The distal cytoplasmic tail of the influenza A M2 protein dynamically extends from the membrane

The influenza A M2 protein is a multifunctional membrane-associated homotetramer that orchestrates several essential events in the viral infection cycle. The monomeric subunits of the M2 homotetramer consist of an N-terminal ectodomain, a transmembrane domain, and a C-terminal cytoplasmic domain. The transmembrane domain forms a four-helix proton channel that promotes uncoating of virions upon host cell entry. The membrane-proximal region of the C-terminal domain forms a surface-associated amphipathic helix necessary for viral budding. The structure of the remaining ~34 residues of the distal cytoplasmic tail has yet to be fully characterized despite the functional significance of this region for influenza infectivity. Here, we extend structural and dynamic studies of the poorly characterized M2 cytoplasmic tail. We used SDSL-EPR to collect site-specific information on the mobility, solvent accessibility, and conformational properties of residues 61–70 of the full-length, cell-expressed M2 protein reconstituted into liposomes. Our analysis is consistent with the predominant population of the C-terminal tail dynamically extending away from the membranes surface into the aqueous medium. These findings provide insight into the hypothesis that the C-terminal domain serves as a sensor that regulates how M2 protein participates in critical events in the viral infection cycle.     

A nuclear localization domain in the hnRNP A1 protein

The heterogeneous nuclear RNP (hnRNP) A1 protein is one of the major pre-mRNA/mRNA binding proteins in eukaryotic cells and one of the most abundant proteins in the nucleus. It is localized to the nucleoplasm and it also shuttles between the nucleus and the cytoplasm. The amino acid sequence of A1 contains two RNP motif RNA-binding domains (RBDs) at the amino terminus and a glycine-rich domain at the carboxyl terminus. This configuration, designated 2x RBD-Gly, is representative of perhaps the largest family of hnRNP proteins. Unlike most nuclear proteins characterized so far, A1 (and most 2x RBD-Gly proteins) does not contain a recognizable nuclear localization signal (NLS). We have found that a segment of ca. 40 amino acids near the carboxyl end of the protein (designated M9) is necessary and sufficient for nuclear localization; attaching this segment to the bacterial protein beta- galactosidase or to pyruvate kinase completely localized these otherwise cytoplasmic proteins to the nucleus. The RBDs and another RNA binding motif found in the glycine-rich domain, the RGG box, are not required for A1 nuclear localization. M9 is a novel type of nuclear localization domain as it does not contain sequences similar to classical basic-type NLS. Interestingly, sequences similar to M9 are found in other nuclear RNA-binding proteins including hnRNP A2.     

Effect of cytoplasmic tail truncations on the activity of the M(2) ion channel of influenza A virus

The M(2) protein of influenza A virus forms a proton channel that is required for viral replication. The M(2) ion channel is a homotetramer and has a 24-residue N-terminal extracellular domain, a 19-residue transmembrane domain, and a 54-residue cytoplasmic tail. We show here that the N-terminal methionine residue is cleaved from the mature protein. Translational stop codons were introduced into the M(2) cDNA at residues 46, 52, 62, 72, 77, 82, 87, and 92. The deletion mutants were designated truncx, according to the amino acid position that was changed to a stop codon. We studied the role of the cytoplasmic tail by measuring the ion channel activity (the current sensitive to the M(2)-specific inhibitor amantadine) of the cytoplasmic tail truncation mutants expressed in oocytes of Xenopus laevis. When their conductance was measured over time, mutants trunc72, trunc77, and trunc92 behaved comparably to wild-type M(2) protein (a decrease of only 4% over 30 min). In contrast, conductance decreased by 28% for trunc82, 27% for trunc62, and 81% for trunc52 channels. Complete closure of the channel could be observed in some cells for trunc62 and trunc52 within 30 min. These data suggest that a role of the cytoplasmic tail region of the M(2) ion channel is to stabilize the pore against premature closure while the ectodomain is exposed to low pH.     

Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-ATPase β1 subunit interacts with M2 proteins of influenza A and B viruses and affects the virus replication

Interplay between the host and influenza virus has a pivotal role for the outcome of infection. The matrix proteins M2/BM2 from influenza (A and B) viruses are small type III integral membrane proteins with a single transmembrane domain, a short amino-terminal ectodomain and a long carboxy-terminal cytoplasmic domain. They function as proton channels, mainly forming a membrane-spanning pore through the transmembrane domain tetramer, and are essential for virus assembly and release of the viral genetic materials in the endosomal fusion process. However, little is known about the host factors which interact with M2/BM2 proteins and the functions of the long cytoplasmic domain are currently unknown. Starting with yeast two-hybrid screening and applying a series of experiments we identified that the β1 subunit of the host Na⁺/K⁺-ATPase β1 subunit (ATP1B1) interacts with the cytoplasmic domain of both the M2 and BM2 proteins. A stable ATP1B1 knockdown MDCK cell line was established and we showed that the ATP1B1 knockdown suppressed influenza virus A/WSN/33 replication, implying that the interaction is crucial for influenza virus replication in the host cell. We propose that influenza virus M2/BM2 cytoplasmic domain has an important role in the virus-host interplay and facilitates virus replication.     

The cytoplasmic tail of the influenza A virus M2 protein plays a role in viral assembly

The viral replication cycle concludes with the assembly of viral components to form progeny virions. For influenza A viruses, the matrix M1 protein and two membrane integral glycoproteins, hemagglutinin and neuraminidase, function cooperatively in this process. Here, we asked whether another membrane protein, the M2 protein, plays a role in virus assembly. The M2 protein, comprising 97 amino acids, possesses the longest cytoplasmic tail (54 residues) of the three transmembrane proteins of influenza A viruses. We therefore generated a series of deletion mutants of the M2 cytoplasmic tail by reverse genetics. We found that mutants in which more than 22 amino acids were deleted from the carboxyl terminus of the M2 tail were viable but grew less efficiently than did the wild-type virus. An analysis of the virions suggested that viruses with M2 tail deletions of more than 22 carboxy-terminal residues apparently contained less viral ribonucleoprotein complex than did the wild-type virus. These M2 tail mutants also differ from the wild-type virus in their morphology: while the wild-type virus is spherical, some of the mutants were filamentous. Alanine-scanning experiments further indicated that amino acids at positions 74 to 79 of the M2 tail play a role in virion morphogenesis and affect viral infectivity. We conclude that the M2 cytoplasmic domain of influenza A viruses plays an important role in viral assembly and morphogenesis.     

The epitope for monoclonal antibody A20 (amino acids 870-890) is located on the luminal surface of the Ca2(+)-ATPase of sarcoplasmic reticulum.

The epitope for monoclonal antibody A20 was mapped to amino acids 870-890 of the Ca2(+)-ATPase of rabbit fast-twitch skeletal muscle sarcoplasmic reticulum. The antibody did not react with the epitope in intact sarcoplasmic reticulum vesicles but reacted with the epitope when the vesicles were solubilized with the detergent C12E8 or made permeable by incubation in a hypotonic medium. By contrast, antibody A52, which binds to a cytoplasmic epitope consisting of amino acids 657-672, reacted with the Ca2(+)-ATPase in vesicular, permeabilized vesicular, and C12E8-solubilized states. These results clearly demonstrate that antibody A20 binds to a luminal epitope and provide the first demonstration that a specific segment of the Ca2(+)-ATPase is located on the luminal surface of the sarcoplasmic reticulum. These results are consistent with, and support, our model for folding of the Ca2(+)-ATPase (Brandl, C. J., Korczak, B., Green, N. M., and MacLennan, D. H. (1986) Cell 44, 597-607) in which residues 657-672 were proposed to form part of the cytoplasmic nucleotide binding domain, while residues 870-890 were proposed to form a luminal loop between proposed transmembrane sequences M7 and M8.     

Intracellular trafficking of hnRNP A2 in oligodendrocytes

Heterogeneous ribonucleoprotein (hnRNP) A2 is a trans-acting factor that mediates intracellular trafficking of specific RNAs containing the A2 response element. HnRNP A2 is localized in the nucleus and also in granules in the perikaryon and processes in oligodendrocytes. The distribution of the cytoplasmic pool of hnRNP A2 is microtubule-dependent. HnRNP A2 is composed of two sequential RNA binding domains (RBDI and RBDII), a glycine-rich domain, and a nuclear import domain (M9). In order to analyze the roles of individual domains in determining the intracellular distribution of hnRNP A2, chimeric mRNAs encoding various domains fused with green fluorescent protein (GFP) were injected into oligodendrocytes, and the subcellular distribution of the GFP hybrid proteins was analyzed by fluorescence microscopy. Chimeric GFP proteins containing the M9 domain were localized to the nucleus. In the absence of the M9 domain, proteins containing the RBDII domain were preferentially concentrated in the distal processes of the cells. Localization of RBDII-containing proteins in the periphery was dependent on the presence of intact microtubules. These data suggest that the RBDII domain of hnRNP A2 targets hnRNP A2 to the periphery of the cell in a microtubule-dependent manner.     

A structural model for the catalytic cycle of Ca(2+)-ATPase

Ca(2+)-ATPase is responsible for active transport of calcium ions across the sarcoplasmic reticulum membrane. This coupling involves an ordered sequence of reversible reactions occurring alternately at the ATP site within the cytoplasmic domains, or at the calcium transport sites within the transmembrane domain. These two sites are separated by a large distance and conformational changes have long been postulated to play an important role in their coordination. To characterize the nature of these conformational changes, we have built atomic models for two reaction intermediates and postulated the mechanisms governing the large structural changes. One model is based on fitting the X-ray crystallographic structure of Ca(2+)-ATPase in the E1 state to a new 6 A structure by cryoelectron microscopy in the E2 state. This fit indicates that calcium binding induces enormous movements of all three cytoplasmic domains as well as significant changes in several transmembrane helices. We found that fluorescein isothiocyanate displaced a decavanadate molecule normally located at the intersection of the three cytoplasmic domains, but did not affect their juxtaposition; this result indicates that our model likely reflects a native E2 conformation and not an artifact of decavanadate binding. To explain the dramatic structural effect of calcium binding, we propose that M4 and M5 transmembrane helices are responsive to calcium binding and directly induce rotation of the phosphorylation domain. Furthermore, we hypothesize that both the nucleotide-binding and beta-sheet domains are highly mobile and driven by Brownian motion to elicit phosphoenzyme formation and calcium transport, respectively. If so, the reaction cycle of Ca(2+)-ATPase would have elements of a Brownian ratchet, where the chemical reactions of ATP hydrolysis are used to direct the random thermal oscillations of an innately flexible molecule.     

Influenza A virus can undergo multiple cycles of replication without M2 ion channel activity

Ion channel proteins are common constituents of cells and have even been identified in some viruses. For example, the M2 protein of influenza A virus has proton ion channel activity that is thought to play an important role in viral replication. Because direct support for this function is lacking, we attempted to generate viruses with defective M2 ion channel activity. Unexpectedly, mutants with apparent loss of M2 ion channel activity by an in vitro assay replicated as efficiently as the wild-type virus in cell culture. We also generated a chimeric mutant containing an M2 protein whose transmembrane domain was replaced with that from the hemagglutinin glycoprotein. This virus replicated reasonably well in cell culture but showed no growth in mice. Finally, a mutant lacking both the transmembrane and cytoplasmic domains of M2 protein grew poorly in cell culture and showed no growth in mice. Thus, influenza A virus can undergo multiple cycles of replication without the M2 transmembrane domain responsible for ion channel activity, although this activity promotes efficient viral replication.     

Chemical ligation of the influenza M2 protein for solid‐state NMR characterization of the cytoplasmic domain

Solid‐state NMR‐based structure determination of membrane proteins and large protein complexes faces the challenge of limited spectral resolution when the proteins are uniformly ¹³C‐labeled. A strategy to meet this challenge is chemical ligation combined with site‐specific or segmental labeling. While chemical ligation has been adopted in NMR studies of water‐soluble proteins, it has not been demonstrated for membrane proteins. Here we show chemical ligation of the influenza M2 protein, which contains a transmembrane (TM) domain and two extra‐membrane domains. The cytoplasmic domain, which contains an amphipathic helix (AH) and a cytoplasmic tail, is important for regulating virus assembly, virus budding, and the proton channel activity. A recent study of uniformly ¹³C‐labeled full‐length M2 by spectral simulation suggested that the cytoplasmic tail is unstructured. To further test this hypothesis, we conducted native chemical ligation of the TM segment and part of the cytoplasmic domain. Solid‐phase peptide synthesis of the two segments allowed several residues to be labeled in each segment. The post‐AH cytoplasmic residues exhibit random‐coil chemical shifts, low bond order parameters, and a surface‐bound location, thus indicating that this domain is a dynamic random coil on the membrane surface. Interestingly, the protein spectra are similar between a model membrane and a virus‐mimetic membrane, indicating that the structure and dynamics of the post‐AH segment is insensitive to the lipid composition. This chemical ligation approach is generally applicable to medium‐sized membrane proteins to provide site‐specific structural constraints, which complement the information obtained from uniformly ¹³C, ¹⁵N‐labeled proteins.     

Regulation of alpha 6 beta 1 integrin laminin receptor function by the cytoplasmic domain of the alpha 6 subunit

The alpha 6 beta 1 integrin is expressed on the macrophage surface in an inactive state and requires cellular activation with PMA or cytokines to function as a laminin receptor (Shaw, L. M., J. M. Messier, and A. M. Mercurio. 1990. J. Cell Biol. 110:2167-2174). In the present study, the role of the alpha 6 subunit cytoplasmic domain in alpha 6 beta 1 integrin activation was examined. The use of P388D1 cells, an alpha 6-integrin deficient macrophage cell line, facilitated this analysis because expression of either the alpha 6A or alpha 6B subunit cDNAs restores their activation responsive laminin adhesion (Shaw, L. S., M. Lotz, and A. M. Mercurio. 1993. J. Biol. Chem. 268:11401-11408). A truncated alpha 6 cDNA, alpha 6-delta CYT, was constructed in which the human cytoplasmic domain sequence was deleted after the GFFKR pentapeptide. Expression of this cDNA in P388D1 cells resulted in the surface expression of a chimeric alpha 6-delta CYT beta 1 integrin that was unable to mediate laminin adhesion or increase this adhesion in response to PMA under normal conditions, i.e., in medium that contained physiological concentrations of Ca++ and Mg++. The alpha 6A-delta CYT transfectants adhered to laminin, however, when Ca++/Mg++ was replaced with 150 microM Mn++. We also assessed the role of serine phosphorylation in the regulation of alpha 6A beta 1 integrin function by site-directed mutagenesis of the two serine residues present in the alpha 6A cytoplasmic domain because this domain is phosphorylated on serine residues in response to stimuli that activate the laminin receptor function of alpha 6 A beta 1. Point mutations were introduced in the alpha 6A cDNA that changed either serine residue #1064 (M1) or serine residue #1071 (M2) to alanine residues. In addition, a double mutant (M3) was constructed in which both serine residues were changed to alanine residues. P388D1 transfectants which expressed these serine mutations adhered to laminin in response to PMA to the same extent as cells transfected with wild-type alpha 6A cDNA. These findings provide evidence for a novel mode of integrin regulation that is distinct from that reported for other regulated integrins (O'Toole, T. E., D. Mandelman, J. Forsyth, S. J. Shattil, E. F. Plow, and M. H. Ginsberg. 1991. Science (Wash. DC). 254:845-847. Hibbs, M. L., H. Xu, S. A. Stacker, and T. A. Springer. 1991. Science (Wash. DC). 251:1611-1613), and they demonstrate that serine phosphorylation of the alpha 6A cytoplasmic domain is not involved in this regulation.     

The influenza C virus CM2 protein can alter intracellular pH, and its transmembrane domain can substitute for that of the influenza A virus M2 protein and support infectious virus production

The influenza C virus CM2 protein and a chimeric influenza A virus M2 protein (MCM) containing the CM2 transmembrane domain were assessed for their ability to functionally replace the M2 protein. While all three proteins could alter cytosolic pH to various degrees when expressed from cDNA, only M2 and MCM could at least partially restore infectious virus production to M2-deficient influenza A viruses. The data suggest that while the CM2 ion channel activity is similar to that of M2, sequences in the extracellular and/or cytoplasmic domains play important roles in infectious virus production.     

Association of the membrane proximal regions of the alpha and beta subunit cytoplasmic domains constrains an integrin in the inactive state

The adhesiveness of integrins is regulated through a process termed "inside-out" signaling. To understand the molecular mechanism of integrin inside-out signaling, we generated K562 stable cell lines that expressed LFA-1 (alpha(L)beta(2)) or Mac-1 (alpha(M)beta(2)) with mutations in the cytoplasmic domain. Complete truncation of the beta(2) cytoplasmic domain, but not a truncation that retained the membrane proximal eight residues, resulted in constitutive activation of alpha(L)beta(2) and alpha(M)beta(2), demonstrating the importance of this membrane proximal region in the regulation of integrin adhesive function. Furthermore, replacement of the alpha(L) and beta(2) cytoplasmic domains with acidic and basic peptides that form an alpha-helical coiled coil caused inactivation of alpha(L)beta(2). Association of these artificial cytoplasmic domains was directly demonstrated. By contrast, replacement of the alpha(L) and beta(2) cytoplasmic domains with two basic peptides that do not form an alpha-helical coiled coil activated alpha(L)beta(2). Induction of ligand binding by the activating cytoplasmic domain mutations correlated with the induction of activation epitopes in the extracellular domain. Our data demonstrate that cytoplasmic, membrane proximal association between integrin alpha and beta subunits, constrains an integrin in the inactive conformation.     

A structural model of the acetylcholine receptor channel based on partition energy and helix packing calculations.

A structural model of the transmembrane portion of the acetylcholine receptor was developed from sequences of all its subunits by using transfer energy calculations to locate transmembrane alpha-helices and to calculate which helical side chains should be in contact with water inside the channel, with portions of other transmembrane helices, or with lipid hydrocarbon chains. "Knobs-into-holes" side chain packing calculations were used with other factors to stack the transmembrane alpha-helices together. In the model each subunit has the following structures in order along the sequence from the NH2 terminus: a large extracellular domain of undetermined structure, a short apolar alpha-helix that lies on the extracellular lipid surface of the membrane; three apolar transmembrane alpha-helices (I, II, and III), a cytoplasmic domain of undetermined structure, an amphipathic transmembrane alpha-helix (L) that forms the channel lining, a short extracellular alpha-helix, another apolar transmembrane alpha-helix (IV), and a small cytoplasmic domain formed by the COOH-terminal end of the chain. Three concentric layers form the pore. A bundle of five amphipathic L helices forms the channel lining. This bundle is surrounded by a bundle of 10 alternating II and III helices. Helices I and IV cover portions of the outer surface of the bundle formed by helices II and III. Positions of disulfide bridges are predicted and a mechanism for opening and closing conformational changes is proposed that requires tilting transmembrane helices and possibly a thiol-disulfide interchange reaction.     

Mechanism of the calcium-dependent multimerization of synaptotagmin VII mediated by its first and second C2 domains

The Ca(2+)-dependent oligomerization activity of the second C2 (C2B) domain of synaptotagmin I (Syt I) has been hypothesized to regulate neurotransmitter release. We previously showed that the cytoplasmic domains of several other Syt isoforms also show Ca(2+)-dependent oligomerization activity (Fukuda, M., and Mikoshiba, K. (2000) J. Biol. Chem. 275, 28180-28185), but little is known about the involvement of their C2 domains in Ca(2+)-dependent oligomerization. In this study, we analyzed the Ca(2+)-dependent oligomerization properties of the first (C2A) and the second C2 (C2B) domains of Syt VII. Unlike Syt I, both C2 domains of Syt VII contribute to Ca(2+)-dependent homo- and hetero-oligomerization with other isoforms. For instance, the Syt VII C2A domain Ca(2+)-dependently binds itself and the C2A domain of Syt VI but not its C2B domain, whereas the Syt VII C2B domain Ca(2+)-dependently binds itself and the C2B domain of Syt II but not its C2A domain. In addition, we showed by gel filtration that a single Syt VII C2 domain is sufficient to form a Ca(2+)-dependent multimer of very high molecular weight. Because of this "two handed" structure, the Syt VII cytoplasmic domain has been found to show the strongest Ca(2+)-dependent multimerization activity in the Syt family. We also identified Asn-328 in the C2B domain as a crucial residue for the efficient Ca(2+)-dependent switch for multimerization by site-directed mutagenesis. Our results suggest that Syt VII is a specific isoform that can cluster different Syt isoforms with two hands in response to Ca(2+).     

Interaction of actin with carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) receptor in liposomes is Ca2+- and phospholipid-dependent

The regulation of binding of G-actin to cytoplasmic domains of cell surface receptors is a common mechanism to control diverse biological processes. To model the regulation of G-actin binding to a cell surface receptor we used the cell-cell adhesion molecule carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1-S) in which G-actin binds to its short cytoplasmic domain (12 amino acids; Chen, C. J., Kirshner, J., Sherman, M. A., Hu, W., Nguyen, T., and Shively, J. E. (2007) J. Biol. Chem. 282, 5749-5760). A liposome model system demonstrates that G-actin binds to the cytosolic domain peptide of CEACAM1-S in the presence of negatively charged palmitoyl-oleoyl phosphatidylserine (POPS) liposomes and Ca(2+). In contrast, no binding of G-actin was observed in palmitoyl-oleoyl phosphatidylcholine (POPC) liposomes or when a key residue in the peptide, Phe-454, is replaced with Ala. Molecular Dynamics simulations on CEACAM1-S in an asymmetric phospholipid bilayer show migration of Ca(2+) ions to the lipid leaflet containing POPS and reveal two conformations for Phe-454 explaining the reversible availability of this residue for G-actin binding. NMR transverse relaxation optimized spectroscopic analysis of (13)C-labeled Phe-454 CEACAM1-S peptide in liposomes plus actin further confirmed the existence of two peptide conformers and the Ca(2+) dependence of actin binding. These findings explain how a receptor with a short cytoplasmic domain can recruit a cytosolic protein in a phospholipid and Ca(2+)-specific manner. In addition, this model system provides a powerful approach that can be applied to study other membrane protein interactions with their cytosolic targets.     

Modular design of Cys-loop ligand-gated ion channels: functional 5-HT3 and GABA rho1 receptors lacking the large cytoplasmic M3M4 loop

Cys-loop receptor neurotransmitter-gated ion channels are pentameric assemblies of subunits that contain three domains: extracellular, transmembrane, and intracellular. The extracellular domain forms the agonist binding site. The transmembrane domain forms the ion channel. The cytoplasmic domain is involved in trafficking, localization, and modulation by cytoplasmic second messenger systems but its role in channel assembly and function is poorly understood and little is known about its structure. The intracellular domain is formed by the large (>100 residues) loop between the alpha-helical M3 and M4 transmembrane segments. Putative prokaryotic Cys-loop homologues lack a large M3M4 loop. We replaced the complete M3M4 loop (115 amino acids) in the 5-hydroxytryptamine type 3A (5-HT(3A)) subunit with a heptapeptide from the prokaryotic homologue from Gloeobacter violaceus. The macroscopic electrophysiological and pharmacological characteristics of the homomeric 5-HT(3A)-glvM3M4 receptors were comparable to 5-HT(3A) wild type. The channels remained cation-selective but the 5-HT(3A)-glvM3M4 single channel conductance was 43.5 pS as compared with the subpicosiemens wild-type conductance. Coexpression of hRIC-3, a protein that modulates expression of 5-HT(3) and acetylcholine receptors, significantly attenuated 5-HT-induced currents with wild-type 5-HT(3A) but not 5-HT(3A)-glvM3M4 receptors. A similar deletion of the M3M4 loop in the anion-selective GABA-rho1 receptor yielded functional, GABA-activated, anion-selective channels. These results imply that the M3M4 loop is not essential for receptor assembly and function and suggest that the cytoplasmic domain may fold as an independent module from the transmembrane and extracellular domains.     

The RGG domain in hnRNP A2 affects subcellular localization

The heterogeneous nuclear ribonucleoproteins (hnRNP) associate with pre-mRNA in the nucleus and play an important role in RNA processing and splice site selection. In addition, hnRNP A proteins function in the export of mRNA to the cytoplasm. Although the hnRNP A proteins are predominantly nuclear, hnRNP A1 shuttles rapidly between the nucleus and the cytoplasm. HnRNP A2, whose cytoplasmic overexpression has been identified as an early biomarker of lung cancer, has been less well studied. Cytosolic hnRNP A2 overexpression has also been noted in brain tumors, in which it has been correlated with translational repression of Glucose Transporter-1 expression. We now examine the role of arginine methylation on the nucleocytoplasmic localization of hnRNP A2 in the HEK-293 and NIH-3T3 mammalian cell lines. Treatment of either cell line with the methyltransferase inhibitor adenosine dialdehyde dramatically shifts hnRNP A2 localization from the nuclear to the cytoplasmic compartment, as shown both by immunoblotting and by immunocytochemistry. In vitro radiolabeling with [(3)H]AdoMet of GST-tagged hnRNP A2 RGG mutants, using recombinant protein arginine methyltransferase (PRMT1), shows (i) that hnRNP A2 is a substrate for PRMT1 and (ii) that methylated residues are found only in the RGG domain. Deletion of the RGG domain (R191-G253) of hnRNP A2 results in a cytoplasmic localization phenotype, detected both by immunoblotting and by immunocytochemistry. These studies indicate that the RGG domain of hnRNP A2 contains sequences critical for cellular localization of the protein. The data suggest that hnRNP A2 may contain a novel nuclear localization sequence, regulated by arginine methylation, that lies in the R191-G253 region and may function independently of the M9 transportin-1-binding region in hnRNP A2.     

A PDZ protein regulates the distribution of the transmembrane semaphorin, M-SemF.

M-SemF is a membrane-associated, neurally enriched member of the semaphorin family of axon guidance signals. We considered whether the cytoplasmic domain of M-SemF might possess a signaling function and/or might control the distribution of M-SemF on the cell surface. We identify a PDZ-containing neural protein as an M-SemF cytoplasmic domain-associated protein (SEMCAP-1). SEMCAP-2 is a closely related nonneuronal protein. SEMCAP-1 has recently also been identified as GIPC, by virtue of its interaction with the RGS protein GAIP in vitro (De Vries, L., Lou, X., Zhao, G., Zheng, B., and Farquhar, M. G. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 12340-12345). Expression studies support the notion that SEMCAP-1(GIPC) interacts with M-SemF, but not GAIP, in brain. Lung SEMCAP-2 and SEMCAP-1(GIPC) are potential partners for both GAIP and M-SemF. The protein interaction requires the single PDZ domain of SEMCAP-1(GIPC) and the carboxyl-terminal four residues of M-SemF, ESSV. While SEMCAP-1(GIPC) also interacts with SemC, it does not interact with other proteins containing a class I PDZ binding motif, nor does M-SemF interact with other class I PDZ proteins. Co-expression of SEMCAP-1(GIPC) induces the redistribution of dispersed M-SemF into detergent-resistant aggregates in HEK293 cells. Thus, SEMCAP-1(GIPC) appears to regulate the subcellular distribution of M-SemF in brain, and SEMCAPs could link M-SemF to G protein signal transduction pathways.     

Engagement of CD46 and alpha5beta1 integrin by group A streptococci is required for efficient invasion of epithelial cells

Membrane cofactor protein (MCP or CD46), a widely distributed complement regulatory human protein, is a cell surface receptor for many pathogens including group A streptococci (GAS). The surface M protein of GAS binds CD46 and mediates GAS adherence to keratinocytes. In the present study, we studied the role of CD46 in GAS invasion of human lung epithelial cells, A549. Anti-CD46 antibody which specifically blocks the domain to which M protein binds inhibited adherence to and invasion of A549 cells by GAS. Moreover, downregulation of CD46 expression on A549 by RNA interference resulted in reduced invasion of these cells by GAS. A mutant form of CD46 with a deletion in the cytoplasmic domain was overexpressed in A549 cells, which resulted in partial inhibition of invasion. This indicates that the cytoplasmic tail is required for CD46 to promote invasion by GAS. Invasion assays with Lactococcus lactis that express M protein demonstrated the dependence of CD46-promoted invasion on interaction with M protein. In addition, CD46-mediated invasion was also found to be dependent on the extracellular matrix protein fibronectin.