Analysis of functional domains of the human tpo receptor encoded by C-MPL

c-Mpl, a member of the hematopoietic cytokine receptor family, is the receptor for thrombopoietin (TPO). To establish a model system for analyzing functional domains of c-Mpl, we searched for a cell line not expressing endogenous c-Mpl. Previously we reported that c-mpl mRNA could not be detected in a human EPO-responsive cell line, UT7/EPO (Blood 82:456,1993; Blood in press). This was confirmed by the reverse transcriptase-polymerase chain reaction (RT-PCR) method. Based on these observations, we prepared an expression vector containingfulllengthc-mp/ cDNA and transfected it into the UT-7/EPO cells. We isolated 3 clones and examined whether these clones exogenously expressed c-Mpl on the surface of the cells by FACS analysis with an anti-c-Mpl antibody. All these transfectants expressed c-Mpl and grew in response to TPO. Moreover, after incubation with TPO for one week, some of the cells became large and multinucleated, and expressed platelet factor-4 mRNA, suggesting that the TPO signalling pathway mediated through c-Mpl is involved in not only the proliferation but also megakaryocytic differentiation of the transfectants. Therefore, this model system may be useful for analyzing the functional domains of human c-Mpl. We are now preparing several kinds of deletion mutants.