No abstract

To identify and characterize the hormone‐binding domain of the thyroid hormone receptor, we analyzed the ligand‐binding capacities of proteins representing chimeras between the normal receptor and P75gag‐v‐erbA, the retrovirus‐encoded form deficient in binding ligand. Our results show that several mutations present in the carboxy‐terminal half of P75gag‐v‐erbA co‐operate in abolishing hormone binding, and that the ligand‐binding domain resides in a position analogous to that of steroid receptors. Furthermore, a point mutation that is located between the putative DNA and ligand‐binding domains of P75gag‐v‐erbA and that renders it biologically inactive fails to affect hormone binding by the c‐erbA protein. These results suggest that the mutation changed the ability of P75gag‐v‐erbA to affect transcription since it also had no effect on DNA binding. Our data also suggest that hormone‐independent activity of P75gag‐v‐erbA provided a selective advantage to the avian erythroblastosis virus during the original selection for a highly oncogenic strain of the virus.

Abstract. The c-erbA proto-oncogenes encode nuclear receptors for thyroid hormone (T3), a hormone intimately involved in mammalian brain maturation. To study thyroid hormone receptor (TR) action on neuronal cells in vitro, we expressed the chicken c-erbA/ TRot-1 as well as its oncogenic variant v-erbA in the adrenal medulla progenitor cell line PC12. In the absence of T3, exogenous TRc~-I inhibits NGF-induced neuronal differentiation and represses neuron-specific gene expression. In contrast, TRo~-I allows normal differentiation and neuronal gene expression to occur in the presence of T3. Finally, TRc~-l-expressing cells become NGF-responsive for proliferation when T3 is absent, but NGF-dependent for survival in presence of T3. A similar differentiation induction by NGF plus T3 was observed in a central nervous system-derived neuronal cell line (E 18) expressing exogenous TRot-1. Together with the finding that TRo~-I constitutively blocked dexamethasone-induced differentiation of PC12 cells into the chromaffin pathway, these results suggest that TRet-1 plays an important role in regulating commitment and maturation of neuronal progenitors.In contrast, the v-erbA oncogene, a mutated, oncogenic version of TRc~-I, partially but constitutively inhibited NGF-induced neuronal differentiation of PC12 cells and potentiated dexamethasone-induced chromaflin differentiation, giving rise to an aberrant "intedineage" cell phenotype.

A retrovirus vector was constructed from the genome of avian erythroblastosis virus ES4. The v-erbA sequences of avian erythroblastosis virus were replaced by those coding for neomycin phosphotransferase, creating a gag-neo fusion protein which provides G418 resistance as a selectable marker. The v-erbB sequences following the splice acceptor were replaced by a cloning linker allowing insertion of foreign genes. The vector has been tested in conjunction with several helper viruses for the transmission of G418 resistance, titer, stability, transcription, and the transduction and expression of foreign genes in both chicken embryo fibroblasts and the QT6 quail cell line. The results show that the vector is capable of producing high titers of Neor virus from stably integrated proviruses. These proviruses express a balanced ratio of genome length to spliced transcripts which are efficiently translated into protein. Using the Escherichia coli beta-galactosidase gene cloned into the vector as a test construct, expression of enzyme activity could be detected in 90 to 95% of transfected target cells and in 80 to 85% of subsequently infected cells. In addition, a cDNA encoding the avian erythrocyte band 3 anion exchange protein has been expressed from the vector in both chicken embryo fibroblasts and QT6 cells and appears to function as an active, plasma membrane-based anion transporter. The ectopic expression of band 3 protein provides a visual marker for vector function in these cells.