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

In our first report, rabbit antibodies directed to recombinant polypeptides of human α-type c-ErbA sequences recognized natural triiodothyronine (T 3 ) 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 T 3 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-α 1 403-410 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 T 3 before antibody addition. This evokes a different conformation of this region after hormone binding. Most interestingly, these anti-α 172-191 antibodies lowered the K a for T 3 and extensively dissociated the adipocyte T 3 -TR complexes; they interfered poorly with the binding of T 3 in liver nuclear extracts. This strongly supports the concept that internal sequences in c-ErbA α, more precisely in a restricted C-terminal part of the D domain, are necessary for efficient T 3 binding, which also need the C-terminal part of domain E