Divergent dimerization properties of mutant beta 1 thyroid hormone receptors are associated with different dominant negative activities.
Syndromes of resistance to thyroid hormones are caused by mutations in the T3-binding domain of the c-erbA beta thyroid hormone receptor gene. The S receptor (deletion of THR332) is a potent dominant negative protein cloned from a kindred with generalized resistance to thyroid hormones. The G-H receptor (ARG311HIS) has compromised dominant negative function and was found in both normal individuals and in a patient with severe pituitary resistance to thyroid hormones. We have investigated the mechanism responsible for the difference in receptor phenotypes by analyzing the binding of S and G-H receptors to thyroid hormone response elements with electrophoretic mobility shift analysis. Wild-type human c-erbA beta 1 (WT), S, and G-H receptors were synthesized in reticulocyte lysate, reacted with a thyroid hormone response element consisting of a direct repeat with 4 base pairs (DR+4; AGGTCA CAGG AGGTCA), and the products analyzed by gel shift. G-H receptor homodimerization was greatly impaired; G-H formed predominantly monomeric complex compared with monomeric and homodimeric WT complexes. The G-H receptor was able to form heterodimeric complexes with cellular thyroid hormone receptor auxiliary protein (TRAP) factors including the human retinoid X receptor-alpha. When TRAP was limiting, the levels of G-H heterodimeric complex were 2- to 3-fold reduced compared with WT receptor. In contrast to the WT and G-H receptors, the S receptor formed almost exclusively homodimeric complex with DR+4; the approximate ratio of S:WT:G-H homodimeric complexes at equivalent concentrations of receptors was 60:20:1. A measurable increase (1.2- to 2.6-fold) in heterodimeric complex formation was observed with the S receptor relative to WT when TRAP was at limiting concentration. As reported previously by others, thyroid hormone significantly reduced the WT homodimeric complex with DR+4. There was no effect on the S homodimeric complex. Finally, the WT, S, and G-H receptors formed different complexes with the element consisting of an inverted repeat with 5 base pairs (IR+5; AGGTCA ACAGT TGACCT) and the IR element (AGGTCA TGACCT), which were differently regulated by thyroid hormone. The S receptor bound as a homodimer with IR+5, whereas the WT receptor bound as a homodimer only with thyroid hormone. No homodimeric complex formed with IR+5 and the G-H receptor. Qualitatively similar results were observed with the IR element. We conclude that the ARG311HIS mutation severely perturbs the homodimerization and, to a much less degree, heterodimerization functions of the c-erbA beta 1 receptor. Furthermore, the THR332 deletion mutation augments homodimerization of the c-erbA beta 1 receptor. These results indicate that different mutations in the c-erbA beta 1 thyroid hormone receptor have divergently affected dimerization activities which seem to influence the level of dominant negative activity in man.
Hao
E
E
Department of Medicine, East Carolina University School of Medicine, Greenville, North Carolina 27858.
Menke
J B
JB
Smith
A M
AM
Jones
C
C
Geffner
M E
ME
Hershman
J M
JM
Wuerth
J P
JP
Samuels
H H
HH
Ways
D K
DK
Usala
S J
SJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002522:Chlorocebus aethiops; D004247:DNA / Q000378:metabolism; D005799:Genes, Dominant; D006801:Humans; D008969:Molecular Sequence Data; D017354:Point Mutation; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011988:Receptors, Thyroid Hormone / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014284:Triiodothyronine / Q000378:metabolism*
D011988:Receptors, Thyroid Hormone; D014284:Triiodothyronine; D004247:DNA