Antibodies against restricted sequences in human c-ErbA hinge domain recognize differentially natural mammalian α- or β-type triiodothyronine receptors and interfere differently with hormone binding

Abstract

Bismuth J, Lenoir C, Teboul M, Daadi M, Giorgilli G, Bonne J, Macchia E, Planells R, Torresani J. Antibodies against restricted sequences in human c-ErbA hinge domain recognize differentially natural mammalian α- or β-type triiodothyronine receptors and interfere differently with hormone binding. Eur J Endocrinol 1995;132:347–56. ISSN 0804–4643 In our first report, rabbit antibodies directed to recombinant polypeptides of human α-type c-ErbA sequences recognized natural triiodothyronine (T3) receptors (TR) in adipocytes (mouse Ob 17 cell line) but not in liver (mouse, rat). Moreover, some of them, directed to the sequence 150–228, markedly interfered with hormone binding to adipocyte T3 receptors. We now raised antibodies against shorter synthetic peptides within this α-type 150–228 c-ErbA sequence, which encompasses part of the hinge (D) domain and N-terminus of the E domain (α-150–166 and α 172–191) and against a β-type c-ErbA sequence (β 204–220 aligned on α 150–166, and differing by eight amino acids). Our present antibodies, which bear the expected c-ErbA α- or β-type specificity, immunoprecipitated the TR in nuclear extracts, with a different pattern between tissues: exclusive precipitation by anti-c-ErbA α antibodies in Ob 17 adipocytes; large but non-exclusive precipitation by anti-cErbA β antibodies in rat or mouse liver, which also expresses some α-type TR. This pattern of discriminative immunoprecipitation, also obtained in parallel analysis using our previously described antibodies to other c-ErbA α or β sequences (anti-α 144–162, anti-α1 403–410 and anti-β 62–82), roughly verifies results of c-erbA mRNA expression in these tissues. Slight differences appeared in the extent of α-type TR recognition by antibodies directed to α 172–191, whether TR were liganded or not to T3 before antibody addition. This evokes a different conformation of this region after hormone binding. Most interestingly, these anti-α 172–191 antibodies lowered the Ka for T3 and extensively dissociated the adipocyte T3–TR complexes; they interfered poorly with the binding of T3 in liver nuclear extracts. This strongly supports the concept that internal sequences in c-ErbA α, more precisely in a restricted Cterminal part of the D domain, are necessary for efficient T3 binding, which also need the C-terminal part of domain E. J. Torresani, INSERM U38, Biochimie Méducake, Faculté de Médecine, 27 Bd J Moulin, 13385 Marseille Cédex 5, France