Prime regulation of blood cell development is exerted through interaction of soluble or membrane-bound hematopoietins with lineage-specific cell surface receptors on committed progenitor cells. Receptor and cytokine cloning has allowed for structure-function analyses of activation mechanisms, and the recent identification of immediate cytosolic effectors. The erythropoietin (EPO) receptor system is an important example since EPO promotes the proliferation of red cell progenitors; is required for the terminal differentiation of erythrocytes; and is valuable in the treatment of renal disease, AIDS. and marrow transplantation. Proposed studies aim firstly to define molecular bases for EPO receptor association with, and activation of, two primary effectors; phosphatidylinositol-3 kinase (Pl3-K) and the Janus protein tyrosine kinase, JAK2. Studies of EPO receptor/Pl3-K interaction will proceed from the delineation of receptor motifs which mediate p85/P13-K binding, to their directed mutagenesis and assay of the putative role for P13-K activation in EPO-induced mitogenesis. Studies of EPO receptor/JAK2 interaction will involve identification of conserved features of the EPO receptor essential cytoplasmic subdomain which mediate JAK2 binding, and activation. Domains of JAK2 involved in EPO receptor association also will be delimited, and a potential role for JAK2 in P13-K activation will be assessed via ectopic expression of dominant negative forms. Direct phosphorylation and allosteric effects exerted by the EPO receptor also will be tested as mechanisms for Pl3-K activation. in related experiments aimed at defining novel primary effectors of EPO action, receptor subdomains which mediate induced late erythroid gene activation will be defined. Here, hybrid receptors comprised of amino-terminal domains of the G-CSF receptor and distal cytoplasmic domains of the EPO receptor will be expressed in erythroid J2E cells, and assayed for the ability to mediate induced transcription of beta-maj, E-gamma, and alpha-globin genes. This model comprises a unique system for studies of molecular pathways of EPO- activated red cell differentiation. Proposed studies should advance an understanding of mechanisms critical to the regulated activation of Class l cytokine receptors, and primary downstream effectors.
