In a previous study we showed that antibodies directed to restricted sequences in the hinge domain of alpha-type triiodothyronine (T3) receptor (TR) (aminoacids 150-166, 172-191, 144-162) selectively recognized and immunoprecipitated alpha-type TR in tissues. Furthermore, antibodies to peptide 172-191 (anti-alpha 172) strongly impaired T3 binding to natural TR alpha. To get more precise informations on the ability of these antibodies to recognize the TR, alter TR properties and/or detect 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 inserted into pTrc99A vector. The recombinant cErb A alpha 1 protein, after solubilization with 5 M guanidine and progressive refolding, presented the main characteristics of TR alpha: unique or largely dominant band of 46 KDa in Western blots with the different anti-c-Erb A alpha antibodies;binding to DNA and to T3. Binding to DNA was markedly attenuated by anti-alpha 144 but not by anti-alpha 150 and anti-alpha 172. Binding to T3 was modified by anti-alpha 150 and -alpha 172 with different characteristics whether recombinant TR was previously bound to T3or not, and with marked differences in comparative studies with natural TR. When liganded to T3, recombinant and natural TR alpha 1 presentthe same pattern of interaction with both antibodies : immunoprecipitation without any dissociation of T3 by anti-alpha 150;marked dissociation of bound T3 by anti-alpha 172. By contrast unliganded recombinantand natural TR are oppositely altered by these antibodies in their ability to bind T3 : strong impairment restricted to anti-alpha 172 for natural TR, and to anti-alpha 150 for recombinant TR. Anti-alpha 144 did not interfere. These results lay emphasis on : II the existence andbiological relevance of different conformational states of TR alpha hinge domain, particularly whether TR is liganded or not to T3 and whether it is in a nuclear environment or bacterially-produced; 2/ an important role of the C-terminal part of hinge domain for efficient hormone binding, this involving a region that overlaps the a 150 and alpha 172 sequences.
Antibodies directed to restricted sequences of the c-erb a α hinge domain interfere with hormone or dna binding to recombinant αtype triiodothyronine receptor (c-erb a α1) and detect structural changes
GIORGILLI, GIANFRANCO;MACCHIA, ENRICO;
1995-01-01
Abstract
In a previous study we showed that antibodies directed to restricted sequences in the hinge domain of alpha-type triiodothyronine (T3) receptor (TR) (aminoacids 150-166, 172-191, 144-162) selectively recognized and immunoprecipitated alpha-type TR in tissues. Furthermore, antibodies to peptide 172-191 (anti-alpha 172) strongly impaired T3 binding to natural TR alpha. To get more precise informations on the ability of these antibodies to recognize the TR, alter TR properties and/or detect 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 inserted into pTrc99A vector. The recombinant cErb A alpha 1 protein, after solubilization with 5 M guanidine and progressive refolding, presented the main characteristics of TR alpha: unique or largely dominant band of 46 KDa in Western blots with the different anti-c-Erb A alpha antibodies;binding to DNA and to T3. Binding to DNA was markedly attenuated by anti-alpha 144 but not by anti-alpha 150 and anti-alpha 172. Binding to T3 was modified by anti-alpha 150 and -alpha 172 with different characteristics whether recombinant TR was previously bound to T3or not, and with marked differences in comparative studies with natural TR. When liganded to T3, recombinant and natural TR alpha 1 presentthe same pattern of interaction with both antibodies : immunoprecipitation without any dissociation of T3 by anti-alpha 150;marked dissociation of bound T3 by anti-alpha 172. By contrast unliganded recombinantand natural TR are oppositely altered by these antibodies in their ability to bind T3 : strong impairment restricted to anti-alpha 172 for natural TR, and to anti-alpha 150 for recombinant TR. Anti-alpha 144 did not interfere. These results lay emphasis on : II the existence andbiological relevance of different conformational states of TR alpha hinge domain, particularly whether TR is liganded or not to T3 and whether it is in a nuclear environment or bacterially-produced; 2/ an important role of the C-terminal part of hinge domain for efficient hormone binding, this involving a region that overlaps the a 150 and alpha 172 sequences.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
