Characterization of the hormone-binding domain of the chicken c-erbA/thyroid hormone receptor protein.

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.

A conserved C-terminal sequence that is deleted in v-ErbA is essential for the biological activities of c-ErbA (the thyroid hormone receptor).

“…The -73 COL-CAT (1) and 2XT3RE-tk-CAT (28) reporters have been described. Construction of the Cl to C5 v-ErbA/c-ErbA chimeras containing gag sequences was previously described (41). To generate the Cl to C5 chimeric expression vectors without gag sequences used in this study, the EcoRI fragments of Cl to C5 were cloned into the EcoRI site of pSG5 (30).…”

“…In vivo T3 binding assays were performed as previously described (41,50,67). For these experiments, primary chick embryo fibroblasts were transfected with the retroviral vectors pNeo-M1 to M4 and pNeo-C1, and neomycin-resistant cells were selected in medium containing G418 (0.8 mg/ml) (19,66).…”

Multiple mutations contribute to repression by the v-Erb A oncoprotein

“…A mutation in the helix 3 domain of v-Erb A impairs T3 binding V-Erb A is significantly impaired in the ability to bind T3 hormone compared to c-Erb A; part of this loss in hormone affinity is due to truncation of helix 12, whereas another component has been mapped to aminoacid differences between v-and c-Erb A in helices 1-3 of the hormone binding domain (Munoz et al, 1988;Damm et al, 1989;Sap et al, 1989;Zenke et al, 1990). We mapped this second determinant responsible for the reduced T3 affinity of v-Erb A to a single amino-acid substitution, K231N, located at the end of helix 3.…”

“…The loss of the C-terminal c-Erb A helix 12 domain from v-Erb A has been proposed to be the major structural basis behind the loss of transcriptional activation by the viral oncoprotein (Munoz et al, 1988;Damm et al, 1989;Sap et al, 1989;Zenke et al, 1990;Saatcioglu et al, 1997). Consistent with this proposal, a c-Erb A protein bearing the v-Erb A Cterminal truncation (CCCV) retained the ability to repress reporter gene expression, but was unable to derepress or activate target genes in response to T3 and functioned as a dominant-negative inhibitor when coexpressed with wild-type c-Erb A over a range of T3 concentrations (Figure 8).…”

“…The C-terminal deletion in v-Erb A removes helix 12 of c-Erb A, resulting in constitutive corepressor binding by v-Erb A and a constitutive transcriptional repression phenotype (Munoz et al, 1988;Zenke et al, 1988;Damm et al, 1989;Sap et al, 1989). As a consequence, v-Erb A can function as a dominant-negative inhibitor of c-Erb A function, and v-Erb A has been proposed to operate in the erythroleukemic cell by interfering with thyroid hormone signaling (Damm et al, 1989;Evans, 1989;Sap et al, 1989;Schroeder et al, 1992b;Samarut, 1996;Bauer et al, 1998;Stunnenberg et al, 1999;Thormeyer and Baniahmad, 1999;Urnov et al, 2000;Rietveld et al, 2001).…”