The junction-associated protein, zonula occludens-1, localizes to the nucleus before the maturation and during the remodeling of cell-cell contacts.

The junction-associated protein zonula occludens-1 (ZO-1) is a member of a family of membrane-associated guanylate kinase homologues thought to be important in signal transduction at sites of cell-cell contact. We present evidence that under certain conditions of cell growth, ZO-1 can be detected in the nucleus. Two different antibodies against distinct portions of the ZO-1 polypeptide reveal nuclear staining in subconfluent, but not confluent, cell cultures. An exogenously expressed, epitope-tagged ZO-1 can also be detected in the nuclei of transfected cells. Nuclear accumulation can be stimulated at sites of wounding in cultured epithelial cells, and immunoperoxidase detection of ZO-1 in tissue sections of intestinal epithelial cells reveals nuclear labeling only along the outer tip of the villus. These results suggest that the nuclear localization of ZO-1 is inversely related to the extent and/or maturity of cell contact. Since cell-cell contacts are specialized sites for signaling pathways implicated in growth and differentiation, we suggest that the nuclear accumulation of ZO-1 may be relevant for its suggested role in membrane-associated guanylate kinase homologue signal transduction.

Regulation of mitogen-activated protein kinases by a calcium/calmodulin-dependent protein kinase cascade.

Membrane depolarization of NG108 cells gives rapid (< 5 min) activation of Ca2+/calmodulin-dependent protein kinase IV (CaM-KIV), as well as activation of c-Jun N-terminal kinase (JNK). To investigate whether the Ca2+-dependent activation of mitogen-activated protein kinases (ERK, JNK, and p38) might be mediated by the CaM kinase cascade, we have transfected PC12 cells, which lack CaM-KIV, with constitutively active mutants of CaM kinase kinase and/or CaM-KIV (CaM-KKc and CaM-KIVc, respectively). In the absence of depolarization, CaM-KKc transfection had no effect on Elk-dependent transcription of a luciferase reporter gene, whereas CaM-KIVc alone or in combination with CaM-KKc gave 7- to 10-fold and 60- to 80-fold stimulations, respectively, which were blocked by mitogen-activated protein (MAP) kinase phosphatase cotransfection. When epitope-tagged constructs of MAP kinases were co-transfected with CaM-KKc plus CaM-KIVc, the immunoprecipitated MAP kinases were activated 2-fold (ERK-2) and 7- to 10-fold (JNK-1 and p38). The JNK and p38 pathways were further investigated using specific c-Jun or ATF2-dependent transcriptional assays. We found that c-Jun/ATF2-dependent transcriptions were enhanced 7- to 10-fold by CaM-KIVc and 20- to 30-fold by CaM-KKc plus CaM-KIVc. In the case of the Jun-dependent transcription, this effect was not due to direct phosphorylation of c-Jun by activated CaM-KIV, since transcription was blocked by a dominant-negative JNK and by two MAP kinase phosphatases. Mutation of the phosphorylation site (Thr196) in CaM-KIV, which mediates its activation by CaM-KIV kinase, prevented activation of Elk-1, c-Jun, and ATF2 by the CaM kinase cascade. These results establish a new Ca2+-dependent mechanism for regulating MAP kinase pathways and resultant transcription.

Differential regulation of beta 1 and beta 2 integrin avidity by chemoattractants in eosinophils.

The CC chemokines regulated on activation normal T expressed and secreted (RANTES) and monocyte chemotactic protein 3 (MCP-3), and the anaphylatoxin C5a, induce activation, degranulation, chemotaxis, and transendothelial migration of eosinophils. Adhesion assays on purified ligands showed differential regulation of beta 1 and beta 2 integrin avidity in eosinophils. Adhesiveness of VLA-4 (alpha 4 beta 1, CD29/CD49d) for vascular cell adhesion molecule 1 or fibronectin was rapidly increased but subsequently reduced by RANTES, MCP-3, or C5a. The deactivation of VLA-4 lead to cell detachment, whereas phorbol 12-myristate 13-acetate induced sustained activation of VLA-4. In contrast, chemoattractants stimulated a prolonged increase in the adhesiveness of Mac-1 (alpha M beta 2, CD11b/CD18) for intercellular adhesion molecule 1. Inhibition by pertussis toxin confirmed signaling via G protein-coupled receptors. Chemoattractants induced transient, while phorbol 12-myristate 13-acetate induced sustained actin polymerization. Disruption of actin filaments by cytochalasins inhibited increases in avidity of VLA-4 but not of Mac-1. Chemoattractants did not upregulate a Mn2+-inducible beta 1 neoepitope defined by the mAb 9EG7, but induced prolonged expression of a Mac-1 activation epitope recognized by the mAb CBRM1/5. This mAb inhibited chemoattractant-stimulated adhesion of eosinophils to intercellular adhesion molecule 1. Thus, regulation of VLA-4 was dependent on the actin cytoskeleton, whereas conformational changes appeared to be crucial for activation of Mac-1. To our knowledge, this is the first demonstration that physiological agonists, such as chemoattractants, can differentially regulate the avidity of a beta 1 and a beta 2 integrin expressed on the same leukocyte.

Random phage mimotopes recognized by monoclonal antibodies against the pyruvate dehydrogenase complex-E2 (PDC-E2).

Dihydrolipoamide acetyltransferase, the E2 component of the pyruvate dehydrogenase complex (PDC-E2), is the autoantigen most commonly recognized by autoantibodies in primary biliary cirrhosis (PBC). We identified a peptide mimotope(s) of PDC-E2 by screening a phage-epitope library expressing random dodecapeptides in the pIII coat protein of fd phage using C355.1, a murine monoclonal antibody (mAb) that recognizes a conformation-dependent epitope in the inner lipoyl domain of PDC-E2 and uniquely stains the apical region of bile duct epithelium (BDE) only in patients with PBC. Eight different sequences were identified in 36 phage clones. WMSYPDRTLRTS was present in 29 clones; WESYPFRVGTSL, APKTYVSVSGMV, LTYVSLQGRQGH, LDYVPLKHRHRH, AALWGVKVRHVS, KVLNRIMAGVRH and GNVALVSSRVNA were singly represented. Three common amino acid motifs (W-SYP, TYVS, and VRH) were shared among all peptide sequences. Competitive inhibition of the immunohistochemical staining of PBC BDE was performed by incubating the peptides WMSYPDRTLRTS, WESYPDRTLRTS, APKTYVSVSGMV, and AALWGVKVRHVS with either C355.1 or a second PDC-E2-specific mAb, C150.1. Both mAbs were originally generated to PDC-E2 but map to distinct regions of PDC-E2. Two of the peptides, although selected by reaction with C355.1, strongly inhibited the staining of BDE by C150.1, whereas the peptide APKTYVSVSGMV consistently inhibited the staining of C355.1 on biliary duct epithelium more strongly than the typical mitochondrial staining of hepatocytes. Rabbit sera raised against the peptide WMSYPDRTLRTS stained BDE of livers and isolated bile duct epithelial cells of PBC patients more intensively than controls. The rabbit sera stained all size ducts in normals, but only small/medium-sized ductules in PBC livers. These studies provide evidence that the antigen present in BDE is a molecular mimic of PDC-E2, and not PDC-E2 itself.

DNA repeats identify novel virulence genes in Haemophilus influenzae.

The whole genome sequence (1.83 Mbp) of Haemophilus influenzae strain Rd was searched to identify tandem oligonucleotide repeat sequences. Loss or gain of one or more nucleotide repeats through a recombination-independent slippage mechanism is known to mediate phase variation of surface molecules of pathogenic bacteria, including H. influenzae. This facilitates evasion of host defenses and adaptation to the varying microenvironments of the host. We reasoned that iterative nucleotides could identify novel genes relevant to microbe-host interactions. Our search of the Rd genome sequence identified 9 novel loci with multiple (range 6-36, mean 22) tandem tetranucleotide repeats. All were found to be located within putative open reading frames and included homologues of hemoglobin-binding proteins of Neisseria, a glycosyltransferase (IgtC gene product) of Neisseria, and an adhesin of Yersinia. These tetranucleotide repeat sequences were also shown to be present in two other epidemiologically different H. influenzae type b strains, although the number and distribution of repeats was different. Further characterization of the IgtC gene showed that it was involved in phenotypic switching of a lipopolysaccharide epitope and that this variable expression was associated with changes in the number of tetranucleotide repeats. Mutation of IgtC resulted in attenuated virulence of H. influenzae in an infant rat model of invasive infection. These data indicate the rapidity, economy, and completeness with which whole genome sequences can be used to investigate the biology of pathogenic bacteria.

A 104-kDa diacylglycerol kinase containing ankyrin-like repeats localizes in the cell nucleus.

The cDNA corresponding to a fourth species of diacylglycerol (DG) kinase (EC 2.7.1.107) was isolated from cDNA libraries of rat retina and brain. This cDNA encoded a 929-aa, 104-kDa polypeptide termed DGK-IV. DGK-IV was different from previously identified mammalian DG kinase species, DGK-I, DGK-II, and DGK-III, in that it contained no EF-hand motifs but did contain four ankyrin-like repeats at the carboxyl terminus. These structural features of DGK-IV closely resemble the recently cloned, eye-specific DG kinase of Drosophila that is encoded by the retinal degeneration A (rdgA) gene. However, DGK-IV was expressed primarily in the thymus and brain with relatively low expression in the eye and intestine. Furthermore, the primary structure of the DGK-IV included a nuclear targeting motif, and immunocytochemical analysis revealed DGK-IV to localize in the nucleus of COS-7 cells transfected with the epitope-tagged cDNA, suggesting an involvement of DGK-IV in intranuclear processes.

Urea-inducible Egr-1 transcription in renal inner medullary collecting duct (mIMCD3) cells is mediated by extracellular signal-regulated kinase activation.

Urea (200-400 milliosmolar) activates transcription, translation of, and trans-activation by the immediate-early gene transcription factor Egr-1 in a renal epithelial cell-specific fashion. The effect at the transcriptional level has been attributed to multiple serum response elements and their adjacent Ets motifs located within the Egr-1 promoter. Elk-1, a principal ternary complex factor and Ets domain-containing protein, is a substrate of the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases. In the renal medullary mIMCD3 cell line, urea (200-400 milliosmolar) activated both ERK1 and ERK2 as determined by in-gel kinase assay and immune-complex kinase assay of epitope-tagged] ERK1 and ERK2. Importantly, urea did not affect abundance of either ERK. Urea-inducible Egr-1 transcription was a consequence of ERK activation because the ERK-specific inhibitor, PD98059, abrogated transcription from the murine Egr-1 promoter in a luciferase reported gene assay. In addition, activators of protein kinase A, including forskolin and 8-Br-cAMP, which are known to inhibit ERK-mediated events, also inhibited urea-inducible Egr-1 transcription. Furthermore, urea-inducible activation of the physiological ERK substrate and transcription factor, Elk-1, was demonstrated through transient cotransfection of a chimeric Elk-1/GAL4 expression plasmid and a GAL4-driven luciferase reporter plasmid. Taken together, these data indicate that, in mIMCD3 cells, urea activates ERKs and the ERK substrate, Elk-1, and that ERK inhibition abrogates urea-inducible Egr-1 transcription. These data are consistent with a model of urea-inducible renal medullary gene expression wherein sequential activation of ERKs and Elk-1 results in increased transcription of Egr-1 through serum response element/Ets motifs.

Propagation of an attenuated virus by design: engineering a novel receptor for a noninfectious foot-and-mouth disease virus.

To gain entry into cells, viruses utilize a variety of different cell-surface molecules. Foot-and-mouth disease virus (FMDV) binds to cell-surface integrin molecules via an arginine-glycine-aspartic acid (RGD) sequence in capsid protein VP1. Binding to this particular cell-surface molecule influences FMDV tropism, and virus/receptor interactions appear to be responsible, in part, for selection of antigenic variants. To study early events of virus-cell interaction, we engineered an alternative and novel receptor for FMDV. Specifically, we generated a new receptor by fusing a virus-binding, single-chain antibody (scAb) to intracellular adhesion molecule 1 (ICAM1). Cells that are normally not susceptible to FMDV infection became susceptible after being transfected with DNA encoding the scAb/ICAM1 protein. An escape mutant (B2PD.3), derived with the mAb used to generate the genetically engineered receptor, was restricted for growth on the scAb/ICAM1 cells, but a variant of B2PD.3 selected by propagation on scAb/ICAM1 cells grew well on these cells. This variant partially regained wild-type sequence in the epitope recognized by the mAb and also regained the ability to be neutralize by the mAb. Moreover, RGD-deleted virions that are noninfectious in animals and other cell types grew to high titers and were able to form plaques on scAb/ ICAM1 cells. These studies demonstrate the first production of a totally synthetic cell-surface receptor for a virus. This novel approach will be useful for studying virus reception and for the development of safer vaccines against viral pathogens of animals and humans.

A sequential dimerization mechanism for erythropoietin receptor activation.

We have probed the interaction of human erythropoietin (EPO) with its receptor (EPO-R) by analyzing a panel of 17 EPO mutants in a variety of in vitro assays. Mutant proteins were expressed in 293s cells and quantified by using an N-terminal epitope tag in conjunction with a surface plasmon resonance assay. Receptor binding was studied using both a soluble form of the EPO-R extracellular domain in an ELISA-format binding competition assay and full-length EPO-R in transfected BaF3 cells. Proliferative activity of the mutants was also determined in the BaF3-derived cell line and was correlated with the results from binding assays. Based on the results of these assays, we identified two distinct receptor binding sites on the EPO molecule. We propose that one site, containing residues Arg-150 and Lys-152, binds initially to EPO receptor on the cell surface. A second site, containing Arg-103 and Ser-104 (and possibly Arg-14), is involved in binding a second EPO-R at the cell surface, thus forming a homodimeric receptor complex. Furthermore, we demonstrate that one EPO mutant (R103A), which has previously been shown to lack proliferative function, is in fact an EPO antagonist. Taken together, these data support a sequential dimerization mechanism of EPO-R activation.

The yeast GAL11 protein binds to the transcription factor IIE through GAL11 regions essential for its in vivo function.

The GAL11 gene encodes an auxiliary transcription factor required for full expression of many genes in yeast. The GAL11-encoded protein (Gal11p) has recently been shown to copurify with the holoenzyme of RNA polymerase II. Here we report that Gal11p stimulates basal transcription in a reconstituted transcription system composed of recombinant or highly purified transcription factors, TFIIB, TFIIE, TFIIF, TFIIH, and TATA box-binding protein and core RNA polymerase II. We further demonstrate that each of the two domains of Gal11p essential for in vivo function respectively participates in the binding to the small and large subunits of TFIIE. The largest subunit of RNA polymerase II was coprecipitated by anti-hemagglutinin epitope antibody from crude extract of GAL11 wild type yeast expressing hemagglutinintagged small subunit of TFIIE. Such a coprecipitation of the RNA polymerase subunit was seen but in a greatly reduced amount, if extract was prepared from gal11 null yeast. In light of these findings, we suggest that Gal11p stimulates promoter activity by enhancing an association of TFIIE with the preinitiation complex in the cell.

The immunodominant major histocompatibility complex class I-restricted antigen of a murine colon tumor derives from an endogenous retroviral gene product.

Tumors express peptide antigens capable of being recognized by tumor-specific cytotoxic T lymphocytes (CTL). Immunization of mice with a carcinogen-induced colorectal tumor, CT26, engineered to secrete granulocyte/macrophage colony-stimulating factor, routinely generated both short-term and long-term CTL lines that not only lysed the parental tumor in vitro, but also cured mice of established tumor following adoptive transfer in vivo. When either short-term or long-term CTL lines were used to screen peptides isolated from CT26, one reverse-phase high performance liquid chromatography peptide fraction consistently sensitized a surrogate target for specific lysis. The bioactivity remained localized within one fraction following multiple purification procedures, indicating that virtually all of the CT26-specific CTL recognized a single peptide. This result contrasts with other tumor systems, where multiple bioactive peptide fractions have been detected. The bioactive peptide was identified as a nonmutated nonamer derived from the envelope protein (gp70) of an endogenous ecotropic murine leukemia provirus. Adoptive transfer with CTL lines specific for this antigen demonstrated that this epitope represents a potent tumor rejection antigen. The selective expression of this antigen in multiple non-viral-induced tumors provides evidence for a unique class of shared immunodominant tumor associated antigens as targets for antitumor immunity.

FRAG1, a gene that potently activates fibroblast growth factor receptor by C-terminal fusion through chromosomal rearrangement.

A constitutively active form of fibroblast growth factor 2 (FGFR2) was identified in rat osteosarcoma (ROS) cells by an expression cloning strategy. Unlike other tyrosine kinase receptors activated by N-terminal truncation in tumors, this receptor, FGFR2-ROS, contains an altered C terminus generated from chromosomal rearrangement with a novel gene, designated FGFR activating gene 1 (FRAG1). While the removal of the C terminus slightly activates FGFR2, the presence of the FRAG1 sequence drastically stimulates the transforming activity and autophosphorylation of the receptor. FGFR2-ROS is expressed as a unusually large protein and is highly phosphorylated in NIH 3T3 transfectants. FRAG1 is ubiquitously expressed and encodes a predicted protein of 28 kDa lacking significant structural similarity to known proteins. Epitope-tagged FRAG1 protein showed a perinuclear localization by immunofluorescence staining. The highly activated state of FGFR2-ROS appears to be attributed to constitutive dimer formation and higher phosphorylation level as well as possibly altered subcellular localization. These results indicate a unique mechanism of receptor activation by a C terminus alteration through a chromosomal fusion with FRAG1.

Agonist-selective endocytosis of mu opioid receptor by neurons in vivo.

Opiate alkaloids are potent analgesics that exert multiple pharmacological effects in the nervous system by activating G protein-coupled receptors. Receptor internalization upon stimulation may be important for desensitization and resensitization, which affect cellular responsiveness to ligands. Here, we investigated the agonist-induced internalization of the mu opioid receptor (MOR) in vivo by using the guinea pig ileum as a model system and immunohistochemistry with an affinity-purified antibody to the C terminus of rat MOR. Antibody specificity was confirmed by the positive staining of human embryonic kidney 293 cells transfected with epitope-tagged MOR cDNA, by the lack of staining of cells transfected with the delta or kappa receptor cDNA, and by the abolition of staining when the MOR antibody was preadsorbed with the MOR peptide fragment. Abundant MOR immunoreactivity (MOR-IR) was localized to the cell body, dendrites, and axonal processes of myenteric neurons. Immunostaining was primarily confined to the plasma membrane of cell bodies and processes. Within 15 min of an intraperitoneal injection of the opiate agonist etorphine, intense MOR-IR was present in vesicle-like structures, which were identified as endosomes by confocal microscopy. At 30 min, MOR-IR was throughout the cytoplasm and in perinuclear vesicles. MOR-IR was still internalized at 120 min. Agonist-induced endocytosis was completely inhibited by the opiate antagonist naloxone. Interestingly, morphine, a high-affinity MOR agonist, did not cause detectable internalization, but it partially inhibited the etorphine-induced MOR endocytosis. These results demonstrate the occurrence of agonist-selective MOR endocytosis in neurons naturally expressing this receptor in vivo and suggest the existence of different mechanisms regulating cellular responsiveness to ligands.

The proteolytic fragments generated by vertebrate proteasomes: structural relationships to major histocompatibility complex class I binding peptides.

Proteasomes are involved in the proteolytic generation of major histocompatibility complex (MHC) class I epitopes but their exact role has not been elucidated. We used highly purified murine 20S proteasomes for digestion of synthetic 22-mer and 41/44-mer ovalbumin partial sequences encompassing either an immunodominant or a marginally immunogenic epitope. At various times, digests were analyzed by pool sequencing and by semiquantitative electrospray ionization mass spectrometry. Most dual cleavage fragments derived from 22-mer peptides were 7-10 amino acids long, with octa- and nonamers predominating. Digestion of 41/44-mer peptides initially revealed major cleavage sites spaced by two size ranges, 8 or 9 amino acids and 14 or 15 amino acids, followed by further degradation of the latter as well as of larger single cleavage fragments. The final size distribution was slightly broader than that of fragments derived from 22-mer peptides. The majority of peptide bonds were cleaved, albeit with vastly different efficiencies. This resulted in multiple overlapping proteolytic fragments including a limited number of abundant peptides. The immunodominant epitope was generated abundantly whereas only small amounts of the marginally immunogenic epitope were detected. The frequency distributions of amino acids flanking proteasomal cleavage sites are correlated to that reported for corresponding positions of MHC class I binding peptides. The results suggest that proteasomal degradation products may include fragments with structural properties similar to MHC class I binding peptides. Proteasomes may thus be involved in the final stages of proteolytic epitope generation, often without the need for downstream proteolytic events.

Induction of cytotoxic T lymphocytes by intramuscular immunization with plasmid DNA is facilitated by bone marrow-derived cells.

Striated muscle is the predominant site of gene expression after i.m. immunization of plasmid DNA, but it is not clear if myocytes or professional antigen-presenting cells (APCs) of hematopoietic origin present the encoded antigens to class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes (CTL). To address this issue, CTL responses were assessed in mice engrafted with immune systems that were partially MHC matched with antigen-producing muscle cells. Spleen cells (sc) from immunocompetent F1 H-2bxd mice were infused into H-2b or H-2d mice carrying the severe combined immunodeficiency (scid) mutation, creating F1sc-->H-2b and F1sc-->H-2d chimeras, respectively. Immunization with DNA plasmids encoding the herpes simplex virus gB or the human immunodeficiency virus gp120 glycoproteins elicited antiviral CTL activity. F1sc-->H-2d chimeras responded to an H-2d-restricted gp120 epitope but not an H-2b restricted gB epitope, whereas F1sc-->H-2b chimeras responded to the H-2b but not the H-2d restricted epitope. This pattern of epitope recognition by the sc chimeras indicated that APCs of recipient (scid) origin were involved in initiation of CTL responses. Significantly, CTL responses against epitopes presented by the mismatched donor class I molecules were elicited if F1 bone marrow cells and sc were transferred into scid recipients before or several days to weeks after DNA immunization. Thus, bone marrow-derived APCs are sufficient for class I MHC presentation of viral antigens after i.m. immunization with plasmid DNA. Expression of plasmid DNA by these APCs is probably not a requirement for CTL priming. Instead, they appear to present proteins synthesized by other host cells.