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R D Klausner

Publications and source records attributed to R D Klausner.

At least 145 records · Page 8Linked to original sources
PubMed1990

The T cell receptor-associated protein is proteolytically cleaved in a pre-Golgi compartment.

The TCR consists of at least seven transmembrane chains: the clonotypic disulfide-linked alpha- and beta-chains, the invariant gamma-, delta-, and epsilon-chains, termed the CD3 complex, and the zeta-zeta homodimer. We have recently described an additional 26-kDa protein, which is transiently associated with newly synthesized mouse CD3 chains in the endoplasmic reticulum. The exact function of this protein, which we called TRAP (for TCR-associated protein) is not yet known; studies suggest however, that it may play a role in the assembly of the TCR complex. Here we report the properties of another protein which has a Mr of 16,000 and, like TRAP, can be coimmunoprecipitated from metabolically labelled murine T cells with antibodies against the chains of the CD3 complex. Kinetic analysis suggests a precursor-product relationship between TRAP and the 16-kDa protein: the latter starts appearing once TRAP begin to disappear. Having reached a maximal level at approximately 1 h after biosynthesis, it is rapidly lost. Agents that slow or block the disappearance of TRAP, delay or prevent the appearance and eventual disappearance of the 16-kDa protein. Incubation of immunoprecipitates containing gamma, epsilon, and TRAP in vitro at 37 degrees C results in the appearance of the 16-kDa protein. Employing HPLC peptide mapping we demonstrate that this 16-kDa protein is structurally related to TRAP. These results suggest that the removal of TRAP from the newly synthesized CD3 chains is accompanied by its proteolytic cleavage in a pre-Golgi compartment.

Animals↗
PubMed1990

Transmembrane helical interactions and the assembly of the T cell receptor complex.

Studies of the subunit interactions of the multicomponent T cell antigen receptor (TCR) revealed that specific pairs of chains have the ability to assemble after transfection into fibroblasts. For one such pair, TCR-alpha and CD3-delta, their ability to assemble was encoded by their transmembrane domains. The specificity of this interaction suggests that well-defined helical interactions in the membrane can explain the assembly of some multichain membrane complexes.

Amino Acid Sequence↗
PubMed1990

Structural mutations of the T cell receptor zeta chain and its role in T cell activation.

T cell hybridomas that express zeta zeta, but not zeta eta, dimers in their T cell receptors (TCRs) produce interleukin-2 (IL-2) and undergo an inhibition of spontaneous growth when activated by antigen, antibodies to the receptor, or antibodies to Thy-1. Hybridomas without zeta and eta were reconstituted with mutated zeta chains. Cytoplasmic truncations of up to 40% of the zeta molecule reconstituted normal surface assembly of TCRs, but antigen-induced IL-2 secretion and growth inhibition were lost. In contrast, cross-linking antibodies to the TCR activated these cells. A point mutation conferred the same signaling phenotype as did the truncations and caused defective antigen-induced tyrosine kinase activation. Thus zeta allows the binding of antigen/major histocompatibility complex (MHC) to alpha beta to effect TCR signaling.

Amino Acid Sequence↗
PubMed1990

Novel post-translational regulation of TCR expression in CD4+CD8+ thymocytes influenced by CD4.

Expression of the multicomponent T-cell antigen receptor (TCR) complex on the surface of thymocytes is developmentally controlled. Most immature CD4-CD8- 'double negative' and CD4+CD8+ 'double positive' thymocytes express either no or few TCR on their surface, and maturation to CD4+CD8- or CD4-CD8+ 'single positive' thymocytes is accompanied by a dramatic increase in the number of surface TCR complexes. Although the initial appearance of TCR during differentiation results from rearrangement and initiation of transcription of TCR genes in the thymus, the mechanisms regulating the quantitative changes in TCR expression during intrathymic differentiation are unknown. Surface TCR levels in T-hybridoma cells can be quantitatively regulated by a series of post-translational processes, including sorting to alternative intracellular compartments and degradation, which ensure that only fully and correctly assembled receptor complexes are efficiently transported to the cell surface. Quantitative increases in TCR expression on the surface of CD4+CD8+ thymocytes occur in vivo in response to anti-CD4 antibody treatment. Here we present evidence that immature CD4+CD8+ thymocytes normally retain and degrade in the endoplasmic reticulum greater than 90% of some endogenously synthesized TCR chains, and that the increased surface TCR expression on immature CD4+CD8+ thymocytes induced by anti-CD4 is due to an increase in the escape of newly synthesized receptor chains from the endoplasmic reticulum, and is not due to increases in RNA levels, translation, or assembly. Post-translational mechanisms therefore control the levels of TCR complexes on CD4+CD8+ thymocytes, and these mechanisms can be modulated by signalling through CD4 surface molecules.

Animals↗
PubMed1990

Microtubule-dependent retrograde transport of proteins into the ER in the presence of brefeldin A suggests an ER recycling pathway.

Characteristics of brefeldin A (BFA)-induced redistribution of Golgi proteins into the endoplasmic reticulum (ER) and its relationship to an ER retrieval pathway were investigated. Retrograde movement of Golgi proteins into the ER occurred via long, tubulovesicular processes extending out of the Golgi along microtubules. Microtubule-disrupting agents (i.e., nocodazole), energy poisons, and reduced temperatures inhibited this pathway. In BFA-treated cells Golgi proteins appeared to cycle between the ER and an intermediate compartment marked by a 53 kd protein. Addition of nocodazole disrupted this dynamic cycle by preferentially inhibiting retrograde movement, causing Golgi proteins to accumulate in the intermediate compartment. In the absence of BFA, such an ER cycling pathway appeared to be followed normally by the 53 kd protein but not by Golgi proteins, as revealed by temperature shift experiments. We propose that BFA induces the interaction of the Golgi with an intermediate "recycling" compartment that utilizes a microtubule-dependent pathway into the ER.

Animals↗
PubMed1990

A peptide sequence confers retention and rapid degradation in the endoplasmic reticulum.

A nonlysosomal pathway exists for the degradation of newly synthesized proteins retained within the endoplasmic reticulum (ER). This pathway is extremely selective: whereas some proteins are rapidly degraded, others survive for long periods in the ER. The question of whether this selectivity is due to the presence within the sensitive proteins of definable peptide sequences that are sufficient to target them for degradation has been addressed. Deletion of a carboxyl-terminal sequence, comprising the transmembrane domain and short cytoplasmic tail of the alpha chain of the T cell antigen receptor (TCR-alpha), prevented the rapid degradation of this polypeptide. Fusion of this carboxyl-terminal sequence to the extracellular domain of the Tac antigen, a protein that is normally transported to the cell surface where it survives long-term, resulted in the retention and rapid degradation of the chimeric protein in the ER. Additional mutagenesis revealed that the transmembrane domain of TCR-alpha alone was sufficient to cause degradation within the ER. This degradation was not a direct consequence of retention in the ER, as blocking transport of newly synthesized proteins out of the ER with brefeldin A did not lead to degradation of the normal Tac antigen. It is proposed that a 23-amino acid sequence, comprising the transmembrane domain of TCR-alpha, contains information that determines targeting for degradation within the ER system.

Amino Acid Sequence↗
PubMed1990

Class II MHC molecules can use the endogenous pathway of antigen presentation.

Models for antigen presentation have divided the world of antigens into two categories, endogenous and exogenous, presented to T cells by class I and class II major histocompatibility complex (MHC) encoded molecules, respectively. Exogenous antigens are though to be taken up into peripheral endosomal compartments where they are processed for binding to class II MHC molecules. Endogenous antigens are either synthesized or efficiently delivered to the cytoplasm before being partially degraded in an as yet undefined way, and complexed with class I MHC molecules. A useful phenotypic distinction between the two pathways has been the sensitivity to weak bases, such as chloroquine, which is a property only of the exogenous pathway. The fungal antibiotic brefeldin A (BFA), which blocks protein transport from the endoplasmic reticulum to the Golgi network, also blocks class I-restricted antigen-presentation, providing us with the corresponding marker of the endogenous pathway. Experiments with influenza virus antigens have supported the view that class II MHC molecules can present exogenous but not endogenous antigen, whereas the observation that class II MHC molecules present measles virus non-membrane antigens by a chloroquine-insensitive pathway suggests that this is not always the case. We show here that influenza A matrix protein can be effectively presented to class II-restricted T cells by two pathways: one of which is chloroquine-sensitive, BFA-insensitive, the other being chloroquine-insensitive and BFA-sensitive. Our results indicate that both class I and class II molecules can complex with antigenic peptides in a pre-Golgi compartment and favour a unified mechanism for MHC-restricted endogenous antigen presentation.

Antigen-Presenting Cells↗
PubMed1990

Translational repression by a complex between the iron-responsive element of ferritin mRNA and its specific cytoplasmic binding protein is position-dependent in vivo.

The interaction of ferritin mRNA is regulated by iron via the interaction of a cytoplasmic binding protein (IRE-BP) with a specific stem-loop structure in the 5' untranslated region (UTR), referred to as the iron-responsive element (IRE). A high affinity RNA-protein complex between the IRE and the IRE-BP functions as a repressor of translation in vivo. Translational repression appears to depend upon the position of the IRE in the 5' UTR of the mRNA. IREs located in the 5' untranslated region 67 nucleotides or more downstream of the 5' terminus of the mRNA fail to mediate iron-dependent translational regulation and give rise to constitutively derepressed transcripts. A model is proposed in which translational regulation of protein biosynthesis involves a position-dependent interference of the IRE/IRE-BP complex with one of the initial steps in translation initiation.

Animals↗
PubMed1990

Association of the fyn protein-tyrosine kinase with the T-cell antigen receptor.

Activation of the T-cell antigen receptor (TCR) results in tyrosine phosphorylation of the TCR zeta chain and other intracellular substrates. Two other T-cell integral membrane proteins, CD4 and CD8, are associated with the protein-tyrosine kinase (PTK), lck. Despite evidence that activation of this enzyme results in TCR-zeta chain phosphorylation, it has not been shown that the TCR activates lck. We have sought evidence that the TCR is associated with a PTK. In this study we use digitonin to solubilize a murine T-cell hybridoma and demonstrate that antibodies binding extracellular but not intracellular domains of the TCR specifically coprecipitate only the fyn PTK and not lck or yes, two other kinases found in these cells. The association of the fyn PTK with the TCR might enable the T cell to independently regulate two PTKs through surface receptors.

Animals↗
PubMed1990

Cloning of the cDNA encoding an RNA regulatory protein--the human iron-responsive element-binding protein.

Iron-responsive elements (IREs) are stemloop structures found in the mRNAs encoding ferritin and the transferrin receptor. These elements participate in the iron-induced regulation of the translation of ferritin and the stability of the transferrin receptor mRNA. Regulation in both instances is mediated by binding of a cytosolic protein to the IREs. High-affinity binding is seen when cells are starved of iron and results in repression of ferritin translation and inhibition of transferrin receptor mRNA degradation. The IRE-binding protein (IRE-BP) has been identified as an approximately 90-kDa protein that has been purified by both affinity and conventional chromatography. In this report we use RNA affinity chromatography and two-dimensional gel electrophoresis to isolate the IRE-BP for protein sequencing. A degenerate oligonucleotide probe derived from a single peptide sequence was used to isolate a cDNA clone that encodes a protein containing 13 other sequenced peptides obtained from the IRE-BP. Consistent with previous characterization of the IRE-BP, the cDNA encodes a protein of 87 kDa with a slightly acidic pI, and the corresponding mRNA of approximately 3.6 kilobases is found in a variety of cell types. The encoded protein contains a nucleotide-binding consensus sequence and regions of cysteine and histidine clusters. This mRNA is encoded by a single gene on human chromosome 9, a finding consistent with previous localization by functional mapping. The protein contains no previously defined consensus motifs for either RNA or DNA binding. The simultaneous cloning of a different, but highly homologous, cDNA suggests that the IRE-BP is a member of a distinct gene family.

Amino Acid Sequence↗
PubMed1990

Tyrosine phosphorylation in T cells is regulated by phosphatase activity: studies with phenylarsine oxide.

Activation of T cells induces rapid tyrosine phosphorylation on the T-cell receptor zeta chain and other substrates. These phosphorylations can be regulated by a number of protein-tyrosine kinases (ATP: protein-tyrosine O-phosphotransferase, EC 2.7.1.112) and protein-tyrosine-phosphatases (protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48). In this study, we demonstrate that phenylarsine oxide can inhibit tyrosine phosphatases while leaving tyrosine kinase function intact. We use this reagent to investigate the effect of tyrosine phosphatase inhibition in a murine T-cell hybridoma. Increasing concentrations of phenylarsine oxide result in an increase in tyrosine phosphate on a number of intracellular substrates in unstimulated T cells, suggesting that a protein-tyrosine kinase is constitutively active in these cells. The effect of phenylarsine oxide on T cells stimulated with an anti-Thy 1 monoclonal antibody is more complex. At low concentrations of drug, there is a synergistic increase in the level of tyrosine phosphate on certain cellular substrates. At higher concentrations, anti-Thy 1-stimulated tyrosine phosphorylation is inhibited. These results indicate that tyrosine phosphorylation in T cells is tightly regulated by tyrosine phosphatases. Partial inhibition of these enzymes results in enhanced substrate phosphorylation. Inhibition of all stimulated tyrosine phosphorylation by high doses of phenylarsine oxide suggests that tyrosine kinase activity is regulated by tyrosine phosphatases.

Animals↗
PubMed1990

Dissociation of a 110-kD peripheral membrane protein from the Golgi apparatus is an early event in brefeldin A action.

Brefeldin A (BFA) has a profound effect on the structure of the Golgi apparatus, causing Golgi proteins to redistribute into the ER minutes after drug treatment. Here we describe the dissociation of a 110-kD cytoplasmically oriented peripheral membrane protein (Allan, V. J., and T. E. Kreis. 1986. J. Cell Biol. 103:2229-2239) from the Golgi apparatus as an early event in BFA action, preceding other morphologic changes. In contrast, other peripheral membrane proteins of the Golgi apparatus were not released but followed Golgi membrane into the ER during BFA treatment. The 110-kD protein remained widely dispersed throughout the cytoplasm during drug treatment, but upon removal of BFA it reassociated with membranes during reformation of the Golgi apparatus. Although a 30-s exposure to the drug was sufficient to cause the redistribution of the 110-kD protein, removal of the drug after this short exposure resulted in the reassociation of the 110-kD protein and no change in Golgi structure. If cells were exposed to BFA for 1 min or more, however, a portion of the Golgi membrane was committed to move into and out of the ER after removal of the drug. ATP depletion also caused the reversible release of the 110-kD protein, but without Golgi membrane redistribution into the ER. These findings suggest that the interaction between the 110-kD protein and the Golgi apparatus is dynamic and can be perturbed by metabolic changes or the drug BFA.

Adenosine Triphosphate↗
PubMed1990

Genetic evidence that ferric reductase is required for iron uptake in Saccharomyces cerevisiae.

The requirement for a reduction step in cellular iron uptake has been postulated, and the existence of plasma membrane ferric reductase activity has been described in both procaryotic and eucaryotic cells. In the yeast Saccharomyces cerevisiae, there is an externally directed reductase activity that is regulated by the concentration of iron in the growth medium; maximal activity is induced by iron starvation. We report here the isolation of a mutant of S. cerevisiae lacking the reductase activity. This mutant is deficient in the uptake of ferric iron and is extremely sensitive to iron deprivation. Genetic analysis of the mutant demonstrates that the reductase and ferric uptake deficiencies are due to a single mutation that we designate fre1-1. Both phenotypes cosegregate in meiosis, corevert with a frequency of 10(-7), and are complemented by a 3.5-kilobase fragment of genomic DNA from wild-type S. cerevisiae. This fragment contains FRE1, the wild-type allele of the mutant gene. The level of the gene transcript is regulated by iron in the same was as the reductase activity. The ferrous ion product of the reductase must traverse the plasma membrane. A high-affinity (Km = 5 microM) ferrous uptake system is present in both wild-type and mutant cells. Thus, iron uptake in S. cerevisiae is mediated by two plasma membrane components, a reductase and a ferrous transport system.

Biological Transport↗
PubMed1990

Coordinate post-transcriptional regulation of ferritin and transferrin receptor expression: the role of regulated RNA-protein interaction.

Excess iron results in an increase in the translation of ferritin mRNA and a decrease in the stability of transferrin receptor (TfR) mRNA. These coordinate regulatory events are mediated by similar sequence/structure motifs that exist within the 5' untranslated region (5'UTR) of the ferritin mRNA and the 3'UTR of the TfR mRNA. We have referred to these motifs as iron-responsive elements (IREs). The IREs from both transcripts interact with a cytoplasmic protein that we have called the IRE-binding protein (IRE-BP). The activity but not the amount of the IRE-BP is dependent on the cellular iron status. The biochemical basis for the altered activity of the IRE-BP appears to be the reversible oxidation-reduction of one or more cysteines in the IRE-BP. The IRE-BP is a 90- to 95-kD cytosolic protein that has been purified to homogeneity by RNA affinity chromatography, and the cDNA corresponding to the IRE-BP has been molecularly cloned. Collectively, our data support a model in which the interaction between the IRE-BP and the ferritin IRE results in attenuation of translation, and similar interaction with TfR mRNA can protect the transcript from rapid degradation mediated by a rapid turnover determinant within the 3'UTR.

Base Sequence↗
PubMed1990

The structure and signalling functions of the invariant T cell receptor components.

The T cell antigen receptor (TCR) is a multisubunit surface molecule on T cells which recognizes foreign antigens. In addition to the clone-specific alpha beta or gamma delta heterodimer antigen recognition element, each TCR has five invariant chains--the CD3-gamma, -delta, and -epsilon chains and a zeta zeta or zeta eta disulfide dimer. Receptor assembly and surface expression requires the presence of all chains except eta. Targetting of partial complexes, however, is determined differently by specific chains with the zeta chain in murine T cells providing safe transport of assembled pentamers from the Golgi complex to the cell surface. TCR signalling involves activation of two kinase pathways--protein kinase C and a non-receptor protein tyrosine kinase. zeta eta-containing TCRs couple preferentially to the PKC pathway by mediating phosphoinositide hydrolysis. We have evidence that the activated protein tyrosine kinase may be fyn, a member of the src family. While specific signalling roles for all invariant chains are not yet defined, we have implicated the zeta chain as uniquely coupling TCR antigen engagement to distal IL-2 signalling, perhaps via activation of the tyrosine kinase pathway.

Animals↗
PubMed1990

Specific high-affinity binding of host cell proteins to the 3' region of rubella virus RNA.

Replication of rubella virus is initiated at the 3' end of the genomic RNA. An inverted repeat sequence of 12 nucleotides that is capable of forming a stem-loop structure is located at the 3' end of the RNA, 59 nucleotides upstream from the poly (A) tail. We screened the 158-bp region of the 3' end of the virus, including the stem-loop structure, for its ability to bind to host-cell proteins. Specific high-affinity binding of three cytosolic proteins with relative molecular masses (Mr) of 61, 63 and 68 kD to the stem-loop structure was observed by UV-induced covalent crosslinking. Altering the stem structure by removal of specific bases abolished the binding interactions. The binding of the host proteins is greatly increased after infection and coincides with the appearance of negative strand RNA synthesis. The increase in binding is dependent on new protein synthesis. The amount of the 61-kD protein that binds varies in uninfected cells and is maximal in cells that are in the stationary phase of growth. All binding activity could be abrogated by alkaline phosphatase treatment of cell lysates. A possible role of these host proteins in the replication of rubella virus is discussed.

Animals↗
PubMed1989

Role of the zeta chain in the expression of the T cell antigen receptor: genetic reconstitution studies.

The zeta (zeta) chain plays a central role in T cell antigen receptor assembly and signal transduction. From previous work in murine T cell hybridomas we have inferred that the zeta subunit is limiting in receptor assembly. Partial receptors made in excess of zeta are assembled in the endoplasmic reticulum, transported through the Golgi, but then rapidly and efficiently degraded in lysosomes. zeta would therefore seem to play a unique role in targeting receptors from the Golgi to the cell surface. To determine directly whether zeta limits receptor assembly we have reconstituted a zeta-deficient T cell line by transfection of the murine zeta cDNA. Transfection results in restoration of expression of surface T cell receptor. In addition, increasing zeta expression results in a commensurate increase in the survival of previously excess subunits. This is reflected in an increased surface expression of complete receptors. Finally, transfection of the zeta cDNA fails to produce detectable zeta-eta heterodimers. The implications of these findings with regard to receptor assembly, and the relationship between zeta and eta, are discussed.

Animals↗