TYROSINE-599 OF THE C-MPL RECEPTOR IS REQUIRED FOR SHC PHOSPHORYLATION AND THE INDUCTION OF CELLULAR-DIFFERENTIATION

Interaction of thrombopoietin (TPO) with its receptor, c-Mpl, triggers cell growth and differentiation responses controlling primitive haemo poietic cell production and megakaryocytopoiesis. To examine the impor tant receptor domains and signal transduction pathways involved in the se cellular responses, c-Mpl cytoplasmic domain truncation and tyrosin e substitution mutants were generated. In the myelomonocytic leukaemia cell lines WEHI3B-D+ and M1, ectopic expression of the wild-type c-Mp l receptor induced TPO-dependent cellular differentiation characterize d by increased cell migration through agar and acquisition of the morp hology and molecular markers of macrophages. Consistent with the conce pt that proliferative and differentiation signals emanate from distinc t receptor domains, the C-terminal 33 amino acids of c-Mpl were dispen sable for a proliferative response in Ba/F3 cells but proved critical for WEHI3B-D+ and M1 differentiation. Finer mapping revealed that subs titution of Tyr599 by phenylalanine within this c-Mpl domain was suffi cient to abolish the normal differentiation response, Moreover, in con trast to the normal c-Mpl receptor, this same mplY599F mutant was also incapable of stimulating TPO-dependent She phosphorylation, the assoc iation of She with Grb2 or c-Mpl and of inducing c-fos expression, Thu s activation of components of the Ras signalling cascade, initiated by interaction of She with c-Mpl Tyr599, may play a decisive role in spe cific differentiation signals emanating from the c-Mpl receptor.