1 and type IV collagen, and form extracellular matrices on cultured Schwann cells by immunofluorescence and electron microscopy. Wild-type laminin and laminin without LG domains polymerized in contrast to laminins with deleted alpha1-, beta1-, or gamma1-LN domains or with duplicated beta1- or alpha1-LN domains. Laminins with a full complement of LN and LG domains accumulated on cell surfaces substantially above those lacking either LN or LG domains and formed a lamina densa. Accumulation of type IV collagen onto the cell surface was found to require laminin with separate contributions arising from the presence of laminin LN domains, nidogen-1, and the nidogen-binding site in laminin. Collectively, the data support the hypothesis that basement membrane assembly depends on laminin self-assembly through formation of alpha-, beta-, and gamma-LN domain complexes and LG-mediated cell surface anchorage. Furthermore, type IV collagen recruitment into the laminin extracellular matrices appears to be mediated through a nidogen bridge with a lesser contribution arising from a direct interaction with laminin.