Differential signalling of NH2-terminal flag-labelled thrombopoietin receptor activated by TPO or anti-FLAG antibodies

In this report, we compared activation of NH2-terminal FLAG-labelled thrombopoietin receptor (Mpl) by anti-FLAG antibodies and by thrombopoietin (TPO). We found that anti-FLAG monoclonal antibodies M1 dimerize FLAG-labelled receptor and trigger proliferation of BaF3/FLAG-Mpl cells. In UT7/FLAG-Mpl cells, activation of the FLAG-Mpl receptor by low TPO concentrations triggered proliferation, while high concentrations triggered differentiation. Activation of FLAG-Mpl receptors in these cells by all tested concentrations of M1 antibodies induced proliferation but not differentiation. Low TPO concentrations induced similar to M1 antibodies level of Jak2, Stat3, Stat5 and Akt phosphorylation. In contrast, only TPO and not M1 antibodies activated Erks phosphorylation. Since the anti-FLAG antibodies do not react with the TPO binding site of the receptor, we hypothesize that they can trigger a distinct signal by dimerizing Mpl in a manner different from that induced by TPO.