Oligomeric binding of T3 receptor is required for maximal T3 response.
Receptors in the thyroid-steroid hormone superfamily bind preferentially as dimers to palindromic response elements containing two hexameric half-sites. The 23-base pair rat growth hormone (rGH) T3 response element (T3RE), however, contains three hexameric binding domains, all of which are required for maximal T3 response. We examined the binding of purified T3 receptor alpha (T3R alpha), overexpressed in Escherichia coli, to wild-type and up and down mutations of the rGH T3RE to evaluate whether transcriptional potency correlates with changes in T3R binding. T3R binds to the rGH T3RE as a monomer, dimer, or higher order oligomer. Cooperative T3R dimer binding was demonstrated to two hexameric domains of the rGH T3RE arranged as either direct or inverted repeats. Decreased binding was seen with point mutations in each domain as well as with mutations which altered hexamer orientation and spacing within the site. These results demonstrate that all three hexamers of the rGH T3RE are involved in binding T3R. Occupancy of all three hexamers by T3R in the gel shift assay was observed with functional up mutations of the T3RE, increasing receptor concentration or addition of nuclear extract. The transcriptional response potencies of T3RE up or down mutants in a transient transfection assay correlated closely with T3R binding. These results confirm our earlier hypothesis that all three hexamers of the rGH T3RE bind T3R in a novel binding arrangement and provide a model for the interaction of T3R and other nuclear proteins with the DNA sequences of thyroid hormone-regulated genes.
Williams
G R
GR
Thyroid Division, Brigham and Women's Hospital, Boston, Massachusetts 02115.
Harney
J W
JW
Forman
B M
BM
Samuels
H H
HH
Brent
G A
GA
D000818:Animals; D001665:Binding Sites; D002645:Chickens; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D006133:Growth Substances / Q000235:genetics / Q000378:metabolism; D008297:Male; D009154:Mutation; D010957:Plasmids; D051381:Rats; D011919:Rats, Inbred Strains; D011988:Receptors, Thyroid Hormone / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D006133:Growth Substances; D011988:Receptors, Thyroid Hormone