Characterization of interaction between nuclear T3 receptors and antiserum against cellular-erb A peptide.

In our previous study, we raised a polyclonal antiserum against a synthetic 15 amino acid peptide encoding the putative hormone binding domain of c-erb A, which not only inhibited T3-binding to rat liver nuclear T3 receptor (NT3R) but also immunoprecipitated [125I]T3-NT3R complex. In the present study, interactions between this antiserum (4 BII-immunoglobulin G (IgG] and NT3R were further characterized. 4BII-IgG interefered T3 from binding to rat liver NT3R both in the nuclear extract and in isolated nuclei in a similar manner. Preincubation with 4BII-IgG decreased the association constant value with no effect on maximal binding capacity of NT3R. Lineweaver-Burk plot revealed competitive inhibition of T3-NT3R binding. Both the inhibition of T3 binding and immunoprecipitation of NT3R by the antiserum were similarly observed with rat liver, kidney, brain, and testis, and human kidney nuclear extract. Since the polypeptide sequence which was used for immunization is highly homologous between c-erb A alpha 1 and beta, it is likely that 4BII-IgG recognizes both c-erb A alpha 1 and beta in various tissues. On the contrary, 4BII-IgG did not interfere with T3 binding to rat liver cytosol T3-binding protein and thyroxine-binding globulin (TBG) prepared from human serum, nor immunoprecipitated these binding proteins labeled with [125I]T3. The binding protein for reverse T3 (NrT3BP) in the rat liver nuclear extract was neither immunoprecipitated nor influenced in its rT3-binding activity by 4BII-IgG. When the immune complex between the nuclear extract and 4BII-IgG was removed by antirabbit IgG goat serum, the remaining T3-binding activity was reduced by 87%, while no rT3-binding activity was immunodepleted, suggesting that NrT3BP is a protein different from NT3R. The data show that the antiserum against c-erb A peptide recognizes NT3R specifically with no distinction between difference in tissues and species. Carboxyl-terminal region of c-erb A/NT3R is critical for T3-binding.