Requirements for cell surface expression of the human TCR/CD3 complex in non-T cells.
The T-cell antigen receptor (TCR) consists of a glycoprotein heterodimer (alpha/beta or gamma/delta) which is non-covalently associated with at least four or five invariant polypeptides (CD3 gamma, delta, epsilon, zeta and eta). In T-cell variants lacking TCR alpha, beta or zeta, it has been shown that incomplete TCR/CD3 complexes are retained within the cell. To examine requirements for cell surface expression of TCR/CD3, we transfected COS monkey kidney cells with cDNAs encoding TCR alpha, beta and CD3 gamma, delta, epsilon and zeta. We report that cell surface appearance of TCR/CD3 on COS cells requires coordinate expression of all six proteins. In the absence of the zeta chain, subcomplexes comprising from two to five chains were readily demonstrable in COS cells, but they failed to reach the cell surface or to acquire N-linked oligosaccharide side chains indicating failure to reach the medial Golgi. Pulse-chase metabolic labelling of transfected COS cells showed that three chains (CD3 gamma, CD3 epsilon, and zeta) were stable while three (TCR alpha, TCR beta and CD3 delta) were rapidly degraded. In two- and three-chain co-transfections specific intracellular subcomplexes were formed between TCR alpha and CD3 gamma, TCR alpha and CD3 delta, or TCR beta and CD3 epsilon. Binary subcomplexes having at least one stable chain (CD3 epsilon - TCR beta) were stable while one formed by two unstable chains (TCR alpha - CD3 delta) was still degraded. Assembly of the TCR/CD3 complex in COS cells thus appears centered around the metabolically stable CD3 gamma and CD3 epsilon proteins. Site-specific mutations of the negatively-charged transmembrane amino acid of residues of the CD3 chains to alanines served to either abolish (for TCR alpha - CD3 delta and TCR beta - CD3 epsilon) or diminish (for TCR alpha -CD3 gamma) these TCR-CD3 interactions. These mutations had no effect, however, on CD3-CD3 interactions or upon synthesis, metabolism, or intracellular distributions of the CD3 proteins. The transmembrane domains of CD3 gamma, delta, and epsilon thus appear to play a major role in associations of CD3 with TCR chains.
Hall
C
C
Laboratory of Molecular Immunology, Dana-Farber Cancer Institute, Boston, MA 02115.
Berkhout
B
B
Alarcon
B
B
Sancho
J
J
Wileman
T
T
Terhorst
C
C
D000818:Animals; D000911:Antibodies, Monoclonal; D000945:Antigens, Differentiation, T-Lymphocyte / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017252:CD3 Complex; D002460:Cell Line; D002462:Cell Membrane / Q000276:immunology; D004247:DNA / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D011948:Receptors, Antigen, T-Cell / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D000911:Antibodies, Monoclonal; D000945:Antigens, Differentiation, T-Lymphocyte; D017252:CD3 Complex; D011948:Receptors, Antigen, T-Cell; D004247:DNA