Cellular trafficking of growth factor receptors, including cross-talk among receptors at the cell surface, may be important for signal transduction in normal hematopoietic cells. To test this idea, the signaling domain of Mpl (the thrombopoietin receptor) was targeted to the plasma membrane, or to the cytoplasm of murine marrow cells, and the ability of the cells to proliferate and differentiate in response to Mpl dimerized at the plasma membrane or free in the cytoplasm was assessed. Constructs encoding the signaling domain of Mpl linked to an FK506 binding protein domain (to permit dimerization by the membrane-permeable ligand AP20187) with or without a myristylation sequence (to target the receptor to the plasma membrane) and a hemagglutinin epitope tag were generated and introduced into murine marrow cells using a murine stem cell virus (MSCV)-based retroviral vector. Both populations of transduced marrow cells proliferated in Iscoves modified Dulbecco medium-10% FCS-100 nM AP20187 without exogenous growth factors for more than 100 days and achieved greater than a 10 7 -fold expansion of cells by day 50 (n ؍ 4 transductions). Growth was dimerizer dependent, and myeloid, erythroid, and megakaryocytic progenitors were generated. Activation of Mpl either at the plasma membrane or in the cytoplasm allowed for the terminal maturation of transduced progenitor cells. Introduction of membrane-targeted or cyto-
IntroductionHematopoietic growth factor receptors are transmembrane glycoproteins located in the plasma membrane of the cell. Binding of growth factor causes receptor dimerization and recruitment and activation of signaling molecules, thus initiating signal transduction. 1 After ligand binding, growth factor receptors cluster in clathrin-coated pits, become internalized, and are targeted for degradation or recycled to the cell surface. The cell surface location of hematopoietic growth factor receptors permits communication with the extracellular milieu. Other attributes of cell surface location of these receptors that may facilitate signal transduction include organization into specific microdomains on the cell surface, 2-5 preassembled signaling complexes, 6,7 cross-talk among heterologous cytokine receptors, [8][9][10] and commencement of normal intracellular trafficking of the receptor-ligand complex. [11][12][13][14] A stringent test of growth factor receptor function is the ability to support the proliferation and differentiation of normal hematopoietic cells. We hypothesized that the location of a hematopoietic growth factor receptor in the plasma membrane was important not only for communication with the extracellular milieu but also for expression of the full repertoire of receptor functions. The availability of membrane-permeable agents that can bind and dimerize FK506 binding protein 12 (FKBP12) domains provided a mechanism to test this hypothesis. [15][16][17]49,50 We linked the 121-amino acid signaling domain of the murine thrombopoietin receptor Mpl to a modified FKBP12 and targeted the constru...