Autonomous roles for the cytoplasmic domains of the CD2 and CD4 T cell surface antigens
Beyers AD, Davis SJ, Cantrell DA, Izquierdo M, Williams AF. (1991), EMBO J. 10, 377-85
CD2 and CD4 are single chain transmembrane T cell surface molecules that are involved in signal transduction. Chimaeric constructs from rat CD2 and CD4 antigens were expressed in the Jurkat human T cell line to examine the role of extracellular, transmembrane and cytoplasmic domains in mediating functions controlled by CD2 and CD4. The results show that the large rise in concentration of cytoplasmic free Ca2+ mediated via CD2 crosslinking is controlled by the cytoplasmic domain and does not require the CD2 transmembrane and extracellular domains. Similarly the CD4 cytoplasmic domain alone was shown to encode the specificity for binding to the p56lck tyrosine kinase and to control down-modulation of CD4 after treatment with phorbol ester. Evidence was obtained that down-modulation of CD4 occurs when p56lck dissociates from the cytoplasmic domain due to phosphorylation of Ser 405.
Effects of crosslinking of various antigens on [Ca2+]i in a rat T cell line and in Jurkat cells transfected with rat CD2 and CD4. The rat MBP-3 T cell line was loaded with fura-2AM and PHA (10 μg/ml final concentration) or the anti-rat CD3 mAb (1F4 ascites, final dilution 200-fold) was added at time points indicated by the ↓ signs. Alternatively, cells were pre-incubated for 1 h on ice in 50 μl of 50 μg/ml of anti-CD2 (OX-34), anti-CD4 (W3/25) or anti-TCR (R73) mAb and then diluted to 2 ml prior to addition of RAM F(ab’)2 at 50 μg/ml. This addition is indicated by the ⇓ symbol and in all other cases this also shows addition of crosslinking antibody. TCR+ve Jurkat cells (clone J5) were transfected with rat CD2 or rat CD4, and TCR-ve Jurkat cells (J.RT3-T3.5) were transfected with rat CD4. Cells were triggered with PHA (10 μg/ml) or with an anti-CD3 mAb (UCHT1, 1 μg/ml final concentration). Cells expressing CD2 were also triggered by a combination of two anti-rat CD2 mAbs, OX-54 and OX-55 (50 μg/ml final concentration each) without crosslinking by a second antibody. Alternatively, cells were pre-incubated in 50 μl of 50 μg/ml of anti-rat CD2 mAbs (OX-34 or OX-54) or anti-rat CD4 mAbs (W3/25 or OX-35) and then diluted to 2 ml before crosslinking with RAM F(ab’)2 (50 μg/ml). Values following the delta (Δ) symbol indicate increases in [Ca2+]i above base lines (70-130 nM) and time is indicated by the horizontal bar. The [Ca2+]i increases are on a non-linear scale.