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

Eur J Endocrinol. 1995 Mar;132(3):347-56. doi: 10.1530/eje.0.1320347.

Abstract

In our first report, rabbit antibodies directed to recombinant polypeptides of human alpha-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 alpha-type 150-228 c-ErbA sequence, which encompasses part of the hinge (D) domain and N-terminus of the E domain (alpha-150-166 and alpha 172-191) and against a beta-type c-ErbA sequence (beta 204-220 aligned on alpha 150-166, and differing by eight amino acids). Our present antibodies, which bear the expected c-ErbA alpha- or beta-type specificity, immunoprecipitated the TR in nuclear extracts, with a different pattern between tissues: exclusive precipitation by anti-c-ErbA alpha antibodies in Ob 17 adipocytes; large but non-exclusive precipitation by anti-cErbA beta antibodies in rat or mouse liver, which also expresses some alpha-type TR. This pattern of discriminative immunoprecipitation, also obtained in parallel analysis using our previously described antibodies to other c-ErbA alpha or beta sequences (anti-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 differences appeared in the extent of alpha-type TR recognition by antibodies directed to alpha 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-alpha 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 alpha, more precisely in a restricted C-terminal part of the D domain, are necessary for efficient T3 binding, which also need the C-terminal part of domain E.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Antibody Specificity
  • Cell Nucleus / metabolism
  • Enzyme-Linked Immunosorbent Assay
  • Humans
  • In Vitro Techniques
  • Mice
  • Molecular Sequence Data
  • Peptide Fragments / immunology
  • Precipitin Tests
  • Rats
  • Receptors, Thyroid Hormone / chemistry
  • Receptors, Thyroid Hormone / immunology*
  • Receptors, Thyroid Hormone / metabolism
  • Triiodothyronine / metabolism*

Substances

  • Peptide Fragments
  • Receptors, Thyroid Hormone
  • Triiodothyronine