The role of the cytoplasmic domain in CD40LG regulation 2769

Abstract Description Although most studies have focused on “forward signaling” by CD40LG, CD40LG interaction with CD40 receptor also leads to increased activation, proliferation, and cytokine secretion by T helper cells, suggesting that “reverse signaling” may constitute an important role in T cell function. To evaluate the role of CD40LG cytoplasmic domain in reverse signaling, we generated Jurkat cells expressing either CD40LG Y5 mutant (Y5m) or cytoplasmic domain deletion mutant (CDm). We found that Y5m cells had comparable levels of CD40LG mRNA and protein expression to wild-type CD40LG cells. In contrast, CDm cells showed reduction of total protein (WB) and surface expression (FC) of CD40LG by 90% with no effects on mRNA levels. Activation of Jurkat cells by PMA and Ionomycin (P&I) increased CD40L surface expression (∼40-fold) and total protein (∼2-fold), and upregulated mRNA (∼2-fold). Similar changes in CD40LG expression in response to P&I stimulation were observed in Y5m cells. P&I stimulation of cells expressing CDm also upregulated CD40LG mRNA levels; however, it did not affect total protein expression. Interestingly, P&I activation upregulated surface expression of CD40LG (∼40-fold) as compared to non-stimulated CDm cells. Our results showed that cytoplasmic domain of CD40LG is required for protein expression, but it is not necessary for mobilization of intracellular CD40LG to the surface. In addition, Y5 mutation did not affect CD40LG protein levels and responses to P&I activation. Topic Categories Immune Response Regulation: Molecular Mechanisms (IRM)