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Disulfide Linkage of the ~ ~d ~ Chins of the T Cell Receptor POSS~LE ~ENTIFICATION OF ~VO S~UC~ C~SSES OF ~CEPTO~" {~ceived for pu~llca~ion, Febru~ 4, I~) ~Hchal Baniyash, Pilaf G~cia-Mo~ai~, Juan S. Bo~facino, Lawrence E. Same~on, ~d Rivh~d D. ~ausner F~m the Cell Biolo~ a~ Met~l~m Brash, Nat~na~ l~ti~te of C~ Health a~ H~ma~ Deve~p~nt, NarWhal l~titutes of He~t~ Bet~sd~ Ma~la~ ~892 The T cell antigen receptor (TCR) is a multisubunit membrane complex. It consists of two dlsulfide-linked polymorphic chains (either ~.-fl or 7-~ heterodimer~) which are noncovalently linked to five invariant chains. The CD3o7 and CD3-5 chains bear ~V-linked carbohydrates and the CD3-~ and ~ chains are nonglyo cosylated. Further analysis of the ~ chain in muriue T cells demonstrates that it can exist as either a homo- dimer or disulfide linked to an additional protein with an apparent Mr of 22,000. The partial peptide map of this 22-kDa protein is different than ~ and all of the CD3 components. Like ~, it has no apparent N-linked carbohydrate chains. We have chosen to refer to this subunit as the, chain of the TCR. Ninety percent of ~ in cloned and nonclonal populations of T celia exist as a homodimer, and the remainder is found linked to the ~ chain. The tight regulation of the ~'o~ to ~=~ ratio suggests an important functional role for these struc- tural components of the TCR. The murlne T cell receptor (TCR)' for antigen has been identified as a complex of at least seven polypeptide chains (1, 2). On the majority of peripheral T cells two glycoproteins, a and #6 (40-45 kDa each), form a clone-specific disulfide- linked heterodimer. These proteins determine the specificity of the receptor for its ligand, an antigen associated with a cell surface molecule encoded by the major histocompatibility complex (3). The a-B hetemdimer is noncovalently associated with five invariant chains: two glycoproteins of 26 and 21 kDa {CD3-~ and CD3-7, respectively), a 25-kDa protein {CD3-~), and a 16-kDa disulfide-linked homodimer (~). Although the function of the invariant chains of the TCR is presently unknown, it is likely that they are involved in signal trans- duction events which follow antigen recognition (4-7). Ana- logues of each of these murine TCR subunits are present in human T cells (8-11). These invariant chains have been termed the CD3 complex. Our laboratory has been interested in studying both the structure of this complex receptor and the early intracellular events that follow engagement of this receptor. Antigen, mi- togen, and a number of stimulatory monoclonal antibodies have been used to demonstrate that two kinase pathways are activated by receptor occupancy (5, 7, 12, 13). These agents "The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore he hereby marked "'adcerti~emeat" in eccvrdance with 18 U.S.C. Section 1734 solely to indicate th~ fact ~ The abbreviations used are: TCR, T cell antigen receptor; e~do- F, endvgtycc~aminldese F; SDS, s~c~um dcd~cyl sulfate; PAGE, l~ly- aerylam~de gel el~ctrcpl~vre~[s; PBS, pb~zphate-ir~ffered saline. lead to turnover of polyphosphoinositides with subsequent activation of protein kinase C and phosphorylation of the 7, and to a lesser degree, e subunits of the antigen receptor. Simultaneously, a protein t~rosine kinase is activated which leeds to phosphorylation of tyrosine residues of a receptor subunit that migrates with an apparent M~ of 20,000-21,000 under reducing conditions on SDS-PAGE. We have previo ously referred to this phosphorylated species as p21 (5}. Stim- ulation of this phaspho~ylation pathway has been observed in murine antigen-specific hybridomas, routine T cell clones, and in human tumor lines and peripheral blood T cells? We have now extended our studies of the structural com- ponents of the TCR in marine T cells. Detailed analysis of ~" reveals that it exists in the receptor complex in two forms. The majority (80-90%) of ~" exists as a homodimer. The rest of ~ is disulfide-linked to a protein with a M~ of 22,000 whose peptide map is unique with respect to all of the other TCR components. We refer to this protein as the, chain of the TCR. Under nonreducing conditions ~" migrates either with a M~ of 32,000 (homodimer) or 38,000 (heterodimer). In a wide variety of T cell clones, tumor cells, hybridomas, and normal splenic or thymic T cells these two forms of ~ are seen. In all of these cells the ratio of homodimer to heterodimer is very similar. These findings suggest the existence of two structural subclasses of TCR. In one, ~'is found uniquely as a homodimer while in the other, representing about 20% of a cell's popula- tion of receptors, ~" exists as port of a heterodlmer. MATERIALS AND METHODS Cells and Antibodies--The antigen-specific T cell hybrid0mas DO 11.10 (14), SKK 9.11 (16), 2B4 and 13.1B6 (16) and C10 (17), the T cell minor line EL4, and the ~ cell hybridoma LK ~5.2 (18) were grown as previously described. The A2B4-2 routine monoclonal an- tibody binds the T cell ~eceptor a chain of 2B4 cells (19). The 2C11- 145 (2C11) hamster monocional antibody binds the routine CD3-~ chain (20}. Anti-CD3-~ polyclonat antibodies reacting with a C- terminal peptide of the routine ~ chain were raised in rabbits (21). Anti-~" polyclonal antibodies (no. 124) were raised in rabbits immu- nized with purified protein (11), The F23.I murlne monoclonal anti- body binds to a determinant of fl chains encoded by the V~8 family and is present in spproxima~ely 10--15% of peripheral T cells {22). For immenoprecipitstion, the antlbod~es were bound to protein A- agarose (Bethesda Research Laboratories). Radi~belin~, Immurmpredphation, and Electr~phores~--Meth- ads for preparation of ~otal cell membranes from a pestnuclear supe~natant and radioiodination of membrane proteins by the lacto- peroxidase-glucose oxidase procedure have been described previously (21}. For stable metabolic labeling atudies, ceils (0.5 × 10~/ml} were labeled for 11 h ~th a mixture of |~]methianine (18 ~C|/ml; tran3- ~S label, ICN, Irvine, CA) and [~S]cy~teine (14 pCi/ml, Amersham Corp.) in methionlne- and c~teine-free RPM] 1~40 medium (Bioflu- ffi M. Baniyash, P. Gareia-Mora~es. J. S. Bon~facino, L. E. Same~n, and R. D. Klausner, unpublished 9374 40000307