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The erythropoietin receptor (EPO-R), a member of the cytokine receptor superfamily, can be activated to signal cell growth by binding either EPO or F-gp55, the Friend spleen focus-forming virus glycoprotein. Activation by F-gp55 results in constitutive EPO-R signalling and the first stage of Friend virus-induced erythroleukemia. We have generated a truncated form of the EPO-R polypeptide [EPO-R(T)] which lacks the critical cytoplasmic signal-transducing domain of the EPO-R required for EPO-or F-gp55-induced cell growth. EPO-R(T) specifically inhibited the EPO-dependent growth of EPO-R-expressing Ba/F3 cells without changing the interleukin-3-dependent growth of these cells. In addition, Ba/F3 cells that coexpressed wild-type EPO-R and EPO-R(T) were resistant to transformation by F-gp55 despite efficient expression of the F-gp55 transforming oncoprotein in infected cells. EPO-R(T) inhibited the EPO-dependent tyrosine phosphorylation of wild-type EPO-R, the tyrosine kinase (JAK2), and the SH2 adaptor protein (Shc). In conclusion, the EPO-R(T) polypeptide is a dominant negative polypeptide which specifically interferes with the early stages of EPO-R-mediated signal transduction and which prevents Friend virus transformation of erythroblasts.The erythropoietin receptor (EPO-R), a 507-amino-acid (aa) membrane protein (12), exerts its biological activity in erythroid precursors through the binding of its 34-kDa glycoprotein ligand, thereby functioning as the primary regulator of erythroid mitogenesis and differentiation. The Friend spleen focus-forming virus (SFFV) glycoprotein (F-gp55) binds to the EPO-R, causing constitutive receptor signalling and the first stage of Friend virus-induced erythroleukemia (2). There is no amino acid similarity between EPO and F-gp55, and each protein appears to bind to a discrete site on the EPO-R (5). Little is known of the mechanisms by which the EPO-R transduces a growth signal. Activation of the EPO-R by EPO or F-gp55 (44) results in the tyrosine phosphorylation of the EPO-R cytoplasmic region (9,15,27), and this phosphorylation correlates with mitogenic activity.Although the EPO-R does not itself contain a tyrosine kinase catalytic domain (13), it contains a critical regulatory domain which interacts with cytoplasmic tyrosine kinases and other cytoplasmic effector molecules. Recent studies demonstrated that EPO activates the cytoplasmic tyrosine kinase (JAK2) (43). The tyrosine kinase Fes has been shown to be tyrosine phosphorylated in response to EPO in TF-1 cells, a human erythroleukemia cell line (18). Other tyrosine kinases may be recruited to the EPO-R, as observed with a variety of cytokine receptors (31); however, their identification remains elusive. EPO stimulation results in the rapid tyrosine phosphorylation of the EPO-R (9, 15, 27), JAK2 (43), and the SH2 adaptor protein (Shc) (8, 10). A number of other biochemical events have been associated with activation of the EPO-R, including an increase in the activities of phosphatidylinositol 3-kinase (11,20,25), p2lras (40),...

The Friend spleen focus-forming virus (SFFV) gp55 glycoprotein binds to the erythropoietin receptor (EPO-R), causing constitutive receptor signaling and the first stage of Friend erythroleukemia. We have used three independent strategies to further define this transforming molecular interaction. First, using a retroviral selection strategy, we have isolated the cDNAs encoding three fusion polypeptides containing regions of both EPO-R and gp55. These fusion proteins, like full-length gp55, transformed the Ba/F3 factor-dependent hematopoietic cell line and localized the transforming activity of gp55 to its transmembrane domain. Second, we have isolated a mutant of gp55 (F-gp55-Ml) which binds, but fails to activate, EPO-R. We have compared the transforming activity of this gp55 mutant with the EPO-R-gp55 fusion proteins and with other variants of gp55, including wild-type polycythemia Friend gp55 and Rauscher gp55. All of the fusion polypeptides and mutant gp55 polypeptides were expressed at comparable levels, and all coimmunoprecipitated with wild-type EPO-R, but only the Friend gp55 and the EPO-R-gp55 fusion proteins constitutively activated wild-type EPO-R. Third, we have examined the specificity of the EPO-R-gp55 interaction by comparing the differential activation of murine and human EPO-R by gp55. Wild-type gp55 had a highly specific interaction with murine EPO-R; gp55 bound, but did not activate, human EPO-R.The Friend and Rauscher murine leukemia virus complexes induce a rapid and fatal erythroleukemia in adult mice (4,30). It is the replication-defective spleen focus-forming virus (SFFV) contained in these complexes that initiates the disease. Both Friend and Rauscher SFFV encode a 55-kDa glycoprotein, gp55, which is necessary and sufficient for the initiation of leukemia (1). Friend SFFV gp55 (F-gp55), encoded by the polycythemia-inducing strain of SFFV, has recently been shown to bind to the normal erythropoietin receptor (EPO-R) expressed in infected erythroblasts, resulting in constitutive activation of EPO-R, EPO-independent growth of the infected cells, and the first stage of Friend erythroleukemia (4,17,22).Several features of the F-gp55 polypeptide affect its leukemogenic activity. F-gp55 is transport defective, with the majority of its expression remaining intracellular (16,19,35,40). Its cell surface form appears to be a prerequisite for its leukemogenicity (21). Also, it is the transmembrane region of F-gp55 that is required for its ability to generate EPOindependent erythroblasts in vivo (8,37,38).The precise molecular interaction between F-gp55 and EPO-R is poorly understood. Most of the EPO-R-gp55 complex is formed in the endoplasmic reticulum of infected cells (41), although cross-linked complexes of EPO-R and F-gp55 have also been detected at the cell surface (7). In addition, the transmembrane region of EPO-R is critical for its interaction with gp55, and at least one binding site for gp55 is present in the extracytoplasmic region of EPO-R (44). Furthermore, the molecular interactions bet...

Our previous studies demonstrated TRH stimulation of TSH beta gene transcription in rat pituitary cell cultures and in transient expression assays, with the TRH-sensitive region located between -1.3 kilobases and -204 basepairs (bp) relative to the major transcriptional start site. Using nuclear runoff and transient expression assays, we have analyzed the interactions among TRH, the phorbol ester 12-myristate 13-acetate (PMA), and the adenylate cyclase activator forskolin on TSH beta gene transcription. In cultured pituitary cells, TSH beta gene transcription was stimulated by 2 h of 10(-9) M TRH (2- to 4-fold), 100 nM PMA (2- to 6-fold), or 2 microM forskolin (1.5- to 2.5-fold) treatment, with additive interactions among all three effectors. Chimeric plasmids containing various 5'-flanking portions of the TSH beta gene and both transcriptional start sites, fused to the chloramphenicol acetyltransferase (CAT) gene, were transfected into the clonal pituitary GH3 cell line to delineate DNA sequences conferring this regulation. Transfected TSH beta CAT constructs containing TSH beta gene sequences from -2100/+27I150, -1295/+27I150, and -520/+27I150 expressed CAT enzyme activity which was stimulated by 24 h of TRH (2- to 3-fold), PMA (3- to 6-fold), or forskolin (1.5- to 3-fold) treatment, similar to observations in normal pituitary cells. In addition, a CAT expression vector construct containing only upstream TSH beta gene sequences from -703 to -85 bp, fused to the heterologous thymidine kinase promoter (tkCAT), exhibited similarly stimulated transcription in a transfection assay in response to TRH, PMA, and forskolin.(ABSTRACT TRUNCATED AT 250 WORDS)

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