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• W1986245836 abstract "Article20 May 2004free access Stoichiometry of the T-cell receptor–CD3 complex and key intermediates assembled in the endoplasmic reticulum Matthew E Call Matthew E Call Department of Cancer Immunology & AIDS, Dana-Farber Cancer Institute, Boston, MA, USA Program in Immunology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Jason Pyrdol Jason Pyrdol Department of Cancer Immunology & AIDS, Dana-Farber Cancer Institute, Boston, MA, USA Search for more papers by this author Kai W Wucherpfennig Corresponding Author Kai W Wucherpfennig Department of Cancer Immunology & AIDS, Dana-Farber Cancer Institute, Boston, MA, USA Program in Immunology, Harvard Medical School, Boston, MA, USA Department of Neurology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Matthew E Call Matthew E Call Department of Cancer Immunology & AIDS, Dana-Farber Cancer Institute, Boston, MA, USA Program in Immunology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Jason Pyrdol Jason Pyrdol Department of Cancer Immunology & AIDS, Dana-Farber Cancer Institute, Boston, MA, USA Search for more papers by this author Kai W Wucherpfennig Corresponding Author Kai W Wucherpfennig Department of Cancer Immunology & AIDS, Dana-Farber Cancer Institute, Boston, MA, USA Program in Immunology, Harvard Medical School, Boston, MA, USA Department of Neurology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Author Information Matthew E Call1,2, Jason Pyrdol1 and Kai W Wucherpfennig 1,2,3 1Department of Cancer Immunology & AIDS, Dana-Farber Cancer Institute, Boston, MA, USA 2Program in Immunology, Harvard Medical School, Boston, MA, USA 3Department of Neurology, Harvard Medical School, Boston, MA, USA *Corresponding author. Department of Cancer Immunology & AIDS, Dana-Farber Cancer Institute, Room Dana-1410, 44 Binney Street, Boston, MA 02115, USA. Tel.: +1 617 632 3086; Fax: +1 617 632 2662; E-mail: [email protected] The EMBO Journal (2004)23:2348-2357https://doi.org/10.1038/sj.emboj.7600245 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The T-cell receptor (TCR)–CD3 complex is critical for T-cell development and function, and represents one of the most complex transmembrane receptors. Models of different stoichiometry and valency have been proposed based on cellular experiments and these have important implications for the mechanisms of receptor triggering. Since determination of receptor stoichiometry in T-cells is not possible due to the presence of previously synthesized, unlabeled receptor components with different half-lives, we examined the stoichiometry of the receptor assembled in endoplasmic reticulum (ER) microsomes of B-cell origin. The stoichiometric relationship among all subunits was directly determined using intact radiolabeled TCR–CD3 complexes that were isolated with a sequential, non-denaturing immunoprecipitation method, and identical results were obtained with two detergents belonging to different structural classes. The results firmly establish that the αβ TCR–CD3 complex assembled in the ER is monovalent and composed of one copy of the TCRαβ, CD3δε, CD3γε and ζ−ζ dimers. Introduction The T-cell receptor (TCR)–CD3 complex is one of the most complex transmembrane (TM) receptor structures that has been identified and serves a critical function in the immune system. Signals delivered through this receptor are required for T-cell development in the thymus, the induction of T-cell-mediated immune responses against infectious agents and differentiation of T-cells into effector and memory populations with discrete functional properties. The αβ TCR–CD3 complex is composed of six different type I single-spanning TM proteins: the TCRα and TCRβ chains that form the TCR heterodimer responsible for ligand recognition, and the non-covalently associated CD3γ, CD3δ, CD3ε and ζ chains, which bear cytoplasmic sequence motifs that are phosphorylated upon receptor activation and recruit a large number of signaling components (Klausner et al, 1990; Exley et al, 1991; Garboczi et al, 1996; Davis et al, 1997; Sun et al, 2001). The complex is formed in the endoplasmic reticulum (ER) by an ordered assembly process driven by interactions among both extracellular and TM portions of the subunits (Alarcon et al, 1988; Bonifacino et al, 1990; Wileman et al, 1993). The signaling components form the CD3γε and CD3δε heterodimers and the ζ−ζ homodimer, which associate with the TCRαβ heterodimer through coordination of ionizable residues in the TM regions (Alcover et al, 1990; Blumberg et al, 1990a; Cosson et al, 1991; Manolios et al, 1991; Call et al, 2002). The mechanism of receptor triggering is not understood and two major models of the TCR–CD3 complex have been proposed, each with unique implications for signal initiation. A model in which a single TCR heterodimer associates with all signaling components (Manolios et al, 1991; Punt et al, 1994; Kearse et al, 1995; Call et al, 2002) implies a triggering mechanism based on a conformational change in individual ligand-engaged receptors and/or recruitment of two or more separate TCR–CD3 complexes into close proximity. In contrast, the model in which two TCR heterodimers are present in a complex (Exley et al, 1995; Jacobs, 1997; San Jose et al, 1998; Fernandez-Miguel et al, 1999) raises the possibility that signaling is initiated by a conformational change in a pre-assembled dimer, as recently described for the erythropoietin receptor (Livnah et al, 1999; Remy et al, 1999) and other cytokine and hormone receptors (Carr et al, 2001; He et al, 2001). The two related but distinct issues of TCR valency (number of TCR heterodimers) and stoichiometry (molar ratios of the different subunits) must therefore be clarified in order to elucidate the mechanism of activation. It has been determined that at least two CD3ε subunits are present in the fully assembled structure, as both human and murine forms could be identified in individual TCR–CD3 complexes from murine T-cells expressing both proteins (Blumberg et al, 1990b; de la Hera et al, 1991). However, no direct assessment of the stoichiometric relationships among all of the subunits has been performed. Two studies of the composition of the TCR–CD3 complex addressed receptor valency using transgenic mice that expressed two distinct αβ TCR (Punt et al, 1994) or two different TCRβ chain sequences with the same TCRα (Fernandez-Miguel et al, 1999). However, these two studies came to opposite conclusions regarding the valency of the receptor, which reflects the experimental difficulties of examining such a complex receptor structure in the available cellular systems. A method for direct determination of the stoichiometry of such a multicomponent receptor structure must meet a number of critical experimental requirements for the conclusions to be valid. All components of the receptor have to be homogenously labeled at a defined number of positions. The stoichiometry among all receptor components can therefore not be determined by labeling of surface receptors with biotin or 125I, since the number of modified sites cannot be determined with certainty. Metabolic labeling is adequate for homogenous labeling of all receptor components, but a number of assembly intermediates and unassembled chains are present in the ER in addition to complete receptor structures. It is thus essential that the receptor is isolated using an approach that yields a population in which each member has the same composition. Finally, analysis of a membrane protein complex requires that proteins be extracted using detergents that can efficiently solubilize the lipid bilayer while simultaneously preserving the non-covalent interactions among the subunits. It is thus critical to demonstrate that the observed stoichiometry does not change with detergent choice. For many complex protein assemblies, absolute subunit stoichiometry is only established when high-resolution structural information becomes available. However, membrane-anchored protein complexes have been generally refractory to the biophysical methods developed to study the structures of water-soluble proteins. Therefore, while complex membrane proteins represent some of the most important structures for basic cellular functions, they are also among the most difficult subjects for structural biologists and protein biochemists. In this study, we addressed both the valency and the stoichiometry of the TCR–CD3 complex using direct biochemical approaches. We employed a method for isolating intact, radiolabeled protein complexes of known composition to first determine the TCR valency, and then to define the stoichiometric relationships among all TCR–CD3 subunits in the complete receptor structure as well as major assembly intermediates. These results define the TCR–CD3 complex as monovalent, with a stoichiometry of TCRαβ–CD3γε–CD3δε–ζζ. These conclusions are supported by a large body of data on TCR–CD3 assembly (Punt et al, 1994; Kearse et al, 1995; Call et al, 2002). Results Valency of the TCR–CD3 complex To examine the valency of the TCR–CD3 complex, we assembled human αβ TCR–CD3 complexes in ER microsomes of B-cell origin. This system has been previously used for a mutational analysis of the polar TM residues whose interaction coordinates TCR–CD3 assembly (Call et al, 2002), and has been shown to reflect accurately the membrane protein interactions observed in cells using metabolic labeling techniques (Ribaudo and Margulies, 1992; Bijlmakers et al, 1994; Huppa and Ploegh, 1997; Hebert et al, 1998). The major strength of this method is that radiolabeled proteins are synthesized only from input mRNAs, and that the presence of individual subunits can therefore be controlled. We reasoned that it would be possible to discern whether one or more TCRαβ heterodimers are present in a complex by performing assembly reactions in the presence of two TCRβ chains that differed only by the sequence and molecular weight of attached affinity tags, the nine-amino-acid HA tag and the 47-amino-acid streptavidin-binding peptide (SBP). Two different affinity tags were used rather than two different TCRβ chain sequences, since TCRα and β pairing efficiency is sequence dependent and highly variable. Figure 1A shows the results of such an experiment for the human MHC class II-restricted HA 1.7 αβ TCR (Lamb et al, 1982; Hennecke et al, 2000). Streptavidin (SA) precipitation of digitonin lysate from a reaction containing an SBP-tagged HA 1.7 TCRβ chain and all other TCR–CD3 subunits precipitated TCRβSBP, the TCRαβSBP disulfide-linked heterodimer, and all four associated polypeptides (lane 1), indicating full assembly of TCR–CD3 complexes. The same result is shown for a reaction with the HA-tagged TCRβ chain (lane 2). When separated by SDS–PAGE under non-reducing conditions, the TCRαβSBP and TCRαβHA heterodimers were clearly resolved based on molecular weight (arrowheads). However, when the two TCRβ mRNAs were co-translated in the same reaction, antibodies to one affinity tag failed to co-precipitate the TCRαβ heterodimer bearing the other affinity tag (lanes 4 and 5), despite equivalent levels of assembly (lane 3). Figure 1.The αβ TCR–CD3 complex assembled in the ER is monovalent. (A) Translation/assembly reactions were performed with mRNAs encoding the TCRα and TCRβ chains of the human HA 1.7 TCR as well as CD3γ, CD3δ, CD3ε and ζ chains. In order to determine whether two TCRβ chains could be incorporated into a TCR–CD3 complex, C-terminal HA or SBP tags were placed onto the HA 1.7 TCRβ chain and either one (lanes 1 and 2) or both β chains (lanes 3–6) were used in the reactions. Reactions were incubated for 15 min at 30°C under reducing conditions to facilitate rapid translation of mRNAs and translocation of radiolabeled proteins into ER microsomes (mouse IVD12; see Materials and methods), followed by a 4-h folding and assembly period under oxidizing conditions, as described in Materials and methods. Single-step IPs were performed for lanes 1–5 as indicated under each gel using SA (SBP tag), anti-HA mAb (HA tag) or mAb UCH-T1 (CD3ε), while two-step snIPs were performed for lanes 6 and 7 in which SBP-tagged complexes were captured onto SA beads, released by competition with biotin and re-precipitated with anti-HA mAb. In these experiments and all others except Figure 2B, final products were digested for 1 h at 37°C with 500 U endoglycosidase H. TCRαβ heterodimers that incorporated TCRβHA or TCRβSBP could be resolved by SDS–PAGE due to the size difference between the two tags. All other components of the complex were precipitated when either TCRβ tag was targeted in the IP (lanes 1 and 2). When the two TCRβ chains were present in the same assembly reaction, both TCRβ chains and TCRαβ heterodimers were precipitated by the anti-CD3ε mAb (lane 3). However, precipitation directly targeting one TCRβ tag failed to co-precipitate the alternate TCRβ chain or TCRαβ heterodimer (lanes 4 and 5). When both TCRβ tags were targeted in a snIP, no radiolabeled proteins were recovered (lane 6). As a control (*) for background signal in the snIP analysis, aliquots of reactions 1 and 2 were mixed after assembly and analyzed in parallel with reaction 6 (lane 7). (B) The experiment in (A) was repeated using constructs encoding the human A6 αβ TCR. In (C, D), the experiments were performed with constructs in which the tags were placed on TCRα rather TCRβ (HA 1.7 TCR in (C), A6 TCR in (D)). In these experiments, the PC tag recognized by the protein C mAb was used in combination with the SBP tag. All experiments demonstrated that a single TCR heterodimer is incorporated into a TCR–CD3 complex. Download figure Download PowerPoint As a second approach, both tags were targeted in a sequential non-denaturing immunoprecipitation (snIP) designed to yield intact complexes only if they contained both TCRβ chain affinity tags. Following the first precipitation step, captured radiolabeled protein complexes were eluted from SA beads by competition with biotin and re-precipitated using a mAb directed against the HA tag. No radiolabeled proteins were recovered in this procedure (lane 6), indicating that complexes containing both TCRβ chains in direct or indirect association were not present. However, the same immunoprecipitation (IP) procedure yielded fully assembled complexes when the tags were placed on TCRβ (SBP) and ζ (HA; see Figures 3 and 4), or on CD3ε (Call et al, 2002), which is known to occur in at least two copies in the assembled receptor (Blumberg et al, 1990b; de la Hera et al, 1991). This method is therefore suitable for addressing the question of receptor valency and does not disrupt intact radiolabeled protein complexes since elution following the first precipitation step was performed under non-denaturing conditions. We controlled for affinity tag placement and sequence effects by shifting the tags to the C-terminus of TCRα, and by using a different second-step affinity tag, which is recognized by a protein C (PC)-specific antibody (C and D). In addition, the experiments were repeated using a second human αβ TCR pair (MHC class I-restricted A6 TCR; Garboczi et al, 1996; Utz et al, 1996), with identical results (panels B and D). In all experiments, the differentially tagged TCRαβ heterodimers associated equally well with CD3 proteins, yet failed to co-precipitate, indicating that only a single TCR heterodimer is present in the TCR–CD3 complex assembled in the ER. Figure 2.T-cell-derived ER membranes contain previously synthesized T-cell-specific proteins and do not induce assembly of multivalent TCR–CD3 complexes. (A) T-cell-derived ER membranes contain endogenous, assembly-competent components of the TCR–CD3 complex. HA-tagged A6 TCRα and SBP-tagged A6 TCRβ mRNAs were translated in the presence of ER microsomes isolated either from TCRβ-deficient Jurkat T-cells (lanes 1–4) or MGAR B-cells (lanes 5–8), without addition of CD3γ, CD3δ, CD3ε or ζ mRNAs. Radiolabeled proteins present in digitonin lysates of membrane fractions were precipitated by targeting TCRαHA (lanes 1 and 5), TCRβSBP (lanes 2 and 6), CD3ε (lanes 3 and 7) and ζ chain (lanes 4 and 8). Anti-CD3ε and anti-ζ antibodies co-precipitated TCR proteins from T-cell membranes (lanes 3 and 4), but not B-cell membranes (lanes 7 and 8). (B) The TCR–CD3 complex assembled in T-cell-derived ER microsomes is monovalent. Assembly reactions with mRNAs encoding A6 TCRα, TCRβSBP and TCRβHA, CD3γ, CD3δ, CD3ε and ζ were carried out in the presence of T-cell- or B-cell-derived ER microsomes. As in Figure 1, the two differentially tagged TCR heterodimers were precipitated by antibodies to TCR-associated signaling chains (lanes 1, 4, 5 and 8), but only one heterodimer was recovered when either of the two TCRβ tags was directly targeted in the precipitation (lanes 2, 3, 6 and 7). Products were not EndoH digested and are therefore observed at different positions compared to the other figures. Download figure Download PowerPoint Figure 3.Direct measurement of subunit stoichiometry in intact radiolabeled TCR–CD3 complexes isolated by snIP. Translation reactions were performed in the presence of [35S]methionine and ER membranes (mouse IVD12) with mRNAs encoding the A6 TCR and all other components of the TCR–CD3 complex. The SBP tag was present on the A6 TCRβ chain and the HA tag on the ζ chain so that complexes containing all subunits could be isolated by TCRβSBP → TCRζHA snIP from 1.0% digitonin lysates. Quadruplicate reactions were subjected to SDS–PAGE (12%, non-reducing conditions) and proteins were transferred to sequencing-grade PVDF membranes overnight (30 V/4°C). Radiolabeled proteins were detected using a phosphor imager and the signal from each lane was quantitated by densitometry using the Wide Line tool in ImageQuant; the histogram to the right of the gel shows the data for lane 1. Raw counts (line a) were normalized by the number of methionine residues present in each polypeptide (line b) to reflect the relative abundance of each subunit (line c). These values were expressed as a ratio with regard to TCRαβ heterodimer (d), which was present in a single copy per TCR–CD3 complex (Figures 1 and 2). The results from two independent experiments (each performed in quadruplicate) were combined and subjected to statistical analysis (line e; numbers expressed as mean±s.d.). Download figure Download PowerPoint Figure 4.The same TCR–CD3 stoichiometry is observed when a detergent belonging to a different structural class is used for membrane solubilization. Intact radiolabeled TCR–CD3 complexes were isolated and analyzed as in Figure 3, with the exception that detergent solubilization and IPs were performed using 0.5% C12E9, a non-ionic polyoxyethylene detergent, instead of 1.0% digitonin, a deoxycholate derivative. Download figure Download PowerPoint These experiments were performed using human B-cell-derived ER microsomes to avoid complications arising from the presence of previously synthesized, unlabeled TCR–CD3 components. Therefore, the possibility remained that additional T-cell-specific factors were required for assembly of a higher-order structure in T-cells. We therefore isolated ER microsomes from a TCRβ-deficient human Jurkat T-cell line, and repeated the analysis using these membranes (Figure 2). T-cell-specific proteins were found to be present in these microsomes and available to participate in assembly (Figure 2A), as illustrated by co-precipitation of newly translated A6 TCRαHA and TCRβSBP chains with antibodies to CD3ε (lane 3) and ζ (lane 4). As mRNAs encoding CD3 and ζ chains were not present, complexes were formed from newly synthesized TCR heterodimers and previously synthesized signaling chains. This result was clearly specific to the T-cell-derived microsomes, since anti-CD3ε and anti-ζ antibodies did not co-precipitate radiolabeled TCR from reactions containing B-cell-derived microsomes (lanes 7 and 8). We therefore tested whether the presence of these or other native T-cell-specific proteins would alter the outcome of the experiments described in Figure 1. To permit parallel analysis in both T-cell- and B-cell-derived membranes, mRNAs for two tagged TCRβ chains (TCRβSBP and TCRβHA) as well as all other components of the complex were included in the reactions, and precipitations were performed by targeting the tag on either TCRβ chain, CD3ε or ζ (Figure 2B). As before, the TCRαβSBP and TCRαβHA heterodimers each assembled with CD3ε and ζ (lanes 1 and 4), yet were not co-precipitated in the same complex (lanes 2 and 3) regardless of whether assembly reactions were performed with membranes from T-cell or B-cell origin (compare lanes 1–4 with lanes 5–8). These results validate that microsomes of B-cell origin are suitable for studying TCR–CD3 assembly, including a direct assessment of receptor stoichiometry. Stoichiometry of the fully assembled TCR–CD3 complex The experiments presented thus far established that the ER-assembled TCR–CD3 complex contains a single TCRαβ heterodimer, but the stoichiometric relationships among all TCR–CD3 subunits has never been directly addressed using a quantitative biochemical approach. The experiment shown in Figure 2A demonstrated as to why it would be difficult to perform a quantitative analysis in T-cells or T-cell-derived membranes, due to the presence of previously synthesized, unlabeled components and partial complexes that are known to differ substantially in their half-life (Bonifacino et al, 1990, 1991). We solved this problem by assembling uniformly 35S-methionine-labeled TCR–CD3 complexes in ER microsomes derived from B-cells, a cell type that is closely related to T-cells but does not synthesize any TCR–CD3 proteins. Complexes of defined composition were isolated by two-step snIP, separated by SDS–PAGE under non-reducing conditions, transferred to polyvinylidene fluoride (PVDF) membranes and quantitated using a phosphor imager. In the experiment shown in Figure 3, fully assembled TCR–CD3 complexes were purified from cleared digitonin lysates by snIP targeting TCRβSBP followed by HA-tagged ζ chain, which is known to be the last subunit to join during assembly (Sussman et al, 1988; Geisler et al, 1989; Weissman et al, 1989). Since the elution following the first precipitation step was performed by competition with biotin, protein complexes were not disrupted. The counts contained in each band were integrated using the Wide Line tool in the ImageQuant software package (Molecular Dynamics). A sample histogram (right panel) is shown for the gel in the left panel (lane 1) with corresponding peak assignments. The raw counts integrated from each peak (line a) were first adjusted according to the known number of methionine positions in the respective polypeptide (line b). This normalized count (line c) represents the relative amounts of each component. Finally, since the experiments described above established that the TCR heterodimer is present in only one copy per complex, we expressed the abundance of each component relative to TCRαβ (line d). The results indicated that each TCRαβ heterodimer is associated with precisely one ζζ homodimer, two CD3ε subunits and one copy of CD3γ as well as CD3δ (line d). These values were highly reproducible (line e), and are consistent with a model in which the complex is built from one copy of each of four distinct modules: the TCRαβ ligand-binding subunit and the non-covalently associated CD3γε, CD3δε and ζζ signaling dimers. We took great care in our choice of detergent, since too harsh a detergent can disrupt non-covalent protein complexes, yet too weak a detergent could leave patches of incompletely solubilized lipid carrying embedded radiolabeled proteins. We initially screened a total of 16 different non-ionic detergents from a variety of structural classes for their effects on the integrity of the TCR–CD3 complex (not shown). Digitonin was clearly the best detergent choice based on three independent criteria: (1) digitonin was among the most effective in extracting proteins from the lipid; (2) digitonin produced the highest yield of non-covalently associated proteins co-precipitating with the IP target; and (3) digitonin lysates did not produce artifactual associations due to incompletely solubilized membrane patches. This last criterion was tested in an experiment where TCR–CD3 components were co-translated with two irrelevant polypeptides that assemble into the HLA-DR1αβ heterodimer. Anti-CD3ε IP from digitonin lysates co-precipitated the associated TCRα and TCRβ polypeptides but not HLA-DR1 α or β (not shown). To further validate our choice and to rule out detergent-specific effects, we repeated these measurements with a second detergent that belonged to a different structural class than digitonin. As shown in Figure 4, analysis of TCR–CD3 complexes extracted using the polyoxyethylene detergent C12E9 (Lubrol) rather than digitonin produced the same result. These measurements were therefore unchanged by an alternative detergent choice, and reflect the actual stoichiometry of the assembled TCR–CD3 complex. Stoichiometry of key assembly intermediates Translation and assembly of protein complexes in ER microsomes allow complete control over the mRNAs that are translated into radiolabeled proteins, thus providing an opportunity to determine the stoichiometry of isolated assembly intermediates that have been shown to represent key steps in the TCR–CD3 assembly process in cells. An analysis of partial complexes formed in the absence of the ζ chain indicated that the stoichiometric relationships among the remaining subunits were unchanged, and that this complex was composed of one TCRαβ heterodimer, one CD3γ, one CD3δ and two CD3ε subunits (Figure 5). This is consistent with the finding that a complex containing TCR and all three CD3 subunits can be recovered from an intracellular compartment in murine T-cells lacking expression of the ζ chain (Sussman et al, 1988; Weissman et al, 1989). The same product was also observed in ER microsome assembly experiments when the TCRα chain carried a point mutation of the arginine in its TM domain that prevented association of the ζζ homodimer (Call et al, 2002). Figure 5.Stoichiometry of a TCR–CD3 assembly intermediate lacking the ζ chain. Assembly reactions were carried out as in previous experiments using mRNAs encoding all subunits with the exception of the ζ chain. Radiolabeled protein complexes containing all five polypeptides were isolated by TCRβSBP → CD3γPC snIP and analyzed as in Figure 4. The small, broad peak between TCRαβSBP and CD3ε may contain non-disulfide-linked TCRα and/or TCRβSBP chains, and was not included in the calculations shown. If assumed to represent TCR, it contributes less than 10% of the total amount of TCR detected. The absence of the ζ chain did not alter the composition or stoichiometry of the remaining subcomplex. Download figure Download PowerPoint TCR–CD3 assembly proceeds in an ordered fashion with a preferred sequence of association: interaction of CD3δε with the TCRα TM domain, followed by association of CD3γε via the TCRβ TM domain, and binding of the ζζ homodimer to the complex via a second, distinct site in the TCRα TM domain (Sussman et al, 1988; Geisler, 1992; Kearse et al, 1995; Call et al, 2002). This last step requires prior association of both CD3δε and CD3γε to TCRαβ since it does not occur when either CD3γ or CD3δ is absent. The TCRα–CD3δε and TCRαβ–CD3δε subcomplexes are formed in the absence of CD3γ and ζ chain (Figure 6; Call et al, 2002), and these early intermediates have been observed in developing thymocytes in pulse-chase experiments (Kearse et al, 1995) as well as in a Jurkat mutant cell line deficient in CD3γ (Geisler, 1992). As illustrated in Figure 6, densitometric analysis of purified TCRαβ–CD3δε complexes reflects the presence of only one copy of CD3δ and CD3ε per TCRαβ heterodimer. Interestingly, the TCRβSBP → CD3ε snIP strategy employed to isolate this partial complex yielded a significant amount of non-disulfide-linked TCRα and TCRβ chains (arrowheads). These two proteins were in a 1:1 stoichiometric relationship with one another, indicating that they most likely represent a non-covalent TCRαβ heterodimer associated with CD3δε. Indeed, when these signals were included in the calculations as additional counts deriving from TCRαβ heterodimer, the relative quantities of the subunits more closely approximated 1:1:1 (TCRαβ:CD3δ:CD3ε). The stoichiometric analysis of assembly intermediates thus allows us to exclude models in which a higher-order structure is formed at a particular assembly step through association of a certain signaling dimer. Rather, these results reflect an ordered assembly process in which a monovalent TCR–CD3 complex is formed by interaction of three dimeric modules with a single TCR heterodimer. Figure 6.The TCRαβ–CD3δε assembly intermediate contains only one copy of CD3ε. Assembly reactions lacking mRNAs for both CD3γ and ζ were carried out as before; complexes containing both TCR and CD3 components were isolated by TCRβSBP → CD3ε snIP. Recovered proteins were analyzed as described for previous experiments. The majority of TCR heterodimers were disulfide-linked, but non-covalently linked TCR heterodimers were also present in these reactions. The results indicate that only one copy of CD3ε is associated with TCR when CD3γ is absent from the reaction. Download figu" @default.
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• W1986245836 title "Stoichiometry of the T-cell receptor–CD3 complex and key intermediates assembled in the endoplasmic reticulum" @default.
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