T-cell antigen receptor assembly and cell surface expression is not affected by treatment with T-cell antigen receptor-alpha chain transmembrane Peptide

A synthetic peptide termed core peptide (CP), which corresponds to a specific sequence of the TCR-alpha chain transmembrane domain, is known to inhibit IL-2 production in antigen stimulated T-cells. The molecular mechanism of the TCR inhibition is not known. This study examined the effects of CP on TCR subunit assembly and TCR cell surface expression in vitro. Co-transfection experiments between TCR-alpha and CD3-delta using COS-7 cells, and the interaction between TCR-alpha and the CD3 proteins in a T-cell line (2B4) were analysed after incubation with CP or its conjugates. Results indicate that CP co-precipitates with CD3-delta and CD3-epsilon in vitro, without any effect on TCR-alpha/CD3-delta dimerisation or TCR multisubunit assembly and cell surface expression.     

Lateral dimerization of the E-cadherin extracellular domain is necessary but not sufficient for adhesive activity

Cadherins are transmembrane glycoproteins involved in Ca(2+)-dependent cell-cell adhesion. Using L cells coexpressing E-cadherin constructs with different epitope tags, we examined the lateral dimerization of E-cadherin and its adhesive activity by co-immunoprecipitation and aggregation assays, respectively. Although the transmembrane domain is required for dimerization, tail-less constructs possessing the transmembrane domain of either N-cadherin or CD45 show dimerization and are active in aggregation assays. Two mutant constructs having either of two amino acid substitutions, W2A or substitutions that disrupt the recognition sequence for endoproteolytic enzymes involved in removal of the precursor segment, cannot form dimers and are inactive in aggregation. These monomeric proteins, like their wild-type dimerizing counterparts, retain their Ca(2+)-dependent resistance to trypsin digestion, suggesting that dimerization per se does not induce a large conformational change. Two other constructs, having either an amino acid substitution, D134A, or a C-terminal deletion of 70 amino acid residues, retain the ability to associate laterally but are inactive in aggregation assays. Staurosporine treatment of cells expressing the latter construct increases aggregation but does not increase the extent of lateral dimerization. Thus, lateral dimerization is necessary, but not sufficient for adhesive activity.     

Alloantigen-specific cytotoxic clones bearing the alpha,beta T cell antigen receptor but not CD4 or CD8 molecules

The vast majority of circulating lymphocytes that express the alpha,beta TCR in association with CD3 also express either CD4 or CD8 molecules, which are thought to act as important accessory structures in HLA class II- and I-restricted T cell functions, respectively. In the current study alpha,beta TCR+ clones devoid of detectable CD4 or CD8 were generated by repeated stimulation of fresh CD3+,CD4-,CD8- cells with an allogeneic lymphoblastoid cell line in the presence of conditioned medium containing IL-2. Except for the absence of CD4 and CD8, which was associated with undetectable levels of CD4 and CD8 mRNA, the clones were phenotypically indistinguishable from classical CD3+,alpha,beta TCR+ cells. Furthermore, they mediated potent cytolysis of their specific stimulator line but did not kill irrelevant LCL or NK-sensitive targets. mAb to CD3 and the alpha,beta TCR inhibited cytolysis, suggesting that the clones use the TCR/CD3 complex to recognize and respond to their targets. mAbs to CD2 and CD11a also inhibited cytolysis, indicating that the clones use these accessory molecules to interact with their targets. Finally, cytolysis was inhibited by an HLA-A,B,C framework-specific mAb (W6/32) as well as a mAb (MA2.1) specific for an HLA-A2 epitope. These results demonstrate that CD3+,alpha,beta TCR+,CD4-,CD8- cytotoxic clones can be generated from the peripheral blood of healthy adults, and use their TCR/CD3 complexes to function in an HLA class I-restricted manner.     

A delta T-cell receptor deleting element transgenic reporter construct is rearranged in alpha beta but not gamma delta T-cell lineages.

T cells can be divided into two groups on the basis of the expression of either alpha beta or gamma delta T-cell receptors (TCRs). Because the TCR delta chain locus lies within the larger TCR alpha chain locus, control of the utilization of these two receptors is important in T-cell development, specifically for determination of T-cell type: rearrangement of the alpha locus results in deletion of the delta coding segments and commitment to the alpha beta lineage. In the developing thymus, a relative site-specific recombination occurs by which the TCR delta chain gene segments are deleted. This deletion removes all D delta, J delta, and C delta genes and occurs on both alleles. This delta deletional mechanism is evolutionarily conserved between mice and humans. Transgenic mice which contain the human delta deleting elements and as much internal TCR delta chain coding sequence as possible without allowing the formation of a complete delta chain gene were developed. Several transgenic lines showing recombinations between deleting elements within the transgene were developed. These lines demonstrate that utilization of the delta deleting elements occurs in alpha beta T cells of the spleen and thymus. These recombinations are rare in the gamma delta population, indicating that the machinery for utilization of delta deleting elements is functional in alpha beta T cells but absent in gamma delta T cells. Furthermore, a discrete population of early thymocytes containing delta deleting element recombinations but not V alpha-to-J alpha rearrangements has been identified. These data are consistent with a model in which delta deletion contributes to the implementation of a signal by which the TCR alpha chain locus is rearranged and expressed and thus becomes an alpha beta T cell.     

The C-terminus of the gamma 2 chain but not of the beta 3 chain of laminin-332 is indirectly but indispensably necessary for integrin-mediated cell reactions

Using a recombinant mini-laminin-332, we showed that truncation of the three C-terminal amino acids of the gamma 2 chain, but not of the C-terminal amino acid of the beta 3 chain, completely abolished alpha 3 beta 1 integrin binding and its cellular functions, such as attachment and spreading. However, a synthetic peptide mimicking the gamma 2 chain C-terminus did not interfere with alpha 3 beta 1 integrin binding or cell adhesion and spreading on laminin-332 as measured by protein interaction assays and electric cell-substrate impedance sensing. Nor was the soluble peptide able to restore the loss of integrin-mediated cell adhesiveness to mini-laminin-332 after deletion of the gamma 2 chain C-terminus. These findings spoke against the hypothesis that the gamma 2 chain C-terminus of laminin-332 is a part of the alpha 3 beta 1 integrin interaction site. In addition, structural studies with electron microscopy showed that truncation of the gamma 2 chain C-terminus opened up the compact supradomain structure of LG1-3 domains. Thus, by inducing or stabilizing an integrin binding-competent conformation or array of the LG1-3 domains, the gamma 2 chain C-terminus plays an indirect but essential role in laminin-332 recognition by alpha 3 beta 1 integrin and, hence, its cellular functions.     

The transmembrane anchor of the T-cell antigen receptor beta chain contains a structural determinant of pre-Golgi proteolysis.

Studies with the T-cell antigen receptor (TCR) have shown that the endoplasmic reticulum, or an organelle closely associated with it, can retain and degrade membrane proteins selectively. The observation that only three (alpha, beta, and delta) of the six (alpha beta gamma delta epsilon zeta) subunits of the TCR are susceptible to proteolysis implies that structural features within the labile proteins mark them for degradation. The TCR beta chain is degraded in the endoplasmic reticulum, and, in this study, we have started to define the domains of the protein that make it susceptible to proteolysis. The experiments show that the transmembrane anchor and short five-amino-acid cytoplasmic tail of the protein contain a dominant determinant of proteolysis. When these residues were removed from the beta chain, the protein became resistant to proteolysis. Even though the resulting ectodomain of the beta chain lacked a transmembrane anchor, it was not secreted by cells and was retained in the endoplasmic reticulum. We conclude that retention in the endoplasmic reticulum alone does not lead to degradation. The results suggest that structural features within the membrane anchor of the protein predispose the beta chain to proteolysis. This was confirmed by replacing the membrane anchor of the interleukin 2 (IL2) receptor, a protein that was stable within the secretory pathway, with that of the TCR beta chain. The unmodified IL2 receptor was transported efficiently to the surface of cells, and an "anchor minus" construct was secreted quantitatively into the culture media. When the membrane anchor of the IL2 receptor was replaced with that of the TCR beta chain, the chimera was unable to reach the Golgi apparatus and was degraded rapidly.     

The achondroplasia mutation does not alter the dimerization energetics of the fibroblast growth factor receptor 3 transmembrane domain

The Gly380 --> Arg mutation in the TM domain of fibroblast growth factor receptor 3 (FGFR3) of the RTK family is linked to achondroplasia, the most common form of human dwarfism. The molecular mechanism of pathology induction is under debate, and two different mechanisms have been proposed to contribute to pathogenesis: (1) Arg380-mediated FGFR3 dimer stabilization and (2) slow downregulation of the activated mutant receptors. Here we show that the Gly380 --> Arg mutation does not alter the dimerization energetics of the FGFR3 transmembrane domain in detergent micelles or in lipid bilayers. This result indicates that pathogenesis in achondroplasia cannot be explained simply by a higher dimerization propensity of the mutant FGFR3 TM domain, thus highlighting the importance of the observed slow downregulation in phenotype induction.     

Early B-cell activation after West Nile virus infection requires alpha/beta interferon but not antigen receptor signaling

The B-cell response against West Nile virus (WNV), an encephalitic Flavivirus of global concern, is critical to controlling central nervous system dissemination and neurological sequelae, including death. Here, using a well-characterized mouse model of WNV infection, we examine the factors that govern early B-cell activation. Subcutaneous inoculation with a low dose of replicating WNV results in extensive B-cell activation in the draining lymph node (LN) within days of infection as judged by upregulation of the surface markers CD69, class II major histocompatibility complex, and CD86 on CD19(+) cells. B-cell activation in the LN but not the spleen was dependent on signals through the type I alpha/beta interferon (IFN-alpha/beta) receptor. Despite significant activation in the draining LN at day 3 after infection, WNV-specific B cells were not detected by immunoglobulin M enzyme-linked immunospot analysis until day 7. Liposome depletion experiments demonstrate that B-cell activation after WNV infection was not affected by the loss of F4/80(+) or CD169(+) subcapsular macrophages. Nonetheless, LN myeloid cells were essential for control of viral replication and survival from infection. Overall, our data suggest that the massive, early polyclonal B-cell activation occurring in the draining LN after WNV infection is immunoglobulin receptor and macrophage independent but requires sustained signals through the type I IFN-alpha/beta receptor.     

Interaction of nectin with afadin is necessary for its clustering at cell-cell contact sites but not for its cis dimerization or trans interaction

We have recently found a novel functional unit of cell-cell adhesion at cadherin-based adherens junctions, consisting of at least nectin, a homophilic cell adhesion molecule, and afadin, an actin filament-binding protein, which connects nectin to the actin cytoskeleton. Here we studied a mechanism of cell-cell adhesion of the nectin-afadin system by use of a cadherin-deficient L cell line stably expressing the intact form of mouse nectin-2alpha, a truncated form of nectin-2alpha incapable of interacting with afadin (nectin-2alpha-DeltaC), or a point-mutated form of nectin-2alpha capable of interacting with afadin and a cadherin-expressing EL cell line, which transiently expressed the point-mutated form of nectin-2alpha. We found that the interaction of nectin-2alpha with afadin was necessary for their clustering at cell-cell contact sites. However, nectin-2alpha-DeltaC showed cis dimerization and trans interaction, both of which did not require the interaction of nectin-2alpha with afadin. We have previously shown in EL cells that the interaction of nectin-1 with afadin is necessary for its recruitment to adherens junctions. We found that the trans interaction of nectin-2alpha was furthermore necessary for this recruitment. On the basis of these observations, we propose a model for the mechanism of cell-cell adhesion of nectin and roles of afadin in this mechanism.     

A free carboxylate oxygen in the side chain of position 674 in transmembrane domain 7 is necessary for TSH receptor activation.

A specific H-bonding network formed between the central regions of transmembrane domain 6 and transmembrane domain 7 has been proposed to be critical for stabilizing the inactive state of glycoprotein hormone receptors. Many different constitutively activating TSH receptor point mutations have been identified in hyperfunctioning thyroid adenomas in the lower portion of transmembrane domain 6. Position D633 in transmembrane domain 6 of the human TSH receptor is the only one in which four different constitutively activating amino acid exchanges have been identified. Further in vitro substitutions led to constitutive activation of the TSH receptor (D633Y, F, C) as well as to the first inactivating TSH receptor mutation in transmembrane domain 6 without changes of membrane expression or TSH binding (D633R). Molecular modeling of this inactivating TSH receptor mutation revealed potential interaction partners of R633 in transmembrane domain 3 and/or transmembrane domain 7, presumably via hydrogen bonds that could be responsible for locking the TSH receptor in a completely inactive state. To further elucidate the H-bond network that most likely maintains the inactive state of the TSH receptor, we investigated these potential interactions by generating TSH receptor double mutants designed to break up possible H bonds. We excluded S508 in transmembrane domain 3 as a possible interaction partner of R633. In contrast, a partial response to TSH stimulation was rescued in a receptor construct with the double-substitution D633R/N674D. Our results therefore confirm the H bond between position 633 in transmembrane domain 6 and 674 in transmembrane domain 7 suggested by molecular modeling of the inactivating mutation D633R. Moreover, the mutagenesis results, together with a three-dimensional structure model, indicate that for TSH receptor activation and G protein-coupled signaling, at least one free available carboxylate oxygen is required as a hydrogen acceptor atom at position 674 in transmembrane domain 7.     

Glycosylation of a membrane protein is restricted to the growing polypeptide chain but is not necessary for insertion as a transmembrane protein.

The membrane glycoprotein of vesicular stomatitis virus (VSV), synthesized in vitro in the presence of pancreatic microsomes, is glycosylated in two distinct steps while its polypeptide chain is nascent (Rothman and Lodish, 1977). We show here that unglycosylated glycoprotein, which accumulates in vivo following treatment of cells with tunicamycin and in vitro as a result of translation in the presence of detergent-treated microsomal membranes, is inserted normally as a transmembrane protein. This means that glycosylation, while normally occurring concurrently with insertion, is not required for insertion. Our experiments also show that the two steps in glycosylation correspond to the sequential transfer of preformed “core” oligosaccharides of typical structure to two Asn residues in the growing chain. The accumulation of unglycosylated glycoprotein in vitro is due to the fact that the completed transmembrane polypeptide cannot be glycosylated. The detergent treatment of microsomes impairs their rate of glycosylation so that chains are frequently completed before they can be glycosylated. This provides a simple explanation for certain types of heterogeneity often found in glycoproteins. We believe that the detergent treatment procedure results in the solubilization of the microsomal membrane followed by reconstitution. This is a prerequisite for the eventual purification of the membrane proteins and lipids involved in insertion and glycosylation of this model membrane protein.     

CD4 molecules are associated with the antigen receptor complex on activated but not resting T cells

To explore the relationship between CD4 and CD3/Ti on the T cell surface, we have studied a panel of Ag-specific Th cell lines and clones, as well as resting and mitogen-activated CD4+ cells. Our results show that exposure of Th cells to their specific antigenic stimuli, but not to irrelevant stimuli, induced the rapid disappearance of approximately 20 to 35% of CD3 and CD4 molecules. The modulation of these molecules was detected in less than 1 h, became maximal at 12 h, and recovered thereafter in parallel. Treatment of Th cells with anti-CD4 antibody prevented Ag-induced modulation of CD3, and treatment with anti-CD3 blocked modulation of CD4. In the absence of Ag, treatment of these cells with an antibody (WT-31) directed at a conformational determinant within CD3/Ti or with the combination of anti-CD3 antibody and goat anti-mouse Ig, also resulted in significant modulation of CD4. Similar treatment of PHA-activated CD4+ T cells with anti-CD3/Ti antibodies also induced CD4 modulation; however, the same antibodies failed to affect CD4 expression on fresh resting T cells. These results indicate that on activated, but not resting T cells, CD4 molecules can be physically associated with CD3/Ti. We postulate that this association is essential for efficient Th cell activation, and further that the ability of anti-CD4 antibodies to inhibit helper functions is due to their prevention of CD4-CD3/Ti interaction on the T cell surface.     

T-cell antigen receptor (TCR)-alpha/beta heterodimer formation is a prerequisite for association of CD3-zeta 2 into functionally competent TCR.CD3 complexes

In order to study the relationship between assembly, surface expression, and signal transduction of the alpha/beta T-cell antigen receptor-CD3 complex (TCR.CD3), a series of T-cell mutants with a partial block in assembly of the complex was generated. By chemical mutagenesis, we produced somatic cell variants of the human T-leukemia cell line, HPB-ALL, which expressed low amounts of TCR.CD3 complexes on their surface. RNA and protein analyses demonstrated that most variants synthesized normal amounts of the individual members of the complex, i.e. TCR-alpha, TCR-beta, CD3-gamma, -delta, -epsilon, and -zeta. In these variants, less than 10% of the TCR.CD3 complexes inside the cell contained the CD3-zeta 2 homodimer due to an intrinsic deficiency in the formation of the TCR-alpha/beta heterodimer. The low level of assembly of CD3-zeta 2 into the TCR.CD3 complex and an additional decrease in the rate of export of the TCR.CD3 complex from the endoplasmic reticulum explained the low level of expression of alpha/beta receptors on the surface of these mutants. Only cells with the complete set of subunits of the TCR.CD3 complex on their surface were capable of transducing CD3-mediated signals. The results presented in this paper indicate that TCR-alpha/beta heterodimer formation is an obligatory requirement for assemblage of CD3-zeta 2 into a functionally competent TCR.CD3 complex.     

Developmental regulation of transmembrane signaling via the T cell antigen receptor/CD3 complex in human T lymphocytes.

We have examined transmembrane signaling events via the TCR/CD3 complex (TCR/CD3) at various stages of T cell development for evidence of developmental regulation. Engagement of TCR/CD3 induced defective activation of phospholipase C (PLC) in thymocytes relative to peripheral blood T lymphocytes. The defect in PLC activation via TCR/CD3 was restricted to immature thymocytes (CD3low, CD4+CD8+). Mature thymocytes (CD3high, CD4+CD8-/CD8+CD4-) were similar to PBL in signaling via TCR/CD3. Both immature and mature thymocytes expressed a similar profile of PLC isoenzyme mRNA species, indicating that the defect in signaling in immature thymocytes was not due to altered expression of PLC isoenzymes. Activation of tyrosine phosphorylation pathways implicated in the coupling of TCR/CD3 to PLC was impaired in immature thymocytes, as evidenced by depressed phosphorylation of CD3 zeta subunit after stimulation with anti TCR/CD3 mAb. This was associated with lower levels of p59fyn tyrosine kinase and minimal or undetectable stimulus-induced kinase activation in immature thymocytes relative to mature thymocytes. We conclude that the capacity to signal via TCR/CD3 is regulated during T cell development by mechanisms acting at the level of TCR/CD3-associated tyrosine phosphorylation pathways.     

The three-dimensional structure of a T-cell antigen receptor V alpha V beta heterodimer reveals a novel arrangement of the V beta domain.

The crystal structure of a mouse T-cell antigen receptor (TCR) Fv fragment complexed to the Fab fragment of a specific anti-clonotypic antibody has been determined to 2.6 A resolution. The polypeptide backbone of the TCR V alpha domain is very similar to those of other crystallographically determined V alphas, whereas the V beta structure is so far unique among TCR V beta domains in that it displays a switch of the c" strand from the inner to the outer beta-sheet. The beta chain variable region of this TCR antigen-binding site is characterized by a rather elongated third complementarity-determining region (CDR3beta) that packs tightly against the CDR3 loop of the alpha chain, without leaving any intervening hydrophobic pocket. Thus, the conformation of the CDR loops with the highest potential diversity distinguishes the structure of this TCR antigen-binding site from those for which crystallographic data are available. On the basis of all these results, we infer that a significant conformational change of the CDR3beta loop found in our TCR is required for binding to its cognate peptide-MHC ligand.     

The amino-terminal domain of bovine viral diarrhea virus Npro protein is necessary for alpha/beta interferon antagonism

The alpha/beta interferon (IFN-alpha/beta) system is the first line of defense against viral infection and a critical link between the innate and adaptive immune responses. IFN-alpha/beta secretion is the hallmark of cellular responses to acute RNA virus infections. As part of their survival strategy, many viruses have evolved mechanisms to counteract the host IFN-alpha/beta response. Bovine viral diarrhea virus (BVDV) (genus Pestivirus) was reported to trigger interferon production in infected cultured cells under certain circumstances or to suppress it under others. Our studies with various cultured fibroblasts and epithelial bovine cells indicated that cytopathic (cp) BVDV induces IFN-alpha/beta very inefficiently. Using a set of engineered cp BVDVs expressing mutant Npro and appropriate controls, we found that the IFN-alpha/beta response to infection was dependent on Npro expression and independent of viral replication efficiency. In order to investigate whether the protease activity of Npro is required for IFN-alpha/beta antagonism, we engineered Npro mutants lacking protease activity by replacement of amino acid E22, H49, or C69. We found that E22 and H49 substitutions abolished the ability of Npro to suppress IFN, whereas C69 had no effect, suggesting that the structural integrity of the N terminus of Npro was more important than its catalytic activity for IFN-alpha/beta suppression. A catalytically active mutant with a change at a conserved Npro region near the N terminus (L8P) in both BVDV biotypes did not antagonize IFN-alpha/beta production, confirming its involvement in this process. Taken together, these results not only provide direct evidence for the role of Npro in blocking IFN-alpha/beta induction, but also implicate the amino-terminal domain of the protein in this function.     

The ligand binding domain of the epidermal growth factor receptor is not required for receptor dimerization.

To examine the role of the ligand binding domain of epidermal growth factor receptor in its dimerization, we studied the dimerization of a truncated form of the receptor that resembles v-erbB in that it lacks a ligand binding domain. Receptor dimerization was determined by sedimentation analysis on sucrose density gradients at different concentrations of Triton X-100. At high concentrations of Triton X-100 (0.2%), the truncated receptor occurred as a monomer and displayed low basal autophosphorylation. By contrast, at low concentrations of Triton X-100 (0.01%), it existed as a dimer and exhibited high basal autophosphorylation. The ability of the truncated receptor to dimerize indicates that the ligand binding domain of the epidermal growth factor receptor is not required for receptor dimerization.