Minami Y, Samelson L E, Klausner R D
Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, Bethesda, Maryland 20892.
J Biol Chem. 1987 Sep 25;262(27):13342-7.
The dynamics of the T cell antigen receptor on a murine antigen specific T cell hybridoma have been analyzed using a monoclonal anti-receptor antibody. When this antibody, A2B4-2, is bound to surface receptors, no internalization is seen at 4 degrees C. Upon warming to 37 degrees C, between 20 and 30% of the antibody molecules are internalized over 20-30 min as measured by sensitivity to external acid. This level of internalization is identical if monovalent Fab fragments are used. In contrast, cross-linking of the anti-receptor antibody with a second antibody leads to rapid internalization of 100% of prebound surface A2B4-2. Phorbol 12-myristate 13-acetate (PMA) leads to the rapid internalization of up to 65% of the surface A2B4-2 or A2B4-2 Fab fragments. This effect requires protein kinase C and can be completely inhibited by depleting this kinase from the cells by long term treatment with high doses of PMA. Pretreatment of the T cells with PMA leads to a 40-50% drop in surface T cell antigen receptor expression. Despite the loss of surface receptors, the uptake of A2B4-2 in PMA-treated cells at 37 degrees C is identical to that seen in control cells. The total uptake of A2B4-2 at 37 degrees C is 25-30% greater than the number of surface receptors in control cells and about 100-150% greater than the number of surface receptors in PMA-treated cells. At steady state the percentage of total A2B4-2 on the cell surface is 75% for control cells and 38% for PMA-treated cells. The good agreement of these numbers with the percent internalization of a cohort of surface receptors suggests that all receptors are constantly cycling. The effect of PMA is to alter the kinetic parameters of this cycling, thus changing the steady state distribution of receptors between the plasma membrane and internal, presumably endosomal compartments. Measurement of initial rates of internalization suggests that the PMA effect can be largely explained by an increase in the internalization rate constant.
