Selection of T cell receptor expression mutants through the functionally linked Ly-6A.
Ly-6A is a glycosyl-phosphatidylinositol (GPI)-anchored molecule that participates in murine T cell activation. Activation of T cell hybridomas with anti-Ly-6A monoclonal antibody (mAb) leads to production of interleukin-2 (IL-2), but also to a paradoxical growth inhibition, which was used to select for signaling mutants. Fifteen subclones derived from two independent mutageneses and anti-Ly-6A selection were characterized. Thirteen subclones responded poorly or not at all to soluble anti-Ly-6A mAb. Although the selective pressure was exerted through Ly-6A, only one mutant did not express the Ly-6A antigen. Interestingly, 10 of the 15 subclones expressed either nondetectable or a very low level of T cell receptor/CD3 complex (TCR/CD3). Preferential expansion of TCR/CD3 expression mutants following anti-Ly-6A selection further established functional linkage between Ly-6A and TCR/CD3 complex. The mechanism of the functional coupling was investigated by analyzing the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2), one of the early events in T cell activation. We showed that PIP2 was not hydrolyzed in response to anti-Ly-6A in TCR/CD3-negative mutants. Aluminum fluoride, which activates G protein directly, did induce PIP2 hydrolysis in these cells. These data suggest that activation signals originated from Ly-6A must be transmitted first to TCR/CD3 complex, which then couples to the G protein/phospholipase C system. A similar requirement also applies to the Thy-1 protein and lectin receptors. Thus, the TCR/CD3 complex plays a central role in the integration and transmission of activation signals that originated from several T cell surface molecules.
Sugiyama
E
E
Medical Services, Massachusetts General Hospital, Harvard Medical School, Boston 02114.
Cantagrel
A
A
Reno
T
T
Stafford-Brady
F
F
Yeh
E T
ET
Bonventre
J V
JV
D000535:Aluminum / Q000494:pharmacology; D017607:Aluminum Compounds; D000818:Animals; D000911:Antibodies, Monoclonal; D000945:Antigens, Differentiation, T-Lymphocyte / Q000096:biosynthesis; D000950:Antigens, Ly / Q000502:physiology*; D000954:Antigens, Surface / Q000502:physiology; D017252:CD3 Complex; D005020:Ethyl Methanesulfonate; D005459:Fluorides / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000502:physiology; D005786:Gene Expression Regulation; D006825:Hybridomas / Q000378:metabolism; D008213:Lymphocyte Activation; D051379:Mice; D016296:Mutagenesis; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols / Q000378:metabolism; D011948:Receptors, Antigen, T-Cell / Q000096:biosynthesis*; D011975:Receptors, Mitogen / Q000502:physiology; D015398:Signal Transduction / Q000276:immunology; D013601:T-Lymphocytes / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D018800:Thy-1 Antigens; D010738:Type C Phospholipases / Q000502:physiology
D017607:Aluminum Compounds; D000911:Antibodies, Monoclonal; D000945:Antigens, Differentiation, T-Lymphocyte; D000950:Antigens, Ly; D000954:Antigens, Surface; D017252:CD3 Complex; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols; D011948:Receptors, Antigen, T-Cell; D011975:Receptors, Mitogen; D018800:Thy-1 Antigens; D005020:Ethyl Methanesulfonate; D000535:Aluminum; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D005459:Fluorides; C032311:aluminum fluoride