Degradation of T-cell receptor chains in the endoplasmic reticulum is inhibited by inhibitors of cysteine proteases.

The endoplasmic reticulum, or an organelle closely associated with it, contains proteases that can be used to remove partially assembled or improperly folded proteins. Very little is known at present about the types of protease that degrade these proteins. The beta chain and cluster of differentiation (CD)3 delta subunit of the human T-cell antigen receptor (TCR) are degraded shortly after synthesis. In this study Chinese hamster ovary (CHO) cells transfected with either beta or delta were incubated with a panel of protease inhibitors, and the rates of degradation of the transfected proteins were followed using chain-specific enzyme-linked immunosorbent assays (ELISAs). Of the protease inhibitors tested, degradation of both chains was highly sensitive to sulfhydryl reagents and peptidyl inhibitors of cysteine proteases. Concentrations of inhibitors that produced near complete inhibition of degradation in the endoplasmic reticulum did not cause gross changes in cellular ATP levels nor did they significantly slow constitutive secretion from CHO cells. The inhibitors did not affect the ability of CHO cells to synthesize and assemble disulphide-linked TCR zeta dimers. We conclude that the protease inhibitors were not toxic to cells and did not affect the biosynthetic activity of the endoplasmic reticulum. Furthermore, they did not alter the ability of the endoplasmic reticulum to deliver its content to the Golgi apparatus. Taken together, these results suggest that the cysteine protease inhibitors slow degradation in the endoplasmic reticulum through an action on cysteine proteases. The results imply that the endoplasmic reticulum contains cysteine proteases that can be used to remove retained proteins.     

Molecular determinants of glycine receptor subunit assembly.

The inhibitory glycine receptor (GlyR) is a pentameric transmembrane protein composed of homologous alpha and beta subunits. Single expression of alpha subunits generates functional homo-oligomeric GlyRs, whereas the beta subunit requires a co-expressed alpha subunit to assemble into hetero-oligomeric channels of invariant stoichiometry (alpha(3)beta(2)). Here, we identified eight amino acid residues within the N-terminal region of the alpha1 subunit that are required for the formation of homo-oligomeric GlyR channels. We show that oligomerization and N-glycosylation of the alpha1 subunit are required for transit from the endoplasmic reticulum to the Golgi apparatus and later compartments, and that addition of simple carbohydrate side chains occurs prior to GlyR subunit assembly. Our data are consistent with both intersubunit surface and conformational differences determining the different assembly behaviour of GlyR alpha and beta subunits.     

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.     

Selective degradation of T cell antigen receptor chains retained in a pre-Golgi compartment

We have examined the fate of newly synthesized T cell antigen receptor (TCR) subunits in a T cell hybridoma deficient in expression of the clonotypic beta chain. Synthesis and assembly of the remaining chains proceed normally but surface expression of TCR chains is undetectable in these cells. A variety of biochemical and morphological techniques has been used to show that the TCR chains in these cells fail to be transported to any of the Golgi cisternae. Instead, they are retained in a pre-Golgi compartment which is either part of or closely related to the endoplasmic reticulum. The CD3-delta chain is degraded by a non- lysosomal process that is inhibited at temperatures at or below 27 degrees C. By contrast, the remaining chains (CD3-epsilon, CD3-gamma, and zeta) are very stable over 7 h. We propose possible mechanisms that may explain the differential fate of TCR chains retained in a pre-Golgi compartment.     

Assembly of the human T cell receptor-CD3 complex takes place in the endoplasmic reticulum and involves intermediary complexes between the CD3-gamma.delta.epsilon core and single T cell receptor alpha or beta chains

Functionally mature human T lymphocytes express a cell-surface receptor for antigen (T cell receptor (TCR)-CD3) composed of at least six polypeptides (TCR-alpha and -beta; T3-gamma, -delta, -epsilon, and -zeta). Immature thymocytes and variants of T cell lines lacking one of the TCR.CD3 polypeptide chains fail to express surface receptor and accumulate the other chains intracellularly. Here we show that the assembly of the TCR.CD3 complex within the endoplasmic reticulum (ER) began with a core of CD3-gamma, -delta, and -epsilon to which TCR-alpha and -beta bound. A recently described intracellular protein, CD3-omega, participated in the assembly since it was found to be associated with the free TCR-alpha or -beta chains or with the CD3 chains. CD3-omega dissociated as TCR.CD3 complexes were formed in the ER. Association of non-disulfide-linked TCR-alpha and -beta chains with CD3 was detected before that of disulfide-bridged TCR-alpha/beta heterodimers. These data suggest that during assembly, the association of TCR-alpha and -beta chains with the CD3 complex precedes the formation of a TCR-alpha/beta dimer. The existence of intermediates consisting of CD3-gamma, -delta, and -epsilon chains and a single TCR-alpha or -beta chain was also confirmed by using a series of variant T cell lines lacking the TCR-beta or -alpha chain, respectively. Once the single TCR-alpha and -beta chains were associated with CD3, disulfide linkages were formed, and a 70-kDa form of the TCR was detected within the ER. This intracellular precursor of the TCR.CD3 complex was subsequently processed into the mature 90-kDa TCR as the TCR.CD3 complex passed through the Golgi apparatus. Assembly of the TCR.CD3 complex is a rather rapid process, whereas export from the ER occurs at a slow rate. After 1 h, 75% of the receptor complex remained within the ER.     

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.     

Degradation from the endoplasmic reticulum: disposing of newly synthesized proteins

We have characterized a pre-Golgi, proteolytic pathway for rapid degradation of newly synthesized T cell receptor (TCR) subunits which is insensitive to drugs that block lysosomal proteolysis. The site of degradation in this pathway is either part of or closely related to the endoplasmic reticulum (ER). This "ER" degradative pathway very likely plays an important role in many cells in the removal of unassembled or incompletely assembled membrane protein complexes from the secretory pathway. It is the sole pathway followed by TCR alpha chains and alpha-beta complexes in transfected fibroblasts. In T cells treated with ionophores, which disrupt transport of the TCR from the ER to the Golgi, all newly synthesized alpha, beta, and delta chains are destroyed by this pathway. A variety of biochemical and morphological techniques have been used to distinguish the "ER" degradative pathway from an alternative, lysosomal pathway.     

The gamma and epsilon subunits of the CD3 complex inhibit pre-Golgi degradation of newly synthesized T cell antigen receptors

The T cell receptor for antigen (TCR) is composed of six different transmembrane proteins. T cells carefully control the intracellular transport of the receptor and allow only complete receptors to reach the plasma membrane. In an attempt to understand how T cells regulate this process, we used c-DNA transfection and subunit-specific antibodies to follow the intracellular transport of five subunits (alpha beta gamma delta epsilon) of the receptor. In particular, we assessed the intracellular stability of each chain. Our results showed that the chains were markedly different in their susceptibility to intracellular degradation. TCR alpha and beta and CD3 delta were degraded rapidly, whereas CD3 gamma and epsilon were stable. An analysis of the N-linked oligosaccharides of the glycoprotein subunits suggested that the chains were unable to reach the medial Golgi during the metabolic chase. This was supported by immunofluorescence micrographs that showed both the stable CD3 gamma and unstable CD3 delta chain localized in the endoplasmic reticulum. To study the effects of subunit associations on intracellular transport we used cotransfection to reconstitute precise combinations of subunits. Associations between stable and unstable subunits expressed in the same cell led to the formation of stable complexes. These complexes were retained in or close to the endoplasmic reticulum. The results suggested that the intracellular transport of the T cell receptor could be regulated by two mechanisms. The TCR alpha and beta and CD3 delta subunits were degraded rapidly and as a consequence failed to reach the plasma membrane. CD3 gamma or epsilon were stable but were retained inside the cell. The results also demonstrated that there was an interplay between the two pathways such that the CD3 gamma and epsilon subunits were able to protect labile chains from rapid intracellular degradation. In this way, they could seed subunit assembly in or close to the endoplasmic reticulum and allow a stable receptor to form before its transport to the plasma membrane.     

Formation of the alpha-bungarotoxin binding site and assembly of the nicotinic acetylcholine receptor subunits occur in the endoplasmic reticulum

During the process by which newly synthesized subunits of the nicotinic acetylcholine receptor (stoichiometry = alpha 2 beta gamma delta) mature and acquire the properties of the fully functional cell surface receptor, they undergo numerous covalent and noncovalent modifications. Using ligand-mediated and subunit-specific immunoprecipitation, four forms in the maturation of the alpha subunit can be detected: the primary translation product; alpha subunit that can bind alpha-bungarotoxin; alpha subunit assembled with the other subunits; and surface receptor. The alpha subunit acquires the ability to bind alpha-bungarotoxin with a t1/2 of approximately 40 min after translation and becomes assembled with a t1/2 of 80 min after translation. Using metabolic labeling and sucrose gradient fractionation, we have determined the subcellular location of alpha subunit when it acquires the ability to bind alpha-bungarotoxin and when it is assembled. Golgi membranes were identified across the gradient by the enzymatic activities UDP-galactose:N-acetylglucosamine galactosyltransferase and alpha-mannosidase. Endoplasmic reticulum membranes were identified by the enzymatic activity glucose-6-phosphatase and by the presence of newly synthesized alpha and beta subunits. Pulse-labeled alpha subunit that bound alpha-bungarotoxin was first detected co-migrating in the gradient with the glucose-6-phosphatase activity. Therefore, the capacity to bind alpha-bungarotoxin was acquired while the alpha subunit was in the endoplasmic reticulum. Assembled alpha subunit was detected by immunoprecipitating with an anti-beta subunit-specific monoclonal antibody. By this method, assembled receptor was first detected 15 min after translation in both the endoplasmic and Golgi portions of the gradient. To validate this method of detecting assembled receptor, we determined the sedimentation coefficient of the receptor subunits in the endoplasmic reticulum. Both unassembled subunits with sedimentation coefficients of 5 S and assembled receptor with a sedimentation coefficient of 9 S were recovered from the endoplasmic reticulum portion of the gradient. Thus, our data concerning the subcellular site of assembly are consistent with assembly occurring in the endoplasmic reticulum followed by rapid transport to the Golgi.     

Assembly, intracellular processing, and expression at the cell surface of the human alpha beta T cell receptor/CD3 complex. Function of the CD3-zeta chain

The TCR/CD3 complex is a multimeric protein complex composed of a minimum of seven transmembrane chains (TCR alpha beta-CD3 gamma delta epsilon zeta 2). Whereas earlier studies have demonstrated that both the TCR-alpha and -beta chains are required for the cell surface expression of the TCR/CD3 complex, the role of the CD3 chains for the TCR/CD3 expression have not been experimentally addressed in human T cells. In this study the function of the CD3-zeta chain for the assembly, intracellular processing, and expression of the TCR/CD3 complex in the human leukemic T cell line Jurkat was investigated. The results indicate that: 1) CD3-zeta is required for the cell surface expression of the TCR/CD3 complex; 2) the pentameric form (TCR alpha beta-CD3 gamma delta epsilon) of the TCR/CD3 complex and single TCR chains associated with CD3 (TCR alpha-CD3 gamma delta epsilon and TCR beta-CD3 gamma delta epsilon) are produced in the endoplasmic reticulum in the absence of CD3-zeta; 3) the CD3-zeta does not associate with TCR alpha-CD3 gamma delta epsilon or TCR beta-CD3 gamma delta epsilon complexes; 4) CD3-zeta associate with the pentameric form of the TCR/CD3 complex in the endoplasmic reticulum to form the heptameric complex (TCR alpha beta-CD3 gamma delta epsilon----TCR alpha beta-CD3 gamma delta epsilon 2); and 5) CD3-zeta is required for the export of the TCR/CD3 complex from the endoplasmic reticulum to the Golgi apparatus for subsequent processing.     

Olfactory receptor trafficking involves conserved regulatory steps

Olfactory receptors are difficult to functionally express in heterologous cells. They are typically retained in the endoplasmic reticulum of cells commonly used for functional expression studies and are only released to the plasma membrane in mature cells of the olfactory receptor neuron lineage. A recently developed olfactory cell line, odora, traffics olfactory receptors to the plasma membrane when differentiated. We found that undifferentiated odora cells do not traffic olfactory receptors to their surface, even though they release the receptors to the Golgi apparatus and endosomes. This behavior differs from other cell lines tested thus far. Differentiated odora cells also properly traffic vomeronasal receptors of the VN1 type, which lack sequence similarity to olfactory receptors. ODR-4, a protein that is necessary for plasma membrane trafficking of a chemosensory receptor in nematodes, facilitates trafficking of rat olfactory receptor U131 in odora and Chinese hamster ovary cells. Olfactory receptor trafficking from the endoplasmic reticulum to the plasma membrane involves at least two steps whose regulation depends on the maturation state of cells in the olfactory receptor neuron lineage. These results also indicate that some components of the regulatory mechanism are conserved.     

T cell receptor assembly and expression in the absence of calnexin

Most subunits of the alphabeta deltaepsilon gammaepsilon zetazeta T cell antigen receptor (TCR) complex associate with the molecular chaperone calnexin shortly after their synthesis in the endoplasmic reticulum, including clonotypic TCRalpha,beta molecules and invariant CD3gamma,delta,epsilon chains. While calnexin interaction is suggested to be important for the stability of newly synthesized TCRalpha subunits, the role of calnexin in the survival and assembly of remaining TCR components is unknown. Here we evaluated the expression of TCR proteins in CEM T cells and the calnexin-deficient CEM variant CEM.NK(R). We found that CEM and CEM.NK(R) cells constitutively synthesized all TCR subunits except for TCRalpha and that CD3gamma,delta,epsilon components and CD3-beta complexes were effectively assembled together in both cell types. The stability and folding of core CD3epsilon chains were similar in CEM and CEM.NK(R) cells. Interestingly, TCRalpha synthesis was differentially induced by phorbol myristate acetate treatment in CEM and CEM.NK(R) cells and TCRalpha proteins synthesized in CEM.NK(R) cells showed reduced survival compared to those made in CEM cells. Importantly, these data show that TCR complexes were inducibly expressed on CEM.NK(R) cells in the absence of calnexin synthesis. These results demonstrate that TCR complexes can be expressed in the absence of calnexin and suggest that the role of calnexin in the quality control of TCR assembly is primarily restricted to the stabilization of newly synthesized TCRalpha proteins.     

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.     

Ultrastructural immunocytochemical localization of the phosphomannosyl receptor in Chinese hamster ovary (CHO) cells

Using ultrastructural immunocytochemistry and antibodies directed against bovine liver phosphomannosyl (PM) receptor, we have localized the receptor in Chinese hamster ovary (CHO) cells. The majority of the receptor was found within the cell. Only a small fraction of the receptor was found on the surface and most of it was clustered in coated pits. Because these cells contain endogenous ligands for the receptor, it was not possible to determine if this clustered state was dependent on occupancy of the receptor. The bulk of the cell's receptor was found in the endoplasmic reticulum, nuclear envelope, and in the Golgi system. Most of the Golgi localization was associated with peripheral Golgi elements, suggesting a possible concentration of receptor in GERL. Very little receptor was found associated with mature lysosomes. PM receptor was also localized in structures that were identified as receptosomes by the presence of alpha 2-macroglobulin (alpha 2M)-gold, a ligand previously shown to enter CHO cells by the coated pit-receptosome pathway. This finding is consistent with the notion that during receptor-mediated endocytosis, receptors accompany ligand from the coated pit into the receptosome. The observation that the majority of the receptor was found in the endoplasmic reticulum and structures similar to GERL raises the possibility that the PM receptor plays an important role in compartmentalization of lysosomal enzymes in the GERL system.     

Posttranslational association of immunoglobulin heavy chain binding protein with nascent heavy chains in nonsecreting and secreting hybridomas

A rat monoclonal antibody specific for immunoglobulin (Ig) heavy chain binding protein (BiP) has allowed the examination of the association of BiP with assembling Ig precursors in mouse B lymphocyte-derived cell lines. The anti-BiP monoclonal antibody immunoprecipitates BiP along with noncovalently associated Ig heavy chains. BiP is a component of the endoplasmic reticulum and binds free intracellular heavy chains in nonsecreting pre-B (mu+, L-) cell lines or incompletely assembled Ig precursors in (H+, L+) secreting hybridomas and myelomas. In the absence of light chain synthesis, heavy chains remain associated with BiP and are not secreted. The association of BiP with assembling Ig molecules in secreting hybridomas is transient and is restricted to the incompletely assembled molecules which are found in the endoplasmic reticulum. BiP loses affinity and disassociates with Ig molecules when polymerization with light chain is complete. We propose that the association of BiP with Ig heavy chain precursors is a novel posttranslational processing event occurring in the endoplasmic reticulum. The Ig heavy chains associated with BiP are not efficiently transported from the endoplasmic reticulum to the Golgi apparatus. Therefore, BiP may prevent the premature escape and eventual secretion of incompletely assembled Ig molecules.     

Studies on the biosynthesis of cartilage proteoglycan in a model system of cultured chondrocytes from the Swarm rat chondrosarcoma

Biosynthesis of cartilage proteoglycan was examined in a model system of cultured chondrocytes from a transplantable rat chondrosarcoma. Extensive modification with the addition of chondroitin sulfate glycosaminoglycan, N-linkcd oligosac-charide, and O-linked oliogosaccharide is required to convert a newly synthesized core protein precursor into a proteoglycan. Kinetic analyses revealed the presence of a large pool of core protein precursor (t1/2 ～ 90 min) awaiting completion into proteoglycan. The large t1/2 of this pool allowed kinetic labeling experiments with a variety of radioactive precursors to distinguish between early biosynthetic events associated primarily with the rough endoplasmic reticulum from late events associated primarily with the Golgi apparatus. The results of a series of experiments indicated that the addition of N-linked oligosaccharide chains occurs early in the biosynthetic process in association with the rough endoplasmic reticulum, whereas the initiation and completion of O-linked oligosaccharides occurs much later, at about the same time as chondroitin sulfate synthesis. This also indicated that keratan sulfate chains, when present in the completed molecule, are added in the Golgi apparatus, as they are probably built on oligosaccharide primers closely related to the O-oligosaccharide chains. Furthermore, when ³H-glucose was used as the precursor, the entry of label into xylose, the linkage sugar between the core protein and the chondroitin sulfate chain, was found to occur within 5 min of the entry of label into galactose and galactosamine in the remainder of the chondroitin sulfate chain. This indicated that the initiation and completion of the chondroitin sulfate chain occurs late in the pathway probably entirely in the Golgi apparatus. Thus, proteoglycan synthesis can be described as occurring in two stages in this system, translation and N-glycosylation of a core protein precursor which has a long half-life in the rough endoplasmic reticulum, followed by extensive rapid modification in the Golgi complex in which the majority of glycosaminoglycan and oligosaccharide chains are added to the core protein precursor with subsequent rapid secretion into the extracellular matrix.     

Immunoglobulin heavy chain-binding protein binds to misfolded mutant insulin receptors with mutations in the extracellular domain.

Cell-surface proteins are transported through the endoplasmic reticulum and Golgi apparatus en route to the plasma membrane. Previously, we have identified three point mutations in the insulin receptor gene that impair transport of the mutant receptors to the cell surface: Asn15----Lys, His209----Arg, and Phe382----Val. Furthermore, these mutations impair post-translational processing steps that normally occur as the receptors are transported through the endoplasmic reticulum and Golgi apparatus. In this study, we have demonstrated that the unprocessed Arg209 and Val382 mutant proreceptors are bound to the immunoglobulin heavy chain-binding protein (BiP) in the endoplasmic reticulum. This was demonstrated by the fact that monoclonal anti-BiP antibody coimmunoprecipitated the mutant proreceptors. Moreover, when ATP was added to the immunoprecipitates, the mutant proreceptors were released from BiP. In contrast, neither the normal human insulin receptor nor the Lys15 mutant proreceptor was coimmunoprecipitated by anti-BiP antibody. It seems likely that the Lys15 receptor also binds BiP, but that the affinity was too low to resist dissociation during the stringent washing of the immunoprecipitate. In conclusion, these observation are consistent with the hypothesis that binding to BiP explains the impaired transport of mutant receptors through the endoplasmic reticulum and Golgi apparatus to the plasma membrane.     

Developmental regulation of alpha beta T cell antigen receptor expression results from differential stability of nascent TCR alpha proteins within the endoplasmic reticulum of immature and mature T cells.

The alpha beta T cell antigen receptor (TCR) that is expressed on most T lymphocytes is a multisubunit transmembrane complex composed of at least six different proteins (alpha, beta, gamma, delta, epsilon and zeta) that are assembled in the endoplasmic reticulum (ER) and then transported to the plasma membrane. Expression of the TCR complex is quantitatively regulated during T cell development, with immature CD4+CD8+ thymocytes expressing only 10% of the number of surface alpha beta TCR complexes that are expressed on mature T cells. However, the molecular basis for low TCR expression in developing alpha beta T cells is unknown. In the present study we report the unexpected finding that assembly of nascent component chains into complete TCR alpha beta complexes is severely impaired in immature CD4+CD8+ thymocytes relative to their mature T cell progeny. In particular, the initial association of TCR alpha with TCR beta proteins, which occurs relatively efficiently in mature T cells, is markedly inefficient in immature CD4+CD8+ thymocytes, even for a matched pair of transgenic TCR alpha and TCR beta proteins. Inefficient formation of TCR alpha beta heterodimers in immature CD4+CD8+ thymocytes was found to result from the unique instability of nascent TCR alpha proteins within the ER of immature CD4+CD8+ thymocytes, with nascent TCR alpha proteins having a median survival time of only 15 min in CD4+CD8+ thymocytes, but > 75 min in mature T cells. Thus, these data demonstrate that stability of TCR alpha proteins within the ER is developmentally regulated and provide a molecular basis for quantitative differences in alpha beta TCR expression on immature and mature T cells. In addition, these results provide the first example of a receptor complex whose expression is quantitatively regulated during development by post-translational limitations on receptor assembly.     

Masking of an endoplasmic reticulum retention signal by its presence in the two subunits of the asialoglycoprotein receptor

Human asialoglycoprotein receptor H1 and H2b subunits assemble into a hetero-oligomer that travels to the cell surface. The H2a variant on the other hand is a precursor of a cleaved soluble form that is secreted. Uncleaved H2a precursor molecules cannot exit the endoplasmic reticulum (ER), a lumenal juxtamembrane pentapeptide being responsible for their retention. Insertion of this pentapeptide into H1 (H1i5) causes its complete ER retention but not fast degradation as happens to H2a. Cotransfection of H2a elicited, by heterodimerization, the Golgi processing of H1i5 and its surface expression. This occurred to a much lesser extent by cotransfection of H2b. Likewise, coexpression of H1i5 and not H1 stabilized H2a and caused its export to the cell surface. Homodimerization of molecules containing the pentapeptide did not cancel the retention. Thus, only when the pentapeptide is present in both subunits is the ER retention efficiently abrogated. The results show the unexpected finding that identical ER retention signals present in two associated chains can mask and cancel each other's effect. This could have important implications as similar abrogation of ER retention of other proteins could eventually be obtained by engineering and coexpressing an associated protein containing the same retention signal.