Protein degradation and increased mRNAs encoding proteins of the ubiquitin-proteasome proteolytic pathway in BC3H1 myocytes require an interaction between glucocorticoids and acidification.

In rats and humans, metabolic acidosis stimulates protein degradation and glucocorticoids have been implicated in this response. To evaluate the importance of glucocorticoids in stimulating proteolysis, we measured protein degradation in BC3H1 myocytes cultured in 12% serum. Acidification accelerated protein degradation but dexamethasone did not augment this response. To reduce the influence of glucocorticoids and other hormones and cytokines in 12% serum that could mediate proteolysis, we studied BC3H1 myocytes maintained in only 1% serum. Acidification of the medium or addition of dexamethasone at pH 7.4 did not significantly increase protein degradation, while acidification plus dexamethasone accelerated proteolysis. The steroid receptor antagonist RU 486 prevented this proteolytic response. Acidification of the medium with 1% serum did increase the mRNAs for ubiquitin and the C2 proteasome subunit, but when dexamethasone was added the mRNAs were increased significantly more. The steroid-receptor antagonist RU 486 suppressed this response to the addition of dexamethasone but the mRNAs remained at the levels measured in cells at pH 7.1 alone. Thus, acidification alone can increase the mRNAs of the ubiquitin-proteasome proteolytic pathway, but both acidosis and glucocorticoids are required to stimulate protein degradation. Since these changes occur without adding cytokines or other hormones, we conclude that the proteolytic response to acidification requires glucocorticoids.

Distinct ligand preferences of Src homology 3 domains from Src, Yes, Abl, Cortactin, p53bp2, PLCgamma, Crk, and Grb2.

Src homology 3 (SH3) domains are conserved protein modules 50-70 amino acids long found in a variety of proteins with important roles in signal transduction. These domains have been shown to mediate protein-protein interactions by binding short proline-rich regions in ligand proteins. However, the ligand preferences of most SH3 domains and the role of these preferences in regulating SH3-mediated protein-protein interactions remain poorly defined. We have used a phage-displayed library of peptides of the form X6PXXPX6 to identify ligands for eight different SH3 domains. Using this approach, we have determined that each SH3 domain prefers peptide ligands with distinct sequence characteristics. Specifically, we have found that the Src SH3 domain selects peptides sharing the consensus motif LXXRPLPXpsiP, whereas Yes SH3 selects psiXXRPLPXLP, Abl SH3 selects PPXthetaXPPPpsiP, Cortactin SH3 selects +PPpsiPXKPXWL, p53bp2 SH3 selects RPXpsiPpsiR+SXP, PLCgamma SH3 selects PPVPPRPXXTL, Crk N-terminal SH3 selects psiPpsiLPpsiK, and Grb2 N-terminal SH3 selects +thetaDXPLPXLP (where psi, theta, and + represent aliphatic, aromatic, and basic residues, respectively). Furthermore, we have compared the binding of phage expressing peptides related to each consensus motif to a panel of 12 SH3 domains. Results from these experiments support the ligand preferences identified in the peptide library screen and evince the ability of SH3 domains to discern subtle differences in the primary structure of potential ligands. Finally, we have found that most known SH3-binding proteins contain proline-rich regions conforming to the ligand preferences of their respective SH3 targets.

Studies of group B streptococcal infection in mice deficient in complement component C3 or C4 demonstrate an essential role for complement in both innate and acquired immunity.

Group B streptococci (GBS) cause sepsis and meningitis in neonates and serious infections in adults with underlying chronic illnesses. Specific antibodies have been shown to be an important factor in protective immunity for neonates, but the role of serum complement is less well defined. To elucidate the function of the complement system in immunity to this pathogen, we have used the approach of gene targeting in embryonic stem cells to generate mice totally deficient in complement component C3. Comparison of C3-deficient mice with mice deficient in complement component C4 demonstrated that the 50% lethal dose for GBS infection was reduced by approximately 50-fold and 25-fold, respectively, compared to control mice. GBS were effectively killed in vitro by human blood leukocytes in the presence of specific antibody and C4-deficient serum but not C3-deficient serum. The defective opsonization by C3-deficient serum in vitro was corroborated by in vivo studies in which passive immunization of pregnant dams with specific antibodies conferred protection from GBS challenge to normal and C4-deficient pups but not C3-deficient pups. These results indicate that the alternative pathway is sufficient to mediate effective opsonophagocytosis and protective immunity to GBS in the presence of specific antibody. In contrast, the increased susceptibility to infection of non-immune mice deficient in either C3 or C4 implies that the classical pathway plays an essential role in host defense against GBS infection in the absence of specific immunity.

A de novo missense mutation of the beta subunit of the epithelial sodium channel causes hypertension and Liddle syndrome, identifying a proline-rich segment critical for regulation of channel activity.

Liddle syndrome is a mendelian form of hypertension characterized by constitutively elevated renal Na reabsorption that can result from activating mutations in the beta or gamma subunit of the epithelial Na channel. All reported mutations have deleted the last 45-76 normal amino acids from the cytoplasmic C terminus of one of these channel subunits. While these findings implicate these terminal segments in the normal negative regulation of channel activity, they do not identify the amino acid residues that are critical targets for these mutations. Potential targets include the short highly conserved Pro-rich segments present in the C terminus of beta and gamma subunits; these segments are similar to SH3-binding domains that mediate protein-protein interaction. We now report a kindred with Liddle syndrome in which affected patients have a mutation in codon 616 of the beta subunit resulting in substitution of a Leu for one of these highly conserved Pro residues. The functional significance of this mutation is demonstrated both by the finding that this is a de novo mutation appearing concordantly with the appearance of Liddle syndrome in the kindred and also by the marked activation of amiloride-sensitive Na channel activity seen in Xenopus oocytes expressing channels containing this mutant subunit (8.8-fold increase compared with control oocytes expressing normal channel subunits; P = 0.003). These findings demonstrate a de novo missense mutation causing Liddle syndrome and identify a critical channel residue important for the normal regulation of Na reabsorption in humans.

Coupling the phosphotransferase system and the methyl-accepting chemotaxis protein-dependent chemotaxis signaling pathways of Escherichia coli.

Chemotactic responses in Escherichia coli are typically mediated by transmembrane receptors that monitor chemoeffector levels with periplasmic binding domains and communicate with the flagellar motors through two cytoplasmic proteins, CheA and CheY. CheA autophosphorylates and then donates its phosphate to CheY, which in turn controls flagellar rotation. E. coli also exhibits chemotactic responses to substrates that are transported by the phosphoenolpyruvate (PEP)-dependent carbohydrate phosphotransferase system (PTS). Unlike conventional chemoreception, PTS substrates are sensed during their uptake and concomitant phosphorylation by the cell. The phosphoryl groups are transferred from PEP to the carbohydrates through two common intermediates, enzyme I (EI) and phosphohistidine carrier protein (HPr), and then to sugar-specific enzymes II. We found that in mutant strains HPr-like proteins could substitute for HPr in transport but did not mediate chemotactic signaling. In in vitro assays, these proteins exhibited reduced phosphotransfer rates from EI, indicating that the phosphorylation state of EI might link the PTS phospho-relay to the flagellar signaling pathway. Tests with purified proteins revealed that unphosphorylated EI inhibited CheA autophosphorylation, whereas phosphorylated EI did not. These findings suggest the following model for signal transduction in PTS-dependent chemotaxis. During uptake of a PTS carbohydrate, EI is dephosphorylated more rapidly by HPr than it is phosphorylated at the expense of PEP. Consequently, unphosphorylated EI builds up and inhibits CheA autophosphorylation. This slows the flow of phosphates to CheY, eliciting an up-gradient swimming response by the cell.

Agonist-induced desensitization of dopamine D1 receptor-stimulated adenylyl cyclase activity is temporally and biochemically separated from D1 receptor internalization.

The regulation of the dopamine D1 receptor was investigated by using c-myc epitope-tagged D1 receptors expressed in Sf9 (fall armyworm ovary) cells. Treatment of D1 receptors with 10 microM dopamine for 15 min led to a loss of the dopamine-detected high-affinity state of the receptor accompanying a 40% reduction in the ability of the receptor to mediate maximal dopamine stimulation of adenylyl cyclase activity. After 60 min of agonist exposure, 45 min after the occurrence of desensitization, 28% of the cell surface receptors were internalized into an intracellular light vesicular membrane fraction as determined by radioligand binding and supported by photoaffinity labeling, immunocytochemical staining, and immunoblot analysis. Pretreatment of cells with concanavalin A or sucrose completely blocked agonist-induced D1 receptor internalization without preventing agonist-induced desensitization, indicating a biochemical separation of these processes. Collectively, these findings indicate that the desensitization of D1 receptor-coupled adenylyl cyclase activity and D1 receptor internalization are temporarily and biochemically distinct mechanisms regulating D1 receptor function following agonist activation.

The intrinsic ability of ribosomes to bind to endoplasmic reticulum membranes is regulated by signal recognition particle and nascent-polypeptide-associated complex.

Signal peptides direct the cotranslational targeting of nascent polypeptides to the endoplasmic reticulum (ER). It is currently believed that the signal recognition particle (SRP) mediates this targeting by first binding to signal peptides and then by directing the ribosome/nascent chain/SRP complex to the SRP receptor at the ER. We show that ribosomes can mediate targeting by directly binding to translocation sites. When purified away from cytosolic factors, including SRP and nascent-polypeptide-associated complex (NAC), in vitro assembled translation intermediates representing ribosome/nascent-chain complexes efficiently bound to microsomal membranes, and their nascent polypeptides could subsequently be efficiently translocated. Because removal of cytosolic factors from the ribosome/nascent-chain complexes also resulted in mistargeting of signalless nascent polypeptides, we previously investigated whether readdition of cytosolic factors, such as NAC and SRP, could restore fidelity to targeting. Without SRP, NAC prevented all nascent-chain-containing ribosomes from binding to the ER membrane. Furthermore, SRP prevented NAC from blocking ribosome-membrane association only when the nascent polypeptide contained a signal. Thus, NAC is a global ribosome-binding prevention factor regulated in activity by signal-peptide-directed SRP binding. A model presents ribosomes as the targeting vectors for delivering nascent polypeptides to translocation sites. In conjunction with signal peptides, SRP and NAC contribute to this specificity of ribosomal function by regulating exposure of a ribosomal membrane attachment site that binds to receptors in the ER membrane.

Double-stranded-RNA-dependent protein kinase and TAR RNA-binding protein form homo- and heterodimers in vivo.

The yeast two-hybrid system and far-Western protein blot analysis were used to demonstrate dimerization of human double-stranded RNA (dsRNA)-dependent protein kinase (PKR) in vivo and in vitro. A catalytically inactive mutant of PKR with a single amino acid substitution (K296R) was found to dimerize in vivo, and a mutant with a deletion of the catalytic domain of PKR retained the ability to dimerize. In contrast, deletion of the two dsRNA-binding motifs in the N-terminal regulatory domain of PKR abolished dimerization. In vitro dimerization of the dsRNA-binding domain required the presence of dsRNA. These results suggest that the binding of dsRNA by PKR is necessary for dimerization. The mammalian dsRNA-binding protein TRBP, originally identified on the basis of its ability to bind the transactivation region (TAR) of human immunodeficiency virus RNA, also dimerized with itself and with PKR in the yeast assay. Taken together, these results suggest that complexes consisting of different combinations of dsRNA-binding proteins may exist in vivo. Such complexes could mediate differential effects on gene expression and control of cell growth.

The VLA4/VCAM-1 adhesion pathway defines contrasting mechanisms of lodgement of transplanted murine hemopoietic progenitors between bone marrow and spleen.

Selective lodgement or homing of transplanted hemopoietic stem cells in the recipient's bone marrow (BM) is a critical step in the establishment of long-term hemopoiesis after BM transplantation. However, despite its biologic and clinical significance, little is understood about the process of homing. In the present study, we have concentrated on the initial stages of homing and explored the functional role in vivo of some of the adhesion pathways previously found to mediate in vitro adhesion of hemopoietic cells to cultured BM stroma. We have found that homing of murine hemopoietic progenitors of the BM of lethally irradiated recipients at 3 h after transplant was significantly reduced after pretreatment of the donor cells with an antibody to the integrin very late antigen 4 (VLA4). This inhibition of marrow homing was accompanied by an increase in hemopoietic progenitors circulating in the blood and an increased uptake of these progenitors by the spleen. Similar results were obtained by treatment of the recipients with an antibody to vascular cell adhesion molecule 1 (VCAM-1), a ligand for VLA4. Furthermore, we showed that administration of the same antibodies (anti-VLA4 or anti-VCAM-1) to normal animals causes mobilization of hemopoietic progenitors into blood. These data suggest that hemopoietic cell lodgement in the BM is a regulatable process and can be influenced by VLA4/VCAM-1 adhesion pathway. Although additional molecular pathways are not excluded and may be likely, our data establish VCAM-1 as a BM endothelial addressin, analogous to the role that mucosal addressin cell adhesion molecule (MAdCAM) plays in lymphocyte homing. Whether splenic uptake of hemopoietic progenitors is passive or controlled through different mechanisms remains to be clarified. In addition, we provide experimental evidence that homing and mobilization are related phenomena involving, at least partly, similar molecular pathways.

Cloning and expression of the cDNA of chicken cation-independent mannose-6-phosphate receptor.

We cloned and sequenced the 8767-bp full-length cDNA for the chicken cation-independent mannose-6-phosphate receptor (CI-MPR), of interest because, unlike its mammalian homologs, it does not bind insulin-like growth factor II (IGF-II). The cDNA encodes a protein of 2470 aa that includes a putative signal sequence, an extracytoplasmic domain consisting of 15 homologous repeat sequences, a 23-residue transmembrane sequence, and a 161-residue cytoplasmic sequence. Overall, it shows 60% sequence identity with human and bovine CI-MPR homologs, and all but two of 122 cysteine residues are conserved. However, it shows much less homology in the N-terminal signal sequence, in repeat 11, which is proposed to contain the IGF-II-binding site in mammalian CI-MPR homologs, and in the 14-aa residue segment in the cytoplasmic sequence that has been proposed to mediate G-protein-coupled signal transduction in response to IGF-II binding by the human CI-MPR. Transient expression in COS-7 cells produced a functional CI-MPR which exhibited mannose-6-phosphate-inhibitable binding and mediated endocytosis of recombinant human beta-glucuronidase. Expression of the functional chicken CI-MPR in mice lacking the mammalian CI-MPR should clarify the controversy over the physiological role of the IGF-II-binding site in mammalian CI-MPR homologs.

Conjugates of folate and anti-T-cell-receptor antibodies specifically target folate-receptor-positive tumor cells for lysis.

High-affinity folate receptors (FRs) are expressed at elevated levels on many human tumors. Bispecific antibodies that bind the FR and the T-cell receptor (TCR) mediate lysis of these tumor cells by cytotoxic T lymphocytes. In this report, conjugates that consist of folate covalently linked to anti-TCR antibodies are shown to be potent in mediating lysis of tumor cells that express either the alpha or beta isoform of the FR. Intact antibodies with an average of five folate per molecule exhibited high affinity for FR+ tumor cells but did not bind to FR- tumor cells. Lysis of FR+ cell lines could be detected at concentrations as low as 1 pM (approximately 0.1 ng/ml), which was 1/1000th the concentration required to detect binding to the FR+ cells. Various FR+ mouse tumor cell lines could be targeted with each of three different anti-TCR antibodies that were tested as conjugates. The antibodies included 1B2, a clonotypic antibody specific for the cytotoxic T cell clone 2C; KJ16, an anti-V beta 8 antibody; and 2C11, an anti-CD3 antibody. These antibodies differ in affinities by up to 100-fold, yet the cytolytic capabilities of the folate/antibody conjugates differed by no more than 10-fold. The reduced size (in comparison with bispecific antibodies) and high affinity of folate conjugates suggest that they may be useful as immunotherapeutic agents in targeting tumors that express folate receptors.

Intracellular polyamines mediate inward rectification of Ca(2+)-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors.

alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors that lack the glutamate receptor GluR2 subunit are Ca(2+)-permeable and exhibit inwardly rectifying current responses to kainate and AMPA. A proportion of cultured rat hippocampal neurons show similar Ca(2+)-permeable inwardly rectifying AMPA receptor currents. Inward rectification in these neurons was lost with intracellular dialysis and was not present in excised outside-out patches but was maintained in perforated-patch whole-cell recordings, suggesting that a diffusible cytoplasmic factor may be responsible for rectification. Inclusion of the naturally occurring polyamines spermine and spermidine in the recording pipette prevented loss of rectification in both whole-cell and excised-patch recordings; Mg2+ and putrescine were without effect. Inward rectification of Ca(2+)-permeable AMPA receptors may reflect voltage-dependent channel block by intracellular polyamines.

Tumor necrosis factors alpha and beta protect neurons against amyloid beta-peptide toxicity: evidence for involvement of a kappa B-binding factor and attenuation of peroxide and Ca2+ accumulation.

In Alzheimer disease (AD) the amyloid beta-peptide (A beta) accumulates in plaques in the brain. A beta can be neurotoxic by a mechanism involving induction of reactive oxygen species (ROS) and elevation of intracellular free calcium levels ([Ca2+]i). In light of evidence for an inflammatory response in the brain in AD and reports of increased levels of tumor necrosis factor (TNF) in AD brain we tested the hypothesis that TNFs affect neuronal vulnerability to A beta. A beta-(25-35) and A beta-(1-40) induced neuronal degeneration in a concentration- and time-dependent manner. Pretreatment of cultures for 24 hr with TNF-beta or TNF-alpha resulted in significant attenuation of A beta-induced neuronal degeneration. Accumulation of peroxides induced in neurons by A beta was significantly attenuated in TNF-pretreated cultures, and TNFs protected neurons against iron toxicity, suggesting that TNFs induce antioxidant pathways. The [Ca2+]i response to glutamate (quantified by fura-2 imaging) was markedly potentiated in neurons exposed to A beta, and this action of A beta was suppressed in cultures pretreated with TNFs. Electrophoretic mobility-shift assays demonstrated an induction of a kappa beta-binding activity in hippocampal cells exposed to TNFs. Exposure of cultures to I kappa B (MAD3) antisense oligonucleotides, a manipulation designed to induce NF-kappa B, mimicked the protection by TNFs. These data suggest that TNFs protect hippocampal neurons against A beta toxicity by suppressing accumulation of ROS and Ca2+ and that kappa B-dependent transcription is sufficient to mediate these effects. A modulatory role for TNF in the neurodegenerative process in AD is proposed.

Molecular characterization of a second melatonin receptor expressed in human retina and brain: the Mel1b melatonin receptor.

A G protein-coupled receptor for the pineal hormone melatonin was recently cloned from mammals and designated the Mel1a melatonin receptor. We now report the cloning of a second G protein-coupled melatonin receptor from humans and designate it the Mel1b melatonin receptor. The Mel1b receptor cDNA encodes a protein of 362 amino acids that is 60% identical at the amino acid level to the human Mel1a receptor. Transient expression of the Mel1b receptor in COS-1 cells results in high-affinity 2-[125I]iodomelatonin binding (Kd = 160 +/- 30 pM). In addition, the rank order of inhibition of specific 2-[125I]iodomelatonin binding by eight ligands is similar to that exhibited by the Mel1a melatonin receptor. Functional studies of NIH 3T3 cells stably expressing the Mel1b melatonin receptor indicate that it is coupled to inhibition of adenylyl cyclase. Comparative reverse transcription PCR shows that the Mel1b melatonin receptor is expressed in retina and, to a lesser extent, brain. PCR analysis of human-rodent somatic cell hybrids maps the Mel1b receptor gene (MTNR1B) to human chromosome 11q21-22. The Mel1b melatonin receptor may mediate the reported actions of melatonin in retina and participate in some of the neurobiological effects of melatonin in mammals.

Identification and characterization of a TFIID-like multiprotein complex from Saccharomyces cerevisiae.

Although the mechanisms of transcriptional regulation by RNA polymerase II are apparently highly conserved from yeast to man, the identification of a yeast TATA-binding protein (TBP)-TBP-associated factor (TAFII) complex comparable to the metazoan TFIID component of the basal transcriptional machinery has remained elusive. Here, we report the isolation of a yeast TBP-TAFII complex which can mediate transcriptional activation by GAL4-VP16 in a highly purified yeast in vitro transcription system. We have cloned and sequenced the genes encoding four of the multiple yeast TAFII proteins comprising the TBP-TAFII multisubunit complex and find that they are similar at the amino acid level to both human and Drosophila TFIID subunits. Using epitope-tagging and immunoprecipitation experiments, we demonstrate that these genes encode bona fide TAF proteins and show that the yeast TBP-TAFII complex is minimally composed of TBP and seven distinct yTAFII proteins ranging in size from M(r) = 150,000 to M(r) = 25,000. In addition, by constructing null alleles of the cloned TAF-encoding genes, we show that normal function of the TAF-encoding genes is essential for yeast cell viability.