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PubMed1998

Structure, organization, and chromosomal mapping of the gene encoding macrosialin, a macrophage-restricted protein.

Murine macrosialin and its human homologue CD68 are heavily glycosylated transmembrane proteins expressed specifically in macrophages and macrophage-related cells. Macrosialin is predominantly a late endosomal protein but is also found on the cell surface where it binds oxidized LDL, an important factor in atherogenesis. We have cloned and sequenced the murine macrosialin gene (Cd68) and localized it by linkage analysis to chromosome 11. The gene is 1908 nucleotides long from the start site of transcription to the end of the 3'UTR. It has six exons, which range in size from 79 to 434 nucleotides. The promoter lacks a classical TATA box but contains other protein binding sites consistent with preferential monocyte/macrophage gene expression. Although the function of macrosialin is unknown, it might play a role in lipoprotein regulation given its binding of oxidized LDL in vitro and its colocalization to a region on chromosome 11 involved in the control of HDL levels.

Animals↗
PubMed2003

Fibroblast growth factor-inducible-14 is induced in axotomized neurons and promotes neurite outgrowth.

For successful nerve regeneration, a coordinated shift in gene expression pattern must occur in axotomized neurons. To identify genes participating in axonal regeneration, we characterized mRNA expression profiles in dorsal root ganglia (DRG) before and after sciatic nerve transection. Dozens of genes are differentially expressed after sciatic nerve injury by microarray analysis. Induction of SOX11, FLRT3, myosin-X, and fibroblast growth factor-inducible-14 (Fn14) mRNA in axotomized DRG neurons was verified by Northern analysis and in situ hybridization. The Fn14 gene encodes a tumor necrosis-like weak inducer of apoptosis (TWEAK) receptor and is dramatically induced in DRG neurons after nerve damage, despite low expression in developing DRG neurons. Fn14 expression in PC12 cells is also upregulated by nerve growth factor treatment. Overexpression of Fn14 promotes growth cone lamelipodial formation and increases neurite outgrowth in PC12 cells. These Fn14 effects are independent of the ligand, TWEAK. Fn14 colocalizes with the Rho family GTPases, Cdc42 and Rac1. Furthermore, Fn14 physically associates with Rac1 GTPase in immunoprecipitation studies. The neurite outgrowth-promoting effect of Fn14 is enhanced by Rac1 activation and suppressed by Rac1 inactivation. These findings suggest that Fn14 contributes to nerve regeneration via a Rac1 GTPase-dependent mechanism.

Animals↗
PubMed1994

Germ plasm formation and germ cell determination in Drosophila.

In organisms as diverse as frogs, worms and flies germline precursor cells are set aside from the somatic cells early in development. It has been proposed that specific molecules, referred to as germ cell determinants, are deposited in the egg and direct the germ cell fate, but the molecular nature and function of these determinants is not fully understood. Genetic and molecular analysis in Drosophila melanogaster indicates that germ cell determination involves not only the synthesis of specific germ cell factors but also the proper localization and assembly of a morphologically distinct germ plasm. A pathway for germ plasm assembly has been established in which the oskar gene has a central role. The amount of oskar product in the embryo controls the number of germ cells formed and mislocalization of oskar RNA and protein in the egg cell leads to the formation of ectopic germ cells in the embryo. In addition to its role in anchoring germ cell-specific signals, the germ plasm also serves as the source of abdomen-specific signal. Such a colocalization of morphogenetic signals involved in germ cell formation and in the specification of the body axis is not unique to Drosophila but is also found in Caenorhabditis elegans and Xenopus.

Animals↗
PubMed1991

Accumulation and localization of two adult acinar cell secretory proteins during development of the rat submandibular gland.

The seromucous acinar cells of the adult rat submandibular gland secrete a characteristic mucin glycoprotein and a family of unusual glutamine/glutamic acid-rich proteins (GRP). Monoclonal antibodies to the mucin and GRP localized in a very few Type III cells in glands of newborn and 1 day-old rats, using light and electron microscopic immunocytochemistry. Both mucin and GRP reactivities were present in the polymorphic Type IIIP granules during the 1st postnatal week. By 9 days after birth, the granules contained both mucin and GRP and were mucous-like in appearance. At earlier stages, however, cells containing only GRP or mucin could be found, indicating that the initiation of GRP and mucin biosynthesis may not be coordinately regulated. No reactivity was seen in the neonatal Type I cells or in duct cells at any age. Northern and Western blot analysis showed GRP mRNA and protein levels to be barely detectable at birth, with marked increases during the first 2 postnatal weeks. In contrast, Western blots of B1-immunoreactive proteins (B1-IP) showed levels highest in the 1st week and markedly decreased in the adult. Immunocytochemical colocalization, using gold particles of different sizes, showed that the B1-IP, mucin, and GRP colocalized in the granules. These results strengthen the hypothesis that the adult acinar cells develop from the neonatal Type III cells. No evidence was obtained for the involvement of Type I cells in the pathway of acinar cell development.

Aging↗
PubMed1995

Dephosphorylation of protein kinase C substrates, neurogranin, neuromodulin, and MARCKS, by calcineurin and protein phosphatases 1 and 2A.

Neurogranin, neuromodulin, and MARCKS are among the most prominent substrates of protein kinase C (PKC) in the mammalian brain. These phosphoproteins were dephosphorylated by three isoforms of rat brain calcineurin, also known as calmodulin (CaM)-dependent protein phosphatase (CaMPP). The three CaMPP isozymes dephosphorylate neurogranin, the most favorable substrate among the three tested, with subtle differences in their responses to divalent metal ions, Mn2+ and Ni2+. Dephosphorylation of neurogranin by all three CaMPP isozymes, CaMPP-1, -2, and -3, were stimulated to a higher extent by Mn2+ than by Ni2+ in the presence of CaM and Ca2+. The Km values of neurogranin in the presence of Mn2+ were lower than those in the presence of Ni2+ for CaMPP-1 and -2, but that for CaMPP-3 was comparable with either divalent metal ion. The Vmax values were higher in the presence of Mn2+ than those of Ni2+ for all three isozymes. Neurogranin and neuromodulin, both phosphorylated by PKC at a single site, were dephosphorylated completely by CaMPP; however, MARCKS, phosphorylated by PKC at three sites, was partially dephosphorylated by this phosphatase. A higher extent of dephosphorylation of MARCKS could be achieved by the combination of CaMPP and protein phosphatase 2A and a complete dephosphorylation of this protein was observed with protein phosphatase 1. Protein phosphatase 1 and 2A were also effective in a complete dephosphorylation of neurogranin and neuromodulin. Amino acid sequence analysis of the tryptic phosphopeptides derived from MARCKS dephosphorylated by CaMPP and protein phosphatase 2A revealed that the former preferentially dephosphorylated Ser155 and the latter Ser162 of rat brain MARCKS. Both phosphatases dephosphorylated poorly of Ser151. Because of the high concentration of CaMPP in the brain and the colocalization of this phosphatase with major PKC substrates in the various brain regions, it is likely that CaMPP is a phosphatase with potential to reverse the action of PKC.

Amino Acid Sequence↗
PubMed2006

Ischemia induces nuclear NOX2 expression in cardiomyocytes and subsequently activates apoptosis.

In previous work we have demonstrated increased expression of NOX2 in cardiomyocytes of infarcted human hearts. In the present manuscript we investigated the functional role of NOX2 in ischemically challenged H9c2 cells, a rat cardiomyoblast cell line, and adult rat cardiomyocytes. Expression of NOX2 in H9c2 cells was confirmed by RT-PCR. In Western-blot experiments, increased NOX2 expression was detected during ischemia, which was inhibited by transcription and translation inhibitors. Surprisingly, under ischemia, in addition to an increased cytosolic expression, NOX2 was localized mainly in the nucleus of apoptotic cardiomyocytes, where it colocalized with nitrotyrosine residues and activated caspase 3. Inhibition of reactive-oxygen-species generation with the flavoenzyme inhibitor diphenylene iodonium (DPI) and the NADPH-oxidase inhibitor apocynin led to a significantly decreased induction of apoptosis as assessed by quantification of caspase-3 activity and by TUNEL analysis. These results demonstrate that NOX2 is expressed in the nucleus of cardiomyocytes during apoptosis and that it likely participates in proapoptotic signaling. To the best of our knowledge, this is the first demonstration of nuclear NOX2 expression and its involvement in cardiomyocyte apoptosis.

Animals↗
PubMed2005

Protein 4.1R expression in normal and dystrophic skeletal muscle.

4.1R pre-mRNA alternative splicing results in multiple mRNA and protein isoforms that are expressed in virtually all tissues. More specifically, isoforms containing the alternative exon 17a, are exclusively expressed in muscle tissues. In this report, we show that these isoforms are preferentially present in the myoplasm of fast myofibres. 4.1R epitopes are also found at the sarcolemma of both slow and fast myofibres in normal muscle. Interestingly, they are absent from dystrophin-deficient sarcolemma of DMD muscle, and colocalize with partially expressed dystrophin in BMD muscle. We also show that alternative splicing of exons 16 and 17a is regulated during muscle differentiation in an asynchronous fashion, with an early inclusion of exon 16 in forming myotubes, and a late inclusion of exon 17a. Consistently, Western blot analysis led to characterize mainly an approximately 96/98-kDa doublet bearing exons 16-17a-encoding peptide, exclusively occurring in the differentiated muscle.

Alternative Splicing↗
PubMed1998

Seizure induced synthesis of fibronectin is rapid and age dependent: implications for long-term potentiation and sprouting.

Extracellular matrix proteins are induced by activity in adult brain but the time course of these responses, and hence the possibility of their involvement in use-dependent synaptic plasticity, is not known. To evaluate this issue, the influence of seizures on fibronectin expression was evaluated in the adult and developing hippocampus. In adult rats, kainic acid-induced seizures increased fibronectin mRNA and immunoreactivity (ir) by about 1 h after the first behavioral seizure. In situ hybridization analysis indicated that fibronectin mRNA was increased in broadly distributed glial cells as well as within discrete neuronal populations that normally express this transcript. Western blots demonstrated that increased fibronectin-ir was evident in both soluble and non-soluble fractions at the same time point. Immunocytochemical colocalization confirmed that fibronectin-ir was indeed elevated in broadly distributed glial fibrillary acidic protein-ir astroglia. Seizures had no detectable effect on fibronectin-ir in the hippocampus of nine day old rats. Together with previous results, the above findings suggest that intense physiological activity triggers a 'matrix response' (i.e., release proteases, activate integrins, secrete matrix proteins) that is sufficiently rapid to participate in the consolidation of long-term potentiation (LTP). The absence of such reactions in the immature hippocampus is in accord with the hypothesis that matrix proteins generated by mature astroglia impose temporal and spatial limitations on axonal remodeling.

Aging↗
PubMed2003

Evaluation of hypoxia-inducible factor-1alpha (HIF-1alpha) as an intrinsic marker of tumor hypoxia in U87 MG human glioblastoma: in vitro and xenograft studies.

PURPOSE: The transcription factor subunit hypoxia-inducible factor-1alpha (HIF-1alpha) is a key regulatory element of the hypoxic response of cells. High protein levels have been linked to poor prognosis in several tumor types, and HIF-1alpha has been suggested as a potential endogenous marker of tumor hypoxia and associated radioresistance. METHODS AND MATERIALS: HIF-1alpha expression following in vitro hypoxia was measured in U87 MG glioblastoma cells by Western blot and flow cytometry. Cell suspensions from U87 MG xenograft tumors grown in SCID mice were assayed by flow cytometry for HIF-1alpha and for pimonidazole as a reference hypoxia marker. After 1 h, 6 h, and 18 h of in vitro hypoxia, a constant increase in HIF-1alpha protein levels with decreasing oxygen concentrations between 20% and <0.02% was observed by both Western blot and flow cytometry, correlating with the pattern of pimonidazole labeling after in vitro hypoxia. In U87 MG xenograft tumors, flow-cytometric analysis of HIF-1alpha and pimonidazole showed a significant correlation of the two markers, but distinction of a HIF-1alpha-positive population was affected by a low dynamic range of the signal. As in published studies for HIF-1alpha and the hypoxic marker EF5, the colocalization of HIF-1alpha and pimonidazole in double-staining experiments was low. CONCLUSIONS: While the in vitro data in U87 MG human glioblastoma cells support the use of HIF-1alpha as an endogenous hypoxia marker, comparison with the standard pimonidazole makes its application to clinical material appear questionable.

Animals↗
PubMed2001

Semaphorin4F interacts with the synapse-associated protein SAP90/PSD-95.

Semaphorins are a family of secreted and membrane-associated proteins involved in growth cone guidance during development. Here, we describe the interaction of Semaphorin4F (Sema4F) with the post-synaptic density protein SAP90/PSD-95. Using the yeast two-hybrid system and coprecipitation assays we were able to show an interaction between the extreme C-terminus of Sema4F and the PDZ domains of SAP90/PSD-95. Heterologous coexpression of a chimeric EphrinB1/Semaphorin4F protein with SAP90/PSD-95 in COS cells leads to translocation of SAP90/PSD-95 from the cytosol to the membrane. Deletion analysis shows that this translocation activity of Sema4F is completely dependent on the presence of the last three C-terminal amino acids. In addition, Sema4F immunoreactivity is present in synaptosome fractions and enriched in post-synaptic density fractions. Consistently, in cultured hippocampal neurons, we demonstrate punctate colocalization of Sema4F and SAP90/PSD-95 in dendrites, furthermore we found colocalization of Sema4F with synapsin1 suggesting a synaptic localization. Our data implicate a new functional context for semaphorins at glutamatergic synapses.

Amino Acid Sequence↗
PubMed2000

Nuclear matrix protein is released from apoptotic white cells during cold (1-6 degrees C) storage of concentrated red cell units and might induce antibody response in multiply transfused patients.

BACKGROUND: A previous study showed that white cells in blood units undergo apoptosis during storage. STUDY DESIGN AND METHODS: The present study attempts to show the release of nuclear matrix protein (NMP) in the supernatants of red cell units and to determine whether antibodies against nuclear components may be present in multiply transfused patients; the methods employed were enzyme-linked immunosorbent assay, flow cytometry, microscopy, immunoblotting, immunofluorescence, and confocal laser-scanning microscopy. RESULTS: NMP is released from white cells in the supernatant of packed red cell units upon cold storage (1-6 degrees C). The concentration of NMP correlates well with the degree of apoptosis, as analyzed by flow cytometry, nuclear dye staining, and DNA gel electrophoresis. Immunofluorescence also shows that white cells undergoing apoptosis (pre-G(1) peak, as seen by propidium iodide staining and flow cytometry) have an NMP content lower than control cells, which confirms an actual release of NMP. Moreover, immunoblotting analysis and immunofluorescent staining showed that, in 4 of 38 multiply transfused patients, autoantibodies against NMPs were present without any clinical or laboratory sign of autoimmune disease. One of the sera, recognizing a 64-kDa NMP, immunostained nuclear dots that were identified as coiled bodies because of their colocalization with p 80 coilin. CONCLUSION: NMP is released in the supernatant of red cell units. The results obtained from patients suggest that nuclear proteins released during apoptosis, once transfused, may induce an immune response in multiply transfused patients.

Antibodies↗
PubMed2000

Cloning and characterization of the mammalian brain-specific, Mg2+-dependent neutral sphingomyelinase.

The enzymatic breakdown of sphingomyelin by sphingomyelinases is considered the major source of the second messenger ceramide. Studies on the contribution of the various described acidic and neutral sphingomyelinases to the signaling pool of ceramide have been hampered by the lack of molecular data on the neutral sphingomyelinases (nSMases). We recently identified a mammalian nSMase, an integral membrane protein with remote similarity to bacterial sphingomyelinases. However, its ubiquitous expression pattern is in contrast to previous findings that sphingomyelinase activity is found mainly in brain tissues. By using an improved database search method, combined with phylogenetic analysis, we identified a second mammalian nSMase (nSMase2) with predominant expression in the brain. The sphingomyelinase activity of nSMase2 has a neutral pH optimum, depends on Mg(2+) ions, and is activated by unsaturated fatty acids and phosphatidylserine. Immunofluorescence reveals a neuron-specific punctate perinuclear staining, which colocalizes with a Golgi marker in a number of cell lines. The likely identity of nSMase2 with cca1, a rat protein involved in contact inhibition of 3Y1 fibroblasts, suggests a role for this enzyme in cell cycle arrest. Both mammalian nSMases are members of a superfamily of Mg(2+)-dependent phosphohydrolases, which also contains nucleases, inositol phosphatases, and bacterial toxins.

Amino Acid Sequence↗
PubMed2004

Human atherosclerotic intima and blood of patients with established coronary artery disease contain high density lipoprotein damaged by reactive nitrogen species.

High density lipoprotein (HDL) is the major carrier of lipid hydroperoxides in plasma, but it is not yet established whether HDL proteins are damaged by reactive nitrogen species in the circulation or artery wall. One pathway that generates such species involves myeloperoxidase (MPO), a major constituent of artery wall macrophages. Another pathway involves peroxynitrite, a potent oxidant generated in the reaction of nitric oxide with superoxide. Both MPO and peroxynitrite produce 3-nitrotyrosine in vitro. To investigate the involvement of reactive nitrogen species in atherogenesis, we quantified 3-nitrotyrosine levels in HDL in vivo. The mean level of 3-nitrotyrosine in HDL isolated from human aortic atherosclerotic intima was 6-fold higher (619 +/- 178 micromol/mol Tyr) than that in circulating HDL (104 +/- 11 micromol/mol Tyr; p < 0.01). Immunohistochemical studies demonstrated striking colocalization of MPO with epitopes reactive with an antibody to 3-nitrotyrosine. However, there was no significant correlation between the levels of 3-chlorotyrosine, a specific product of MPO, and those of 3-nitrotyrosine in lesion HDL. We also detected 3-nitrotyrosine in circulating HDL, and linear regression analysis demonstrated a strong correlation between the levels of 3-chlorotyrosine and levels of 3-nitrotyrosine. These observations suggest that MPO promotes the formation of 3-chlorotyrosine and 3-nitrotyrosine in circulating HDL but that other pathways also produce 3-nitrotyrosine in atherosclerotic tissue. Levels of HDL isolated from plasma of patients with established coronary artery disease contained twice as much 3-nitrotyrosine as HDL from plasma of healthy subjects, suggesting that nitrated HDL might be a marker for clinically significant vascular disease. The detection of 3-nitrotyrosine in HDL raises the possibility that reactive nitrogen species derived from nitric oxide might promote atherogenesis. Thus, nitrated HDL might represent a previously unsuspected link between nitrosative stress, atherosclerosis, and inflammation.

Arteries↗
PubMed2000

Observing FcepsilonRI signaling from the inside of the mast cell membrane.

We have determined the membrane topography of the high-affinity IgE receptor, FcstraightepsilonRI, and its associated tyrosine kinases, Lyn and Syk, by immunogold labeling and transmission electron microscopic (TEM) analysis of membrane sheets prepared from RBL-2H3 mast cells. The method of Sanan and Anderson (Sanan, D.A., and R.G.W. Anderson. 1991. J. Histochem. Cytochem. 39:1017-1024) was modified to generate membrane sheets from the dorsal surface of RBL-2H3 cells. Signaling molecules were localized on the cytoplasmic face of these native membranes by immunogold labeling and high-resolution TEM analysis. In unstimulated cells, the majority of gold particles marking both FcepsilonRI and Lyn are distributed as small clusters (2-9 gold particles) that do not associate with clathrin-coated membrane. Approximately 25% of FcepsilonRI clusters contain Lyn. In contrast, there is essentially no FcepsilonRI-Syk colocalization in resting cells. 2 min after FcepsilonRI cross-linking, approximately 10% of Lyn colocalizes with small and medium-sized FcepsilonRI clusters (up to 20 gold particles), whereas approximately 16% of Lyn is found in distinctive strings and clusters at the periphery of large receptor clusters (20-100 gold particles) that form on characteristically osmiophilic membrane patches. While Lyn is excluded, Syk is dramatically recruited into these larger aggregates. The clathrin-coated pits that internalize cross-linked receptors bud from membrane adjacent to the Syk-containing receptor complexes. The sequential association of FcstraightepsilonRI with Lyn, Syk, and coated pits in topographically distinct membrane domains implicates membrane segregation in the regulation of FcstraightepsilonRI signaling.

Animals↗
PubMed2004

Functional correlation of P-glycoprotein expression and genotype with expression of the human immunodeficiency virus type 1 coreceptor CXCR4.

The aim of this study was to investigate the relationship between lymphocyte P-glycoprotein (P-gp) expression and genotype in vivo and the expression of lymphocyte receptors critical in the life cycle of human immunodeficiency virus type 1 (HIV-1), i.e., CD4, CCR5, and CXCR4. Using flow cytometry to quantify each membrane receptor/transporter, we demonstrate a highly significant correlation between P-gp protein expression and the expression of CXCR4 (rho = 0.874; P < 0.0001). Furthermore, confocal microscopy showed colocalized expression of CXCR4 and P-gp in the lymphocyte membrane. This significant relationship was also apparent at the mRNA level by use of reverse transcription-PCR (rho = 0.61; P < 0.005) and was present in both phytohemagglutinin-stimulated and unstimulated peripheral blood mononuclear cells. Genotypic analysis of the C3435T single-nucleotide polymorphism of P-gp confirmed significantly higher levels of P-gp in C (range, 2.45 to 11.00 relative fluorescence units [RFU])- than in T (range, 0.25 to 5.00 RFU)-homozygous individuals (P = 0.0088; 95% confidence interval [95% CI], 0.7 to 6.3 RFU). An equivalent association between CXCR4 levels and C (range, 12.7 to 44.1 RFU) versus T (range, 3 to 18.9 RFU) genotype was also demonstrated (P = 0.0019; 95% CI, 5.4 to 23.7). Functionally, although these correlates had no impact on HIV-1 production from either X4- or R5-tropic virus, expression correlated significantly with the activity of the HIV-1 protease inhibitor (PI) saquinavir for both P-gp (rho = 0.75; P = 0.0019) and CXCR4 (rho = 0.71; P = 0.0041). This study defines an association between P-gp (expression and genotype) and CXCR4 that may have implications for the selection of viral tropism and the access of drugs to protease for specific tropic types. The interplay between these two proteins may also influence the viral genotypes which escape effective chemotherapy and which therefore have the opportunity to evolve resistance to PIs.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2004

Sequestosome 1/p62 is a polyubiquitin chain binding protein involved in ubiquitin proteasome degradation.

Herein, we demonstrate that the ubiquitin-associated (UBA) domain of sequestosome 1/p62 displays a preference for binding K63-polyubiquitinated substrates. Furthermore, the UBA domain of p62 was necessary for aggregate sequestration and cell survival. However, the inhibition of proteasome function compromised survival in cells with aggregates. Mutational analysis of the UBA domain reveals that the conserved hydrophobic patch MGF as well as the conserved leucine in helix 2 are necessary for binding polyubiquitinated proteins and for sequestration-aggregate formation. We report that p62 interacts with the proteasome by pull-down assay, coimmunoprecipitation, and colocalization. Depletion of p62 levels results in an inhibition of ubiquitin proteasome-mediated degradation and an accumulation of ubiquitinated proteins. Altogether, our results support the hypothesis that p62 may act as a critical ubiquitin chain-targeting factor that shuttles substrates for proteasomal degradation.

Adaptor Proteins, Signal Transducing↗
PubMed1995

Expression of multiple connexins in cultured neonatal rat ventricular myocytes.

Three gap junction proteins have been identified in mammalian cardiac myocytes: connexin43 (Cx43), connexin45 (Cx45), and connexin40 (Cx40). These proteins form channels with different electrophysiological properties and have different distributions in cardiac tissues with disparate conduction properties. We characterized the expression, phosphorylation, turnover, and subcellular distribution of these connexins in primary cultures of neonatal rat ventricular myocytes. Cx43, Cx45, and Cx40 mRNA were specifically detected in RNA blots. Immunofluorescent staining with antibodies specific for Cx43 and Cx45 revealed punctate labeling at appositional membranes, but no immunoreactive Cx40 was detected. Double-label immunofluorescence confocal microscopy of cultured myocytes revealed colocalization of Cx43 and Cx45. Cx43 and Cx45 were both identified by immunoprecipitation from [35S]methionine-labeled cultures, but anti-Cx40 antibodies did not precipitate any radiolabeled protein. Phosphorylated forms of both Cx45 and Cx43 were immunoprecipitated from cultures metabolically labeled with [32P]orthophosphate. Phosphoamino acid analysis demonstrated that Cx45 was modified on serine residues, and Cx43 was phosphorylated on serine and threonine residues. Pulse-chase labeling experiments demonstrated that the half-lives of Cx43 and Cx45 were 1.9 and 2.9 hours, respectively. Thus, both Cx43 and Cx45 turn over relatively rapidly, suggesting that myocardial gap junctions have the potential for dynamic remodeling. The results implicate multiple mechanisms of gap junction regulation that may differ for different connexins.

Animals↗
PubMed2003

Pinin/DRS/memA interacts with SRp75, SRm300 and SRrp130 in corneal epithelial cells.

PURPOSE: Pinin (Pnn/DRS/memA) is a cell-adhesion-related and nuclear protein that has been identified as central in the establishment and maintenance of corneal epithelial cell-cell adhesion. To begin the elucidation of the role of Pnn within the nucleus of corneal epithelial cells, this study was undertaken to identify the proteins that bind to Pnn. METHODS: Yeast two-hybrid analyses were performed. A human cDNA library in the pGAD-10 vector and C-terminal region of human Pnn (465-717) in a pAS2-1 vector were cotransformed into the PJ69-4A yeast strain, containing the lacZ, HIS3, and ADE2 reporter genes. To dissect domains of Pnn responsible for mediating the interaction with the identified proteins, PNN fragments were ligated with the DNA-binding domain of the pAS2-1 vector. Human corneal epithelial cells (HCE-T, RCB1384) and HEK-293 cells were cotransfected with mammalian expression vectors containing Pnn with identified interacting partners and subsequently immunostained and immunoblotted to determine expressed and endogenous proteins. RESULTS: Pnn colocalized and copurified with serine-arginine (SR) proteins. Three SR-rich proteins were identified that interact with the C-terminus of Pnn: SRp75 and SRm300, known components of spliceosome machinery, and a novel 130-kDa nuclear protein, SRrp130. All of these proteins colocalized and coimmunoprecipitated with one another and exhibited speckled nuclear distribution that aligned with components of the pre-mRNA splicing machinery. The cDNA for SRrp130 encoded a protein of 805 amino acid residues and contained multiple arginine-serine (RS) repeats but had no RNA recognition motif. Analysis of the Pnn motifs using two-hybrid system assays demonstrated that the polyserine/RS motif within Pnn plays a central but not exclusive role in mediating molecular interactions with identified SR-rich proteins. CONCLUSIONS: The results suggest that Pnn and SR-rich proteins may be part of a multiprotein complex within the nucleus and may be involved in pre-mRNA processing.

Amino Acid Sequence↗