CD30/TNF receptor-associated factor interaction: NF-kappa B activation and binding specificity.

CD30 is a member of the tumor necrosis factor (TNF) receptor superfamily. CD30 is expressed on normal activated lymphocytes, on several virally transformed T- or B-cell lines and on neoplastic cells of Hodgkin's lymphoma. The interaction of CD30 with its ligand induces pleiotropic effects on cells resulting in proliferation, differentiation, or death. The CD30 cytoplasmic tail interacts with TNF receptor-associated factors (TRAFs), which have been shown to transduce signals mediated by TNF-R2 and CD40. We demonstrate here that TRAF2 also plays an important role in CD30-induced NF-kappa B activation. We also show that TRAF2-mediated activation of NF-kappa B plays a role in the activation of HIV transcription induced by CD30 cross-linking. Detailed site-directed mutagenesis of the CD30 cytoplasmic tail reveals that there are two independent binding sites for TRAF, each interacting with a different domain of TRAF. Furthermore, we localized the TRAF-C binding site in CD30 to a 5-7 amino acid stretch.

Disruption of epithelial gamma delta T cell repertoires by mutation of the Syk tyrosine kinase.

Chimeric mice in which lymphocytes are deficient in the Syk tyrosine kinase have been created. Compared with Syk-positive controls, mice with Syk -/- lymphocytes display substantial depletion of intraepithelial gamma delta T cells in the skin and gut, with developmental arrest occurring after antigen receptor gene rearrangement. In this dependence on Syk, subsets of intraepithelial gamma delta T cells are similar to B cells, but distinct from splenic gamma delta T cells that develop and expand in Syk-deficient mice. The characteristic associations of certain T-cell receptor V gamma/V delta gene rearrangements with specific epithelia are also disrupted by Syk deficiency.

Mutations affecting conserved cysteine residues within the extracellular domain of Neu promote receptor dimerization and activation.

Overexpression of the Neu/ErbB-2 receptor tyrosine kinase has been implicated in the genesis of human breast cancer. Indeed, expression of either activated or wild-type neu in the mammary epithelium of transgenic mice results in the induction of mammary tumors. Previously, we have shown that many of the mammary tumors arising in transgenic mice expressing wild-type neu occur through somatic activating mutations within the neu transgene itself. Here we demonstrate that these mutations promote dimerization of the Neu receptor through the formation of disulfide bonds, resulting in its constitutive activation. To explore the role of conserved cysteine residues within the region deleted in these altered Neu proteins, we examined the transforming potential of a series of Neu receptors in which the individual cysteine residues were mutated. These analyses indicated that mutation of certain cysteine residues resulted in the oncogenic activation of Neu. The increased transforming activity displayed by the altered receptors correlated with constitutive dimerization that occurred in a disulfide bond-dependent manner. We further demonstrate that addition of 2-mercaptoethanol to the culture medium interfered with the specific transforming activity of the mutant Neu receptors. These observations suggest that oncogenic activation of Neu results from constitutive disulfide bond-dependent dimerization.

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.

RXR gamma null mice are apparently normal and compound RXR alpha +/-/RXR beta -/-/RXR gamma -/- mutant mice are viable.

The RXR gamma (RXR, retinoid X receptor) gene was disrupted in the mouse. Homozygous mutant mice developed normally and were indistinguishable from their RXR gamma +/- or wild-type littermates with respect to growth, fertility, viability, and apparent behavior in the animal facility. Moreover, RXR alpha -/-/RXR gamma -/- and RXR beta -/-/RXR gamma -/- mutant phenotypes were indistinguishable from those of RXR alpha -/- and RXR beta -/- mutants, respectively. Strikingly, RXR alpha +/-/RXR beta -/-/RXR gamma -/- triple mutants were viable. Thus, it appears that RXR gamma does not exert any essential function that cannot be performed by RXR alpha or RXR beta, and one copy of RXR alpha is sufficient to perform most of the functions of the RXRs.

Activation of Jak/STAT proteins involved in signal transduction pathway mediated by receptor for interleukin 2 in malignant T lymphocytes derived from cutaneous anaplastic large T-cell lymphoma and Sezary syndrome.

Signaling through the interleukin 2 receptor (IL-2R) involves phosphorylation of several proteins including Jak3, STAT5, and, in preactivated cells, STAT3. In the present study, we examined the functional status of the IL-2R-associated Jak/STAT pathway in malignant T lymphocytes from advanced skin-based lymphomas: anaplastic large T-cell lymphoma (ALCL) and Sezary syndrome (SzS). Proliferation of three ALCL cell lines (PB-1, 2A, and 2B) was partially inhibited by rapamycin, a blocker of some of the signals mediated by IL-2R, but not by cyclosporin A, FK-506, and prednisone, which suppress signals mediated by the T-cell receptor. All the cell lines expressed on their surface the high-affinity IL-2R (alpha, beta, and gamma c chains). They showed basal, constitutive phosphorylation, and coassociation of Jak3, STAT5, and STAT3. Weak basal phosphorylation of IL-2R gamma c was also detected. In regard to SzS, peripheral blood mononuclear cells from 10 of 14 patients showed basal phosphorylation of Jak3, accompanied by phosphorylation of STAT5 in 9 patients, and STAT3 in 4 patients. However, in vitro overnight culture of SzS cells without exogenous cytokines resulted in markedly decreased Jak3 and STAT5 phosphorylation, which could be reversed by stimulation with IL-2. This indicates that the basal phosphorylation of Jak3 and STAT5 in freshly isolated SzS cells is induced rather than constitutive. The basal activation of the Jak/STAT pathway involved in IL-2R signal transduction in ALCL and SzS cells reported here suggests that this pathway may play a role in the pathogenesis of cutaneous T-cell lymphomas, although the mechanism (induced versus constitutive) may vary between different lymphoma types.

Evidence for an insulin receptor substrate 1 independent insulin signaling pathway that mediates insulin-responsive glucose transporter (GLUT4) translocation.

Interaction of the activated insulin receptor (IR) with its substrate, insulin receptor substrate 1 (IRS-1), via the phosphotyrosine binding domain of IRS-1 and the NPXY motif centered at phosphotyrosine 960 of the IR, is important for IRS-1 phosphorylation. We investigated the role of this interaction in the insulin signaling pathway that stimulates glucose transport. Utilizing microinjection of competitive inhibitory reagents in 3T3-L1 adipocytes, we have found that disruption of the IR/IRS-1 interaction has no effect upon translocation of the insulin-responsive glucose transporter (GLUT4). The activity of these reagents was demonstrated by their ability to block insulin stimulation of two distinct insulin bioeffects, membrane ruffling and mitogenesis, in 3T3-L1 adipocytes and insulin-responsive rat 1 fibroblasts. These data suggest that phosphorylated IRS-1 is not an essential component of the metabolic insulin signaling pathway that leads to GLUT4 translocation, yet it appears to be required for other insulin bioeffects.

Cellular adherence elicits ligand-independent activation of the Met cell-surface receptor.

Cell adhesion has a fundamental role in the proliferation and motility of normal cells and the metastasis of tumor cells. To identify signaling pathways activated by the adherence of tumor cells, we analyzed the tyrosine phosphorylation of proteins in mouse melanoma cells before and after attachment to substrata. We discovered that cellular adherence activated the protein-tyrosine kinase of the cell surface receptor Met, whose ligand is hepatocyte growth factor and scatter factor. The activation was exceedingly prompt, affected the great majority of Met in the cells, persisted so long as the cells remained adherent, and was rapidly reversed as soon as the cells were detached from substrata. Activation of Met required that cells be adherent but not that they spread on the substratum, and it occurred in the absence of any apparent ligand for the receptor. Ligand-independent activation of Met occurred in several varieties of tumor cells but not in normal endothelial cells that express the receptor. The activation of Met described here may represent a means by which cells respond to mechanical as opposed to biochemical stimuli.

Evidence for a preformed transducer complex organized by the B cell antigen receptor.

The B cell antigen receptor (BCR) consists of the membrane-bound immunoglobulin (mIg) molecule and the Ig-alpha/Ig-beta heterodimer, which functions as signaling subunit of the receptor. Stimulation of the BCR activates protein tyrosine kinases (PTKs) that phosphorylate a number of substrate proteins, including the Ig-alpha/Ig-beta heterodimer of the BCR itself. How the PTKs become activated after BCR engagement is not known at present. Here, we show that BCR-negative J558L cells treated with the protein tyrosine phosphatase inhibitor pervanadate/H2O2 display only a weak substrate phosphorylation. However, in BCR-positive transfectants of J558L, treatment with pervanadate/H2O2 induces a strong phosphorylation of several substrate proteins. Treatment with pervanadate/H2O2 does not result in receptor crosslinking, yet the pattern of protein phosphorylation is similar to that observed after BCR stimulation by antigen. The response requires cellular integrity because tyrosine phosphorylation of most substrates is not visible in cell lysates. Cells that express a BCR containing an Ig-alpha subunit with a mutated immunoreceptor tyrosine-based activation motif display a delayed response. The data suggest that, once expressed on the surface, the BCR organizes protein tyrosine phosphatases, PTKs, and their substrates into a transducer complex that can be activated by pervanadate/H202 in the absence of BCR crosslinking. Assembly of this preformed complex seems to be a prerequisite for BCR-mediated signal transduction.

Reduction of two functional gamma-globin genes to one: an evolutionary trend in New World monkeys (infraorder Platyrrhini).

Nucleotide sequences were determined for the gamma1- and gamma2-globin loci from representatives of the seven anciently separated clades in the three extant platyrrhine families (Atelidae, Pitheciidae, and Cebidae). These sequences revealed an evolutionary trend in New World monkeys either to inactivate the gamma1 gene or to fuse it with the gamma2 gene, i.e. to have only one functional fetally expressed gamma gene. This trend is clearly evident in six of the seven clades: (i) it occurred in atelids by deletion of most of the gamma1 gene in the basal ancestor of this clade; (ii-iv) in pitheciid titi, saki, and cebid capuchin monkeys by potentially debilitating nucleotide substitutions in the proximal CCAAT box of the gamma1 promoters and (v and vi) in cebid owl and squirrel monkeys by crossovers that fused 5' sequence from gamma1 with 3' sequence from gamma2. In the five clades with gamma1 and gamma2 loci separated by intergenic sequences (the fifth clade being the cebid marmosets), the gamma2 genes retained an unaltered proximal CCAAT motif and their gamma2 promoters accumulated fewer nucleotide substitutions than did the gamma1 promoters. Thus, phylogenetic considerations indicate that the stem platyrrhines, ancestral to all New World monkeys, had gamma2 as the primary fetally expressed gamma gene. A further inference is that when the earlier stem anthropoid gamma gene duplicated, gamma2 (at its greater downstream distance from epsilon) could evade embryonic activation by the locus control region but could be fetally activated once released by regulatory mutations from fetal repressors.

Different functions for the interleukin 8 receptors (IL-8R) of human neutrophil leukocytes: NADPH oxidase and phospholipase D are activated through IL-8R1 but not IL-8R2.

Two monoclonal antibodies, anti-IL8R1 and anti-IL8R2, raised against both interleukin 8 receptors (IL-8R) of human neutrophils, IL-8R1 and IL-8R2, were used to study individual receptor functions after stimulation with IL-8, GRO alpha, or NAP-2. Efficacy and selectivity of the antibodies were tested in Jurkat cells transfected with cDNA coding for one or the other receptor. The binding of 125 I labeled IL-8 and IL-8-induced changes of the cytosolic free Ca2+ concentration were inhibited by anti-IL8RI in cells expressing IL-8R1 and by anti-IL8R2 in cells expressing IL-8R2. In human neutrophils, release of elastase was observed after stimulation with IL-8 or GRO alpha. The response to IL-8 was inhibited slightly by anti-IL8R1 and more substantially when both monoclonal antibodies were present, while the response to GRO alpha was inhibited by anti-IL8R2 but was not affected by anti-IL8R1. These results indicate that both IL-8 receptors can signal independently for granule enzyme release. Superoxide production, a measure of the respiratory burst, was obtained with increasing concentrations of IL-8 with maximum effects at 25 to 50 nM, but no response was observed upon challenge with GRO alpha or NAP-2 up to 1000 nM. The superoxide production induced by IL-8 was inhibited by anti-IL8R1, but was not affected by anti-IL8R2. Stimulation of neutrophils with IL-8, in contrast to GRO alpha or NAP-2, also elicited phospholipase D activity. The effect of IL-8 was again inhibited by anti-IL-8R1 but not by anti-IL8R2, indicating that this response, like the respiratory burst, was mediated by IL-8R1. Taken together, our results show that IL-8R1 and IL-8R2 are functionally different. Responses, such as cytosolic free Ca2+ changes and the release of granule enzymes, are mediated through both receptors, whereas the respiratory burst and the activation of phospholipase D depend exclusively on stimulation through IL-8R1.

Characterization of a murine Ahr null allele: involvement of the Ah receptor in hepatic growth and development.

The Ah receptor (AHR) is a ligand-activated transcription factor that mediates a pleiotropic response to environmental contaminants such as benzo[a]pyrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin. In an effort to gain insight into the physiological role of the AHR and to develop models useful in risk assessment, gene targeting was used to inactivate the murine Ahr gene by homologous recombination. Ahr-/- mice are viable and fertile but show a spectrum of hepatic defects that indicate a role for the AHR in normal liver growth and development. The Ahr-/- phenotype is most severe between 0-3 weeks of age and involves slowed early growth and hepatic defects, including reduced liver weight, transient microvesicular fatty metamorphosis, prolonged extramedullary hematopoiesis, and portal hypercellularity with thickening and fibrosis.

Receptor stimulation causes slow inhibition of IRK1 inwardly rectifying K+ channels by direct protein kinase A-mediated phosphorylation.

Strongly rectifying IRK-type inwardly rectifying K+ channels are involved in the control of neuronal excitability in the mammalian brain. Whole-cell patch-clamp experiments show that cloned rat IRK1 (Kir 2.1) channels, when heterologously expressed in mammalian COS-7 cells, are inhibited following the activation of coexpressed serotonin (5-hydroxytryptamine) type 1A receptors by receptor agonists. Inhibition is mimicked by internal perfusion with GTP[gamma-S] and elevation of internal cAMP concentrations. Addition of the catalytic subunits of protein kinase A (PKA) to the internal recording solution causes complete inhibition of wild-type IRK1 channels, but not of mutant IRK1(S425N) channels in which a C-terminal PKA phosphorylation site has been removed. Our data suggest that in the nervous system serotonin may negatively control IRK1 channel activity by direct PKA-mediated phosphorylation.

Cell-type and promoter-context dependent retinoic acid receptor (RAR) redundancies for RAR beta 2 and Hoxa-1 activation in F9 and P19 cells can be artefactually generated by gene knockouts.

By using RAR type (alpha, beta, or gamma)-specific synthetic retinoids and a pan-retinoic X receptor (RXR)-specific ligand, we have investigated the contribution of RARs and RXRs in the activation of RA target genes and the differentiation of embryonal carcinoma cells. We demonstrate cell-type- and promoter context-dependent functional redundancies that differ between the three RAR types for mediating the induction of RARbeta2 and Hoxa-1 in wild-type, RARgamma-/- and RARalpha-/- F9 cells and in P19 cells. The extent of redundancy between RARs is further modulated by the synergistic activation of RXRs with a pan-RXR agonist. We also demonstrate that the expression of RARbeta2 is auto-inducible in RARgamma-/- but not in wild-type F9 cells, indicating that the functional redundancies observed between RARs in gene disruption studies can be artefactually generated. Thus, even though all three RARs can functionally substitute each other for inducing the expression of RA target genes and cell differentiation, one RAR can cell-specifically override the activity of the other RARs. Interestingly, only RARgamma can mediate the retinoic acid-induced differentiation of wild-type F9 cells, whereas the differentiation of P19 cells can be mediated by either RARalpha or RARgamma.

Dexamethasone responsiveness of a major glucocorticoid-inducible CYP3A gene is mediated by elements unrelated to a glucocorticoid receptor binding motif.

Elements responsible for dexamethasone responsiveness of CYP3A23, a major glucocorticoid-inducible member of the CYP3A gene family, have been identified. DNase I footprint analysis of the proximal promoter region revealed three protected sites (sites A, B, and C) within the sequence defined by -167 to -60. Mutational analysis demonstrated that both sites B and C were necessary for maximum glucocorticoid responsiveness and functioned in a cooperative manner. Interestingly, neither site contained a glucocorticoid responsive element. Embedded in site C was an imperfect direct repeat (5'-AACTCAAAGGAGGTCA-3'), showing homology to an AGGTCA steroid receptor motif, typically recognized by the estrogen receptor family, while site B contained an ATGAACT direct repeat; these core sequences were designated dexamethasone response elements 1 and 2 (DexRE-1 and -2), respectively. Neither element has previously been associated with a glucocorticoid-activated transcriptional response. Conversion of the DexRE-1 to either a perfect thyroid hormone or vitamin D3 responsive element further enhanced induction by dexamethasone. Gel-shift analysis demonstrated that glucocorticoid receptor did not associate with either DexRE-1 or -2; hence, glucocorticoid receptor does not directly mediate glucocorticoid induction of CYP3A23. These unusual features suggest an alternate pathway through which glucocorticoids exert their effects.