Enhanced clathrin-dependent endocytosis in the absence of calnexin

Background

Calnexin, together with calreticulin, constitute the calnexin/calreticulin cycle. Calnexin is a type I endoplasmic reticulum integral membrane protein and molecular chaperone responsible for the folding and quality control of newly-synthesized (glyco)proteins. The endoplasmic reticulum luminal domain of calnexin is responsible for lectin-like activity and interaction with nascent polypeptide chains. The role of the C-terminal, cytoplasmic portion of calnexin is not clear.

Methodology/Principal Findings

Using yeast two hybrid screen and immunoprecipitation techniques, we showed that the Src homology 3-domain growth factor receptor-bound 2-like (Endophilin) interacting protein 1 (SGIP1), a neuronal specific regulator of endocytosis, forms complexes with the C-terminal cytoplasmic domain of calnexin. The calnexin cytoplasmic C-tail interacts with SGIP1 C-terminal domains containing the adaptor complexes medium subunit (Adap-Comp-Sub) region. Calnexin-deficient cells have enhanced clathrin-dependent endocytosis in neuronal cells and mouse neuronal system. This is reversed by expression of full length calnexin or calnexin C-tail.

Conclusions/Significance

We show that the effects of SGIP1 and calnexin C-tail on clathrin-dependent endocytosis are due to modulation of the internalization of the receptor-ligand complexes. Enhanced clathrin-dependent endocytosis in the absence of calnexin may contribute to the neurological phenotype of calnexin-deficient mice.     

Quaternary structure and assembly process of the T cell receptor.

The human immune system contains T and B lymphocytes which respond in an antigen-specific manner to foreign antigens. These foreign antigens are recognized by multimeric receptors expressed on the cell surface of T and B lymphocytes. The subunits that make up the T and B cell receptor complexes have been identified, but their stoichiometries and positions in the complex remain to be resolved. Elucidation of the quaternary structures is necessary to understand the molecular basis of signal transduction events which follow antigen recognition and will contribute to the design of drugs that can modulate T and B cell responses. Here, I will discuss recent insights into the quaternary structures of the TCR and BCR and the striking similarities between the two, both in the structures of the subunits and in the overall quaternary structures. In addition, the intracellular assembly processes of these receptor complexes will be discussed, as well as the recent realization that these processes appear to be mediated by house-keeping proteins that transiently bind to partial TCR and BCR complexes. Similar to the role of BiP which mediates assembly processes of B cell immunoglobulins, a recently identified intracellular protein of 90 kD, called IP90, appears to play a role in TCR and BCR assembly processes. Analyses of the IP90 protein might contribute not only insight into the folding and assembly processes in lymphocytes, but also into those of newly synthesized proteins in many different cell types.     

Role of CD3 gamma in T cell receptor assembly

The T cell receptor (TCR) consists of the Ti alpha beta heterodimer and the associated CD3 gamma delta epsilon and zeta 2 chains. The structural relationships between the subunits of the TCR complex are still not fully known. In this study we examined the role of the extracellular (EC), transmembrane (TM), and cytoplasmic (CY) domain of CD3 gamma in assembly and cell surface expression of the complete TCR in human T cells. A computer model indicated that the EC domain of CD3 gamma folds as an Ig domain. Based on this model and on alignment studies, two potential interaction sites were predicted in the EC domain of CD3 gamma. Site-directed mutagenesis demonstrated that these sites play a crucial role in TCR assembly probably by binding to CD3 epsilon. Mutagenesis of N-linked glycosylation sites showed that glycosylation of CD3 gamma is not required for TCR assembly and expression. In contrast, treatment of T cells with tunicamycin suggested that N-linked glycosylation of CD3 delta is required for TCR assembly. Site-directed mutagenesis of the acidic amino acid in the TM domain of CD3 gamma demonstrated that this residue is involved in TCR assembly probably by binding to Ti beta. Deletion of the entire CY domain of CD3 gamma did not prevent assembly and expression of the TCR. In conclusion, this study demonstrated that specific TCR interaction sites exist in both the EC and TM domain of CD3 gamma. Furthermore, the study indicated that, in contrast to CD3 gamma, glycosylation of CD3 delta is required for TCR assembly and expression.     

鸡T细胞受体γ链基因位点重新测序和组装

动物T细胞受体(T cell receptor,TCR)基因由多个不同的高度同源的基因家族组成,通过全基因组测序很难获得准确的基因序列和排列位置。文章通过在NCBI中发布的鸡TCR的γ链(TCRγ或TRG)基因片段序列定位了鸡TRG基因所在区域,并确定了与鸡TRG基因位点对应的细菌人工染色体(BAC)克隆(CH261-174P24)。对该克隆进行高通量的重新测序和组装后,得到含有10个scaffolds的基因组草图,较完整地覆盖了鸡TRG基因位点及两侧区域。通过PCR扩增和测序证明了scaffold内部结构的正确性,校正了鸡参考基因组TRG基因位点一个可变基因和一个缺口序列(gap)附近各一处错误序列,以及可变基因区多处序列错误。文章通过校正鸡参考基因组TRG基因位点的序列,为鸡TRA/D和TRB基因位点的基因组序列分析提供了新方法。     

T cell chemokine receptor expression in aging

Changes in chemokine receptor expression are important in determining T cell migration and the subsequent immune response. To better understand the contribution of the chemokine system in immune senescence we determined the effect of aging on CD4(+) T cell chemokine receptor function using microarray, RNase protection assays, Western blot, and in vitro chemokine transmigration assays. Freshly isolated CD4(+) cells from aged (20-22 mo) mice were found to express a higher level of CCR1, 2, 4, 5, 6, and 8 and CXCR2-5, and a lower level of CCR7 and 9 than those from young (3-4 mo) animals. Caloric restriction partially or completely restored the aging effects on CCR1, 7, and 8 and CXCR2, 4, and 5. The aging-associated differences in chemokine receptor expression cannot be adequately explained by the age-associated shift in the naive/memory or Th1/Th2 profile. CD4(+) cells from aged animals have increased chemotactic response to stromal cell-derived factor-1 and macrophage-inflammatory protein-1alpha, suggesting that the observed chemokine receptor changes have important functional consequences. We propose that the aging-associated changes in T cell chemokine receptor expression may contribute to the different clinical outcome in T cell chemokine receptor-dependent diseases in the elderly.     

N-linked glycosylation is required for nicotinic receptor assembly but not for subunit associations with calnexin

We investigated how asparagine (N)-linked glycosylation affects assembly of acetylcholine receptors (AChRs) in the endoplasmic reticulum (ER). Block of N-linked glycosylation inhibited AChR assembly whereas block of glucose trimming partially blocked assembly at the late stages. Removal of each of seven glycans had a distinct effect on AChR assembly, ranging from no effect to total loss of assembly. Because the chaperone calnexin (CN) associates with N-linked glycans, we examined CN interactions with AChR subunits. CN rapidly associates with 50% or more of newly synthesized AChR subunits, but not with subunits after maturation. Block of N-linked glycosylation or trimming did not alter CN-AChR subunit associations nor did subunit mutations prevent N-linked glycosylation. Additionally, CN associations with subunits lacking N-linked glycans occurred without subunit aggregation or misfolding. Our data indicate that CN associates with AChR subunits without N-linked glycan interactions. Furthermore, CN-subunit associations only occur early in AChR assembly and have no role in events later that require N-linked glycosylation.     

The biosynthesis and assembly of T cell receptor alpha- and beta-chains with the CD3 complex

The biosynthesis, processing, and assembly of the TCR alpha- and beta-chains with each other and with the CD3 complex were investigated on both cell surface positive (TCR+CD3-) and negative (TCR-CD3-) cell lines. The results indicate that 1) in cell surface TCR-CD3- cell lines (MOLT 3, CCRF-CEM), TCR-beta, but not alpha-chains are present intracellularly. TCR-beta-CD3 complexes are readily found in these cell lines, but no evidence for final processing or cell surface expression of such incomplete TCR-CD3 complexes is observed. 2) In the cell surface TCR+CD3+ cell line HPB-ALL, both alpha- and beta-chains are present intracellularly. Whereas non-glycosylated forms of TCR-beta chain can be detected, only more mature forms of TCR alpha-chains are detected indicating that the alpha-chains are more rapidly glycosylated than the beta-chains. 3) The large majority of the intracellular alpha- and beta-chains is not disulfide linked and a small fraction of these is associated with CD3. 4) Only small amounts of the total intracellular TCR chains are found as CD3-associated disulfide-linked alpha beta-heterodimers. 5) Final processing of TCR chains for cell surface expression takes place after formation of these TCR-alpha beta-CD3 complexes. Thus, both the TCR alpha- and beta-chains are over-produced and only relatively small amounts of these chains form CD3-associated heterodimers that are processed for cell surface expression. Analogous results were obtained with a non-leukemic CTL clone. Based on these observations, a model for the biosynthesis and assembly of the TCR-CD3 complex is presented.     

T cell differentiation model based on the expression of T cell receptor chains and genes--study in the human leukemia/lymphoma cell lines

A total of 33 human leukemia/lymphoma cell lines were classified into 4 groups with respect to the pattern of cell membrane (sm) expression of the CD3 and T cell receptor (TCR) molecules; (i) smCD3+TCR alpha beta (16 cell lines), (ii) smCD3+TCR beta delta (1 cell line), (iii) smCD3+TCR gamma delta (3 cell lines) amd (iv) smCD3-TCR- (13 cell lines), respectively. Using monoclonal antibodies (MoAbs) specific to CD3 (NU-T3), TCR alpha chain (alpha F1), TCR beta chain (beta F1), and TCR gamma chain (C gamma M1), respectively, cytoplasmic (cy) expression of these molecules was determined by immunofluorescence test. Expression of cyCD3 was present in all cell lines regardless of groups. In group (i), all 16 cell lines expressed both TCR alpha and beta chains. While only TCR beta chain was expressed in group (ii), TCR gamma chain was expressed in all 3 cell lines of group (iii). One (PEER) of the three in group (iii) expressed TCR beta chain as well. In group (iv), we found 8 cell lines with cyTCR alpha expression, 11 cell lines with cyTCR beta expression, and 10 cell lines with cyTCR gamma expression, respectively. For TCR genes, except 1 cell line all cell lines were found to present rearranged C beta gene and its mRNA, including all 3 TCR gamma/delta cell lines of group (iii). One of the TCR alpha beta cell lines exhibited rearranged C delta and J delta genes as well as its mRNA. Two cell lines of the 13 CD3-TCR- of group (iv) exhibited rearranged C delta and J delta and its mRNA. An NK-like activity and IL-2 production were induced in the TCR beta delta and gamma delta cell lines [group (ii) and (iii)] by treatment with PHA and PMA.     

Transport and secretion of truncated T cell receptor beta-chain occurs in the absence of association with CD3

The T cell receptor (TCR) beta-chain is produced in the endoplasmic reticulum where it associates with the TCR alpha-chain and the members of the CD3 complex to form the complete receptor. When the other chains of the complex are not available, the beta-chain is rapidly degraded within the endoplasmic reticulum. When incomplete TCR.CD3 complexes are formed, they are transported through the Golgi apparatus and degraded in lysosomes. In this study, a truncated form of the TCR beta-chain has been made by removal of the transmembrane and cytoplasmic segments. Unlike the normal beta-chain, the truncated molecule is stable and is transported through the Golgi apparatus and secreted. This process occurs at a similar rate in both T and B cells, indicating that it is not affected by the presence or absence of CD3 components. These data suggest that an element in the transmembrane or cytoplasmic region of the beta-chain confers sensitivity to the degradative control mechanisms that regulate TCR expression.     

Clonal anergy is induced in vitro by T cell receptor occupancy in the absence of proliferation.

Murine Th1 clones that receive signals through their TCR in the absence of APC-derived co-stimulatory signals do not produce IL-2 and instead become anergic, i.e., they are subsequently unable to produce IL-2 in response to Ag and normal APC. The critical cellular event required to prevent the induction of this anergic state appears to be T cell proliferation. Anergy was induced when T cell clones were stimulated under conditions where both TCR occupancy and costimulatory signals were provided but where proliferation in response to the IL-2 produced was prevented. Once induced, anergy could be reversed if the T cells were allowed to undergo multiple rounds of cell division. These results show that anergy is induced as a consequence of TCR occupancy in the absence of cell division; this can be achieved either by limiting IL-2 production because of deficient provision of co-stimulatory signals or by preventing response to IL-2.     

Associations between subunit ectodomains promote T cell antigen receptor assembly and protect against degradation in the ER

The T cell antigen receptor (TCR) is an oligomeric protein complex made from at least six different integral membrane proteins (alpha beta gamma delta epsilon and zeta). The TCR is assembled in the ER of T cells, and correct assembly is required for transport to the cell surface. Single subunits and partial receptor complexes are retained in the ER where TCR alpha, beta, and CD3 delta chains are degraded selectively. The information required for the ER degradation of the TCR beta chain is confined to the membrane anchor of the protein (Wileman et al., 1990c; Bonifacino et al., 1990b). In this study we show that the rapid degradation of the TCR beta chain is inhibited when it assembles with single CD3 gamma, delta, or epsilon subunits in the ER, and have started to define the role played by transmembrane anchors, and receptor ectodomains, in the masking proteolytic targeting information. Acidic residues within the membrane spanning domains of CD3 subunits were essential for binding to the TCR beta chain. TCR beta chains and CD3 subunits therefore interact via transmembrane domains. However, when sites of binding were restricted to the membrane anchor of the TCR beta chain, stabilization by CD3 subunits was markedly reduced. Interactions between membrane spanning domains were not, therefore, sufficient for the protection of the beta chain from ER proteolysis. The presence of the C beta domain, containing the first 150 amino acids of the TCR ectodomain, greatly increased the stability of complexes formed in the ER. For assembly with CD3 epsilon, stability was further enhanced by the V beta amino acids. The results showed that the efficient neutralization of transmembrane proteolytic targeting information required associations between membrane spanning domains and the presence of receptor ectodomains. Interactions between receptor ectodomains may slow the dissociation of CD3 subunits from the beta chain and prolong the masking of transmembrane targeting information. In addition, the close proximity of TCR and CD3 ectodomains within the ER may provide steric protection from the action of proteases within the ER lumen.     

Failure to synthesize the CD3-gamma chain. Consequences for T cell antigen receptor assembly, processing, and expression.

The TCR consists of the Ti alpha beta heterodimer and the associated CD3 chains, CD3 gamma delta epsilon zeta 2 or zeta eta. The structural relationships between the subunits of the Ti/CD3 complex are still not fully understood. To explore the roles of the individual CD3 chains for the assembly, intracellular processing, and expression of the TCR, mutants of the T cell line Jurkat were isolated. One variant, JGN, was found to produce all the Ti/CD3 components with the exception of CD3-gamma. The results indicate that: 1) the tetrameric form (Ti alpha beta-CD3 delta epsilon) of the Ti/CD3 complex is produced in the endoplasmic reticulum in the absence of CD3-gamma; 2) CD3-zeta does not associate with the Ti alpha beta-CD3 delta epsilon complex; 3) the Ti alpha beta-CD3 delta epsilon complex is not exported from the endoplasmic reticulum to the Golgi apparatus; and 4) CD3-gamma is required for cell surface expression of the Ti/CD3 complex. Transfection of the wild-type CD3-gamma gene into JGN reconstituted expression of functional Ti/CD3 complexes, and analysis of T cell lines producing different amounts of CD3-gamma indicated that CD3-gamma and CD3-delta competed for the binding to CD3-epsilon.     

V gamma 3 T cell receptor rearrangement and expression in the adult thymus.

Rearrangement and expression of the V gamma 3-J gamma 1 TCR has been found in murine dendritic epidermal cells (DEC) and fetal thymus. By using the polymerase chain reaction technique, V gamma 3-J gamma 1 rearrangements and RNA expression were detected in the murine adult thymus. Individual genomic and cDNA junctions were cloned and sequenced. In genomic DNA, 55% (16/29) of V gamma 3-J gamma 1 junctional sequences had N regions ranging in length from 1 to 12 nucleotides resulting in considerable junctional diversity. Only 5% (2/42) of cDNA sequences had N regions. The canonical DEC sequence represented 36% (15/42) of the cDNA sequences. Thus, fetal-type V gamma 3-J gamma 1 rearrangements lacking N regions were preferentially expressed in adult thymocytes, some of which may be DEC precursors. The developmental stages in which V gamma 3-J gamma 1 rearrangements are generated were studied by using polymerase chain reaction to detect circular rearrangement products. Active V gamma 3-J gamma 1 rearrangement was detected in thymuses from fetal, newborn, and 2-wk-old mice but not in 5-wk or 8-wk-old (adult) mice. V gamma 2, one of the most common V gamma rearrangements in the adult, was found to be actively rearranging to J gamma 1 in the adult thymus. However, V gamma 2-V gamma 3 replacement rearrangement was not found. These results support the hypotheses that adult thymocytes with rearranged V gamma 3-J gamma 1 are persistent from earlier developmental stages and represent a separate lineage from those with V gamma 2-J gamma 1 rearrangements.     

T cell receptor genes and T cell development in virus-transformed early T cell lines

We have derived T cell lines from mice inoculated with Gross leukemia virus, which appear to represent early T cell developmental stages and to reflect normal T cell development. These cell lines may provide a breakthrough in the study of T cell development as Abelson transformants have done for the study of B cell development. Analysis of the TCR gene expression in these cell lines reveals that the sequence of rearrangement and expression of each TCR gene is not strictly ordered. Expression of RNA for the TCR alpha and -beta genes appears to be coordinated with rearrangement at the alpha and beta loci. This is not the case for gamma gene expression. Availability of the homogeneous populations of cells represented in these cells lines allows for a more detailed molecular analysis of T cell development than was previously possible.