Integrins and inside-out signal transduction: converging signals from PKC and PIP3.

Recent studies have identified molecules that interact with integrins and appear to participate in the signaling pathways that regulate integrin adhesiveness. Clues provided by studies of these molecules point to the integration by integrins of signal transduction pathways implicated in cell division and activation.

Endoplasmic reticulum chaperone gp96 is required for innate immunity but not cell viability.

Chaperone proteins are thought to promote the correct folding and assembly of newly synthesized proteins and to facilitate restoration of the folded state under environmental conditions that favour protein denaturation. They are among the most ubiquitous and highly conserved of all proteins. The eukaryotic endoplasmic reticulum (ER) chaperone gp96 in particular has long been thought to be indispensable for cell survival. Here we report that a screen for genes required for the immune response to bacterial endotoxins has identified a B-cell line deficient in gp96. Absence of gp96 is compatible with cellular survival even under stress conditions and causes a defect in the formation of only a small subset of cell surface receptors. Toll-like receptors are retained intracellularly in the absence of gp96, explaining the unresponsiveness of the mutant to microbial stimuli.

Analysis of two cDNA clones encoding the B lymphocyte antigen CD20 (B1, Bp35), a type III integral membrane protein.

Two cDNA clones encoding the pan-B cell CD20 antigen were isolated from a COS cell expression library. The two clones bear identical coding sequences and differ only in the length of the 3' untranslated region. The predicted CD20 sequence is 297 residues long and contains three hydrophobic domains, one of which is long enough to span the membrane twice. COS cells transfected with either CD20 clone express an immunoreactive protein of 33 kD.

CD19, the earliest differentiation antigen of the B cell lineage, bears three extracellular immunoglobulin-like domains and an Epstein-Barr virus-related cytoplasmic tail.

The isolation and expression of a full-length cDNA clone encoding the B cell-specific glycoprotein CD19 is reported. The sequence of the cDNA predicts a glycosylated integral membrane protein with a precursor molecular weight of 51.8 x 10(3) and an extracellular domain organized into three contiguous Ig-like sub-domains. The cytoplasmic domain bears significant relatedness to two proteins encoded by the Epstein-Barr virus and the int-1 oncogene. CD19 transcripts are restricted to members of the B cell lineage, being most abundant in pre-B cell lines and least abundant in plasmacytomas.

The lymphocyte glycoprotein CD6 contains a repeated domain structure characteristic of a new family of cell surface and secreted proteins.

The isolation, characterization, and expression of a full-length cDNA encoding the human T cell glycoprotein CD6 is described. COS cells transfected with the CD6 clone express a 90-kD protein that reacts with all available anti-CD6 monoclonal antibodies. RNA blot hybridization analysis indicates that CD6 transcripts are predominantly restricted to cells in the T lineage. The predicted CD6 sequence is 468 amino acids long, with the typical features of a type I integral membrane protein. The cytoplasmic domain of CD6 contains two serine residues, one or both of which are substrates for phosphorylation during T cell activation. The extracellular domain of CD6 is significantly related to the extracellular domain of the human and mouse T cell antigen CD5, the cysteine-rich domain of the bovine and mouse type I macrophage scavenger receptor, the extracellular domain of the sea urchin spermatozoa protein that crosslinks the egg peptide speract, the mammalian complement factor 1, and the human lung tumor antigen L3. These molecules, therefore, constitute a new gene superfamily that is well conserved across species boundaries.

The primary structure of the human leukocyte antigen CD37, a species homologue of the rat MRC OX-44 antigen.

Comparison of NH2-terminal protein sequence from the rat OX-44 antigen with the sequence of the human CD37 antigen deduced from a cDNA clone shows that these antigens are species homologues. The CD37 sequence is 244 amino acids in length and lacks a conventional leader sequence. The molecule is likely to have an NH2-terminal cytoplasmic domain followed by three transmembrane sequences that lie within the first 110 amino acids. The rest of the molecule is hydrophillic and contains three sites for N-linked glycosylation.

Isolation and expression of functional high-affinity Fc receptor complementary DNAs.

Human and murine mononuclear phagocytes express a high-affinity receptor for immunoglobulin G that plays a central role in macrophage antibody-dependent cellular cytotoxicity and clearance of immune complexes. The receptor (FcRI) may also be involved in CD4-independent infection of human macrophages by human immunodeficiency virus. This report describes the isolation of cDNA clones encoding the human FcRI by a ligand-mediated selection technique. Expression of the cDNAs in COS cells gave rise to immunoglobulin G binding of the expected affinity and subtype specificity. RNA blot analysis revealed expression of a 1.7-kilobase transcript in macrophages and in cells of the promonocytic cell line U937 induced with interferon-gamma. The extracellular region of FcRI consists of three immunoglobulin-like domains, two of which share homology with low-affinity receptor domains.

Recognition by ELAM-1 of the sialyl-Lex determinant on myeloid and tumor cells.

Endothelial leukocyte adhesion molecule-1 (ELAM-1) is an endothelial cell adhesion molecule that allows myeloid cells to attach to the walls of blood vessels adjacent to sites of inflammation. ELAM-1 recognizes the sialyl-Lewis X (sialyl-Lex) determinant, NeuAc alpha 2-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc-, a granulocyte carbohydrate also found on the surface of some tumor cell lines. Binding of myeloid cells to soluble ELAM-1 is inhibited by a monoclonal antibody recognizing sialyl-Lex or by proteins bearing sialyl-Lex, some of which may participate in humoral regulation of myeloid cell adhesion. Stimulated granulocytes also release an inhibitor of ELAM-1 binding that can be selectively adsorbed by monoclonal antibody to sialyl-Lex.

Endothelial leukocyte adhesion molecule 1: an inducible receptor for neutrophils related to complement regulatory proteins and lectins.

Focal adhesion of leukocytes to the blood vessel lining is a key step in inflammation and certain vascular disease processes. Endothelial leukocyte adhesion molecule-1 (ELAM-1), a cell surface glycoprotein expressed by cytokine-activated endothelium, mediates the adhesion of blood neutrophils. A full-length complementary DNA (cDNA) for ELAM-1 has now been isolated by transient expression in COS cells. Cells transfected with the ELAM-1 clone express a surface structure recognized by two ELAM-1 specific monoclonal antibodies (H4/18 and H18/7) and support the adhesion of isolated human neutrophils and the promyelocytic cell line HL-60. Expression of ELAM-1 transcripts in cultured human endothelial cells is induced by cytokines, reaching a maximum at 2 to 4 hours and decaying by 24 hours; cell surface expression of ELAM-1 protein parallels that of the mRNA. The primary sequence of ELAM-1 predicts an amino-terminal lectin-like domain, an EGF domain, and six tandem repetitive motifs (about 60 amino acids each) related to those found in complement regulatory proteins. A similar domain structure is also found in the MEL-14 lymphocyte cell surface homing receptor, and in granule-membrane protein 140, a membrane glycoprotein of platelet and endothelial secretory granules that can be rapidly mobilized (less than 5 minutes) to the cell surface by thrombin and other stimuli. Thus, ELAM-1 may be a member of a nascent gene family of cell surface molecules involved in the regulation of inflammatory and immunological events at the interface of vessel wall and blood.

Cloning and expression of the human interleukin-6 (BSF-2/IFN beta 2) receptor.

Interleukin-6 (IL-6/BSF-2/IFN beta 2) is a multifunctional cytokine that regulates the growth and differentiation of various tissues, and is known particularly for its role in the immune response and acute phase reactions. A complementary DNA encoding the human IL-6 receptor (IL-6-R) has now been isolated. The IL-6-R consists of 468 amino acids, including a signal peptide of approximately 19 amino acids and a domain of approximately 90 amino acids that is similar to a domain in the immunoglobulin (Ig) superfamily. The cytoplasmic domain of approximately 82 amino acids lacks a tyrosine/kinase domain, unlike other growth factor receptors.

Myelinated, synapsing cultures of murine spinal cord--validation as an in vitro model of the central nervous system.

Research in central nervous system (CNS) biology and pathology requires in vitro models, which, to recapitulate the CNS in vivo, must have extensive myelin and synapse formation under serum-free (defined) conditions. However, finding such a model has proven difficult. The technique described here produces dense cultures of myelinated axons, with abundant synapses and nodes of Ranvier, that are suitable for both morphological and biochemical analysis. Cellular and molecular events were easily visualised using conventional microscopy. Ultrastructurally, myelin sheaths were of the appropriate thickness relative to axonal diameter (G-ratio). Production of myelinated axons in these cultures was consistent and repeatable, as shown by statistical analysis of multiple experimental repeats. Myelinated axons were so abundant that from one litter of embryonic mice, myelin was produced in amounts sufficient for bulk biochemical analysis. This culture method was assessed for its ability to generate an in vitro model of the CNS that could be used for both neurobiological and neuropathological research. Myelin protein kinetics were investigated using a myelin fraction isolated from the cultures. This fraction was found to be superior, quantitatively and qualitatively, to the fraction recovered from standard cultures of dissociated oligodendrocytes, or from brain slices. The model was also used to investigate the roles of specific molecules in the pathogenesis of inflammatory CNS diseases. Using the defined conditions offered by this culture system, dose-specific, inhibitory effects of inflammatory cytokines on myelin formation were demonstrated, unequivocally. The method is technically quick, easy and reliable, and should have wide application to CNS research.

Codon usage limitation in the expression of HIV-1 envelope glycoprotein.

BACKGROUND: The expression of both the env and gag gene products of human immunodeficiency virus type 1 (HIV-1) is known to be limited by cis elements in the viral RNA that impede egress from the nucleus and reduce the efficiency of translation. Identifying these elements has proven difficult, as they appear to be disseminated throughout the viral genome. RESULTS: Here, we report that selective codon usage appears to account for a substantial fraction of the inefficiency of viral protein synthesis, independent of any effect on improved nuclear export. The codon usage effect is not specific to transcripts of HIV-1 origin. Re-engineering the coding sequence of a model protein (Thy-1) with the most prevalent HIV-1 codons significantly impairs Thy-1 expression, whereas altering the coding sequence of the jellyfish green fluorescent protein gene to conform to the favored codons of highly expressed human proteins results in a substantial increase in expression efficiency. CONCLUSIONS: Codon-usage effects are a major impediment to the efficient expression of HIV-1 genes. Although mammalian genes do not show as profound a bias as do Escherichia coli genes, other proteins that are poorly expressed in mammalian cells can benefit from codon re-engineering.

Membrane compartmentation and the response to antigen.

Antigen-receptor engagement is a complex, highly orchestrated rearrangement of signaling molecules at, and adjacent to, the plasma membrane. Recent discoveries have shown that the plasma membrane itself is differentiated into microdomains of distinct lipid constituents and that the affiliation of lipid-modified proteins with those domains has important implications for antigen-receptor function. Disruption of lipid microdomains by a variety of methods attenuates TCR signal transduction.

The Epstein-Barr virus oncoprotein latent membrane protein 1 engages the tumor necrosis factor receptor-associated proteins TRADD and receptor-interacting protein (RIP) but does not induce apoptosis or require RIP for NF-kappaB activation.

A site in the Epstein-Barr virus (EBV) transforming protein LMP1 that constitutively associates with the tumor necrosis factor receptor 1 (TNFR1)-associated death domain protein TRADD to mediate NF-kappaB and c-Jun N-terminal kinase activation is critical for long-term lymphoblastoid cell proliferation. We now find that LMP1 signaling through TRADD differs from TNFR1 signaling through TRADD. LMP1 needs only 11 amino acids to activate NF-kappaB or synergize with TRADD in NF-kappaB activation, while TNFR1 requires approximately 70 residues. Further, LMP1 does not require TRADD residues 294 to 312 for NF-kappaB activation, while TNFR1 requires TRADD residues 296 to 302. LMP1 is partially blocked for NF-kappaB activation by a TRADD mutant consisting of residues 122 to 293. Unlike TNFR1, LMP1 can interact directly with receptor-interacting protein (RIP) and stably associates with RIP in EBV-transformed lymphoblastoid cell lines. Surprisingly, LMP1 does not require RIP for NF-kappaB activation. Despite constitutive association with TRADD or RIP, LMP1 does not induce apoptosis in EBV-negative Burkitt lymphoma or human embryonic kidney 293 cells. These results add a different perspective to the molecular interactions through which LMP1, TRADD, and RIP participate in B-lymphocyte activation and growth.

Hypoxia and acidosis independently up-regulate vascular endothelial growth factor transcription in brain tumors in vivo.

Hypoxia and acidosis are hallmarks of tumors as well as critical determinants of response to treatments. They can upregulate vascular endothelial growth factor (VEGF) in vitro. However, the relationship between tissue oxygen partial pressure (pO(2))/pH and VEGF transcription in vivo is not known. Thus, we developed a novel in vivo microscopy technique to simultaneously measure VEGF promoter activity, pO(2), and pH. To monitor VEGF expression in vivo, we engineered human glioma cells that express green fluorescent protein (GFP) under the control of the VEGF promoter. These cells were implanted into the cranial windows in severe combined immunodeficient mice, and VEGF promoter activity was assessed by GFP imaging. Tissue pO(2) and pH were determined by phosphorescence quenching microscopy and ratio imaging microscopy, respectively. These techniques have allowed us to show, for the first time, that VEGF transcription in brain tumors is independently regulated by the tissue pO(2) and pH. One week after tumor implantation, significant angiogenesis was observed, with increased GFP fluorescence throughout the tumor. Under hypoxic or neutral pH conditions, VEGF-promoter activity increased, with a decrease in pO(2) and independent of pH. Under low pH or oxygenated conditions, VEGF-promoter activity increased, with a decrease in pH and independent of pO(2). In agreement with the in vivo findings, both hypoxia and acidic pH induced VEGF expression in these cells in vitro and showed no additive effect for combined hypoxia and low pH. These results suggest that VEGF transcription in brain tumors is regulated by both tissue pO(2) and pH via distinct pathways.

A comparison of postmigration and migration-coupled mismatch correction mechanisms for branch migration-mediated gene conversion.

We examine several models for nonreciprocal recombination based on the formation of heteroduplex DNA by bidirectional branch migration. We show that the consistency of genetic maps resulting from postmigration mismatch correction is disrupted by the presence of a favored site for initiation of strand exchange, with local inversion of the genetic and physical maps a predicted consequence. We also examine models that allow mismatch correction to take place concurrently with the process of strand exchange and show that the genetic maps resulting from such migration-coupled conversions are substantially free of the anomalies expected of postmigration conversions.

Making agonists of antagonists.

Cell-surface receptors can be divided into three classes, depending on whether they transmit information by allosteric conformational change, by receptor dimerization, or by receptor aggregation. So far, only the first class of receptors has proven readily accessible to chemical techniques; this is no accident, but times are changing.

Developments in expression cloning.

Recent years have seen a dramatic expansion in the range of applications of expression cloning techniques. New vectors and detection methods promise to further broaden the applicability of function-based screening approaches to problems in gene discovery. A major theme in the past year has been the introduction of engineered reporter cells that heighten the sensitivity with which clones expressing cDNAs can be identified.