PRV-1 Interacts with the Cytoplasmic Domain of Mpl and Interferes with TPO-Induced Signaling Cascades.

Polycythemia rubra vera-1 (PRV-1) is a member of the uPAR/CD59/Ly6 family of cell surface receptors. An increase in the expression level of PRV-1 mRNA has been shown in patients with polycythemia vera and essential thrombocythemia, however, the ligand and function of PRV-1 remains unclear. Here, we show for the first time that PRV-1 expression alters the TPO-mediated Mpl receptor-signaling pathway. We also report a novel interaction between PRV-1 and the cytoplasmic region of Mpl (121 amino acids) using a yeast two-hybrid screen. Further characterization of this interaction in yeast containing truncated versions of the Mpl receptor cytoplasmic region indicates that the binding of PRV-1 to Mpl is regulated at the distal end of the cytoplasmic region. Deletion analysis utilizing Mpl receptors truncated after 53 (T53) or 69 (T69) cytoplasmic amino acid residues failed to show an association with PRV-1. Further mapping of the cytoplasmic domain of Mpl showed that truncations equal or greater than 111 cytoplasmic residues (T111) restored the interaction with PRV-1. To examine the physiological relevance of this interaction, we co-expressed Mpl and PRV-1 in BaF3 cells. Western blot analysis with an anti-phosphotyrosine antibody on TPO-stimulated BaF3/Mpl/PRV-1 whole cell lysates demonstrated a noticeable change in intracellular phosphorylation when compared to BaF3/Mpl. We found no affect on Jak2 and Mpl tyrosine phosphorylation. In contrast, there were higher levels of tyrosine-phosphorylated STAT5 and STAT3 in BaF3/Mpl cells expressing PRV-1. We then further investigated the effect of PRV-1 expression on MAPK signaling. We found that TPO-stimulation of BaF3/Mpl/PRV-1 cells resulted in a reduction of MAPK phosphorylation compared with cells expressing Mpl alone. These results indicate a novel function for PRV-1 as a signaling molecule in cytokine signaling cascades and may lead to a greater understanding of the mechanism of overexpression of PRV-1 in myeloproliferative disorders.