A constitutively activated chimeric cytokine receptor confers factor- independent growth in hematopoietic cell lines

The high-affinity receptor for granulocyte-macrophage colony- stimulating factor (GMR) comprises at least 2 distinct subunits, alpha and beta common (beta c), whereas the normal erythropoietin receptor (nEpoR) comprises only one known subunit. An arginine to cysteine (R129C) mutation of the extracytoplasmic domain of the murine EpoR leads to Epo-independent growth in transduced cells (cEpoR). To investigate the proliferative functions of the cytoplasmic regions of each GMR subunit separately and the potential of the R129C EpoR mutation to induce factor-independent growth through heterologous receptor regions, we constructed four hybrid receptors: the extracellular region of either murine nEpoR or cEpoR linked to the transmembrane and cytoplasmic regions of either the human GMR alpha or beta c subunit (nE alpha, nE beta, cE alpha, and cE beta). We then expressed them in an interleukin-3-dependent murine cell line, Ba/F3. Expression of nE beta led to Epo-dependent growth, whereas expression of cE beta conferred factor-independent growth. Surprisingly, expression of cE alpha also resulted in factor-independent cell growth, whereas nE alpha did not respond to Epo. Furthermore, the functional hybrid receptors showed Epo-dependent (nE beta) or constitutive (cE alpha and cE beta) tyrosine phosphorylation of the cytoplasmic kinases JAK1 and JAK2. We reasoned that the proliferative signal of cE alpha was transduced either through the alpha tail itself or through an accessory protein such as the endogenous murine beta common subunit (mu beta c). To distinguish these possibilities, the chimeric receptor cE alpha was expressed in the interleukin-2-dependent murine cell line, CTLL-2, that does not express mu beta c. cE alpha did not induce cell growth in CTLL-2; however, when mu beta c was coexpressed with cE alpha in CTLL-2, factor-independent growth was reconstituted. In conclusion, the cytoplasmic domain of the GMR alpha subunit requires a beta chain for transduction of a proliferative signal. Furthermore, the R129C EpoR mutation can constitutively activate heterologous receptors to mediate factor-independent proliferation.     

Soluble thrombopoietin receptor (Mpl) and granulocyte colony- stimulating factor receptor directly stimulate proliferation of primitive hematopoietic progenitors of mice in synergy with steel factor or the ligand for Flt3/Flk2

In an effort to establish the specificity of the thrombopoietin (TPO) effects on murine multipotential progenitors, we tested the effects of soluble TPO receptor (sTPOR; sMpl) on multilineage colony formation that was supported by a combination of TPO and steel factor (SF). Surprisingly, sTPOR did not suppress colony formation from primitive progenitors. This led to the discovery that sTPOR synergizes with SF or Flt3/Flk2 ligand (FL) to support the formation of various types of hematopoietic colonies including multilineage colonies. The colonies supported by the combination of sTPOR and SF were capable of expressing both myeloid and B-lymphoid potentials. Studies using micromanipulation and serum-free culture showed that the effects of sTPOR and SF on the primitive progenitors are direct, not mediated by contaminating stromal cells, and not dependent on factors present in the serum. TPOR belongs to the cytokine receptor group that includes granulocyte colony- stimulating factor receptor (G-CSFR) and erythropoietin receptor (EPOR). Therefore, we tested the effects of sG-CSFR and sEPOR on primitive progenitors. sG-CSFR, but not sEPOR, was able to synergize with SF or FL in supporting the proliferation of primitive progenitors. The direct effects of the soluble receptors appear to be mediated through interactions with their respective membrane-bound receptors expressed on the primitive hematopoietic progenitors.