ANTIBODIES DIRECTED TO RESTRICTED SEQUENCES OF THE C-ERB-A-ALPHA HINGE DOMAIN INTERFERE WITH HORMONE OR DNA-BINDING TO RECOMBINANT ALPHA-TYPE TRIIODOTHYRONINE RECEPTOR (C-ERB-A-ALPHA-1) AND DETECT STRUCTURAL-CHANGES

In a previous study we showed that antibodies directed to restricted s equences in the hinge domain of alpha-type triiodothyronine (T3) recep tor (TR) (aminoacids 150-166, 172-191, 144-162) selectively recognized and immunoprecipitated alpha-type TR in tissues. Furthermore, antibod ies to peptide 172-191 (anti-alpha 172) strongly impaired T3 binding t o natural TR alpha. To get more precise informations on the ability of these antibodies to recognize the TR, alter TR properties and/or dete ct conformational changes in TR, TR alpha 1 was produced in E. coli as a non-mutated, non-fusion protein from a rat c-erb A alpha 1 cDNA ins erted into pTrc99A vector. The recombinant cErb A alpha 1 protein, aft er solubilization with 5 M guanidine and progressive refolding, presen ted the main characteristics of TR alpha: unique or largely dominant b and of 46 KDa in Western blots with the different anti-c-Erb A alpha a ntibodies;binding to DNA and to T3. Binding to DNA was markedly attenu ated by anti-alpha 144 but not by anti-alpha 150 and anti-alpha 172. B inding to T3 was modified by anti-alpha 150 and -alpha 172 with differ ent characteristics whether recombinant TR was previously bound to T3 or not, and with marked differences in comparative studies with natura l TR. When liganded to T3, recombinant and natural TR alpha 1 present the same pattern of interaction with both antibodies : immunoprecipita tion without any dissociation of T3 by anti-alpha 150;marked dissociat ion of bound T3 by anti-alpha 172. By contrast unliganded recombinant and natural TR are oppositely altered by these antibodies in their abi lity to bind T3 : strong impairment restricted to anti-alpha 172 for n atural TR, and to anti-alpha 150 for recombinant TR. Anti-alpha 144 di d not interfere. These results lay emphasis on : II the existence and biological relevance of different conformational states of TR alpha hi nge domain, particularly whether TR is liganded or not to T3 and wheth er it is in a nuclear environment or bacterially-produced; 2/ an impor tant role of the C-terminal part of hinge domain for efficient hormone binding, this involving a region that overlaps the a 150 and alpha 17 2 sequences.