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Mutations in the KIT transmembrane protein-tyrosine kinase receptor affect erythropoiesis, resulting in fewer committed late progenitors (colony-forming unit erythroid, CFU-E) in the fetal liver. As the survival and proliferation of CFU-Es depend absolutely on erythropoietin (EPO), these results suggest that CFU-Es cannot proliferate or mature further unless both the KIT and EPO receptor signalling pathways are functional. How KIT affects proliferation or differentiation of CFU-Es is not clear. Here we show that the KIT ligand SCF (for stem-cell factor) can replace EPO in supporting the growth and survival of HCD57 cells, an EPO-dependent erythroid-progenitor cell line expressing high levels of KIT. SCF supports the proliferation of 32D cells that express KIT only if they also express the EPO receptor. In HCD57 cells, SCF rapidly induces tyrosine phosphorylation of the EPO receptor, and KIT physically associates with the extended box 2 region in the cytoplasmic domain of the EPO receptor. Our results indicate that KIT may activate the EPO receptor by tyrosine phosphorylation to induce further proliferation and maturation of CFU-Es.

The receptors for erythropoietin and other cytokines constitute a new superfamily. They have no tyrosine-kinase or other enzyme motif and their signal-transducing mechanism is unclear. Here we describe two classes of activating mutations in the erythropoietin receptor (EPOR). A single point mutation in the exoplasmic domain enables it to induce hormone-independent cell growth and tumorigenesis after expression in nontumorigenic, interleukin-3-dependent haematopoietic cells. A C-terminal truncation in the cytoplasmic domain of the EPOR renders the receptor hyperresponsive to erythropoietin, but is insufficient to induce hormone-independent growth or tumorigenicity. The activating point mutation retards intracellular transport and turnover of the receptor. These alterations in metabolism and tumorigenicity caused by the EPOR with activating point mutations are similar to those observed in erythropoietin-independent activation of the wild type EPOR by association with gp55, the Friend spleen focus-forming virus glycoprotein.

Friend spleen focus-forming virus (SFFV) is a defective murine C-type retrovirus which causes a multi-stage erythroleukaemia in mice and erythroblastosis in bone marrow cultures. The SFFV env gene encodes a membrane glycoprotein, gp55, which is located on the cell surface and in the rough endoplasmic reticulum and is essential both for the induction of leukaemia in vivo and erythroblast proliferation in vitro. The mechanism by which gp55 causes increased erythroblastosis and ultimately leukaemia is unknown, but a reasonable suggestion is that gp55 can mimic the action of erythropoietin by binding to its receptor (Epo-R), thereby triggering prolonged proliferation of erythroid cells. To test this possibility, we have co-expressed gp55 and the murine Epo-R in a fibroblast cell line. We show here that in such cells, the SFFV glycoprotein binds directly to Epo-R. Furthermore, when an interleukin-3 (IL-3)-dependent lymphoid cell line was co-infected by SFFV and a virus that carries the Epo-R gene, it could grow without IL-3. We suggest that through direct binding to Epo-R, gp55 can stimulate the receptor and by-pass the normal requirement for Epo, causing prolonged proliferation of infected erythroid cells. This could be the first step of leukaemogenesis induced by Friend virus.

The amino-acid sequence of murine band 3, deduced from the nucleotide sequence of a complementary DNA clone, confirms that this integral membrane glycoprotein is composed of two major structural domains which correlate with its dual functions as the anchor for the erythrocyte cytoskeleton and as a plasma membrane anion antiporter. This latter activity resides within a highly hydrophobic domain that crosses the plasma membrane at least 12 times.

Most eukaryotic secretory and membrane proteins insert co-translationally into the membrane of the rough endoplasmic reticulum (RER), and are targeted there by one or more NH2-terminal or internal signal sequences. However, little is known about the actual translocation and membrane integration processes. In particular, any energy requirements for targeting and integration have remained obscure because of the inability to uncouple the processes from concomitant protein synthesis. We recently showed that the human glucose transporter (GT), an integral membrane glycoprotein, can insert post-translationally into dog pancreatic microsomes with low but demonstrable efficiency in vitro, and that a fragment corresponding to the NH2-terminal 340 amino acids and 8 of the 12 membrane-spanning alpha-helixes of GT (GT-N) can insert with significantly greater efficiency. We report here that post-translational insertion of GT-N into pancreatic microsomes requires energy in the form of a phosphodiester bond, and suggest that co-translational insertion of proteins into the RER may also require energy independent of that used for polypeptide synthesis.

Studies of the synthesis and incorporation of the vesicular stomatitis virus glycoprotein into membranes in a synchronised cell-free system demonstrate a tight coupling between polypeptide synthesis and membrane insertion, as a result of which the nascent chain crosses the membrane. The studies reveal a surprisingly precise sequence by which the nascent chain of this membrane glycoprotein is glycosylated in two steps. These findings have important implications for the mechanisms of membrane assembly.

The 5'-terminus 7-methylguanosine of vesicular stomatitis virus mRNA is not essential for translation of the mRNA. The 7-methylguanosine seems to have a mediating rather than an obligatory role in ribosome binding by the mRNA and in mRNA translation.

The stability of mRNA is an important facet of the regulation of protein synthesis. In mammalian cells most mRNAs have long half-lives (5-15 hours) but a substantial fraction are much less stable. There are few examples where the stability of a particular mRNA or class of mRNAs is specifically affected by environmental or developmental stimuli. Certain hormones cause specific stabilization of mRNAs species and preferential mRNA stability is important in the accumulation of globin and myosin mRNAs during the terminal stages of erythropoesis or myogenesis, respectively. Disaggregation of Dictyostelium discoideum aggregates induces the specific destabilization of a large class of developmentally regulated mRNAs; thus, this system is an excellent one in which to determine how such controls are effected. Here we show that addition of cyclic AMP to disaggregated cells specifically prevents the destabilization of these mRNAs.

Upon starvation, the cellular slime mould Dictyostelium discoideum initiates a 24-h programme of differentiation. Within 6 h, cells move towards aggregation centres in response to pulsatile synthesis and secretion of cyclic AMP. At about 12 h, aggregates of 10(5) cells are formed, held together by newly made surface adhesion molecules. The cells then differentiate into the two principal types found in the terminal stage of development, spores and stalks. Here we show that the chemotaxis and aggregation stages of this developmental programme can be described as a series of sequential events in which these extracellular signals--starvation, cyclic AMP and cell-cell contact--induce specific, sequential changes in the pattern of gene expression.

In eukaryotic cells, secretory proteins and glycoproteins migrate from the rough endoplasmic reticulum, their site of synthesis, through Golgi vesicles before being released from the cell. Cellular and viral integral plasma membrane glycoproteins are co-translationally inserted into the rough endoplasmic reticulum membrane and follow a similar pathway to the cell surface. Previous studies using endoglycosidase H (Endo H) suggested that in rat hepatoma cells the vesicular stomatitis virus (VSV) G protein, albumin and transferrin migrate from the rough endoplasmic reticulum to the Golgi apparatus at different rates. Here we show directly that in human hepatoma HepG2 cells, five secreted proteins mature from the rough endoplasmic reticulum to Golgi vesicles at characteristic rates which differ at least threefold. The results are incompatible with bulk-phase movement of the luminal contents of the endoplasmic reticulum, and suggest that there is a membrane-bound receptor that selectively mediates the transport of secretory proteins from the rough endoplasmic reticulum to the Golgi.