ANTIBODIES AGAINST RESTRICTED SEQUENCES IN HUMAN C-ERBA HINGE DOMAIN RECOGNIZE DIFFERENTIALLY NATURAL MAMMALIAN ALPHA-TYPE OR BETA-TYPE TRIIODOTHYRONINE RECEPTORS AND INTERFERE DIFFERENTLY WITH HORMONE-BINDING

In our first report, rabbit antibodies directed to recombinant polypep tides of human alpha-type c-ErbA sequences recognized natural triiodot hyronine (T-3) receptors (TR) in adipocytes (mouse Ob 17 cell line) bu t not in liver (mouse, rat). Moreover, some of them, directed to the s equence 150-228, markedly interfered with hormone binding to adipocyte T-3 receptors. We now raised antibodies against shorter synthetic pep tides within this alpha-type 150-228 c-ErbA sequence, which encompasse s part of the hinge (D) domain and N-terminus of the E domain (alpha-1 50-166 and alpha 172-191) and against a beta-type c-ErbA sequence (bet a 204-220 aligned on alpha 150-166, and differing by eight amino acids ). Our present antibodies, which bear the expected c-ErbA alpha- or be ta-type specificity, immunoprecipitated the TR in nuclear extracts, wi th a different pattern between tissues: exclusive precipitation by ant i-c-ErbA alpha antibodies in Ob 17 adipocytes; large but non-exclusive precipitation by anti-cErbA beta antibodies in rat or mouse liver, wh ich also expresses some alpha-type TR. This pattern of discriminative immunoprecipitation, also obtained in parallel analysis using our prev iously described antibodies to other c-ErbA alpha or beta sequences (a nti-alpha 144-162, anti-alpha(1) 403-410 and anti-beta 62-82), roughly verifies results of c-erbA mRNA expression in these tissues. Slight d ifferences appeared in the extent of alpha-type TR recognition by anti bodies directed to alpha 172-191, whether TR were liganded or not to T -3 before antibody addition. This evokes a different conformation of t his region after hormone binding. Most interestingly, these anti-alpha 172-191 antibodies lowered the K-a for T-3 and extensively dissociate d the adipocyte T-3-TR complexes; they interfered poorly with the bind ing of T-3 in liver nuclear extracts. This strongly supports the conce pt that internal sequences in c-ErbA alpha, more precisely in a restri cted C-terminal part of the D domain, are necessary for efficient T-3 binding, which also need the C-terminal part of domain E.