Leucine at codon 428 in the ninth heptad of thyroid hormone receptor beta1 is necessary for interactions with the transcriptional cofactors and functions regardless of dimer formations.
Structure/function studies of the thyroid hormone receptor (TR) beta(1) have demonstrated that single amino acid substitutions in either position 428 or 429 in the ligand-binding domain (LBD) can alter heterodimerizations and homodimerizations, respectively. A leucine at 428 is located in a highly conserved region corresponding to the putative ninth heptad repeat of a leucine-zipper-like motif in the LBD of TRbeta(1). To investigate how the side chain of amino acids at 428 affect receptor characteristics, gel-shift mobility shift assays and yeast two-hybrid assays were analyzed. The neutral status amino acids such as a leucine (wild-type) or a glutamine at 428 preferred heterodimerization with RXR. Furthermore, a positively charged side chain of amino acids at 428 such as an arginine or a lysine, preserved homodimer formation. Irrespective of charge, ninth heptad mutant receptors did not bind the ligand and were not able to interact with either corepressor or coactivating proteins. Limited trypsinization assays revealed no major conformational change in the ninth heptad mutant receptors. Together, these findings suggested that a leucine at 428 was a critical amino acid for both interaction with the thyroid hormone receptor associated proteins and ligand-independent and -dependent functions regardless of dimer formations.
Monden
Tsuyoshi
T
First Department of Internal Medicine, Gunma University School of Medicine, Maebashi, Japan. tmonden@showa.gunma-u.ac.jp
Yamada
Masanobu
M
Ishii
Sumiyasu
S
Hosoya
Takeshi
T
Satoh
Teturo
T
Wondisford
Fredric E
FE
Hollenberg
Anthony N
AN
Mori
Masatomo
M
D019943:Amino Acid Substitution; D000596:Amino Acids / Q000737:chemistry / Q000235:genetics; D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D003062:Codon / Q000235:genetics*; D019281:Dimerization; D024202:Electrophoretic Mobility Shift Assay; D007930:Leucine / Q000737:chemistry / Q000235:genetics*; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism; D056971:Nuclear Receptor Co-Repressor 1; D011487:Protein Conformation; D018168:Receptors, Retinoic Acid / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism; D020218:Response Elements / Q000235:genetics; D047488:Retinoid X Receptors; D012441:Saccharomyces cerevisiae / Q000235:genetics; D037042:Thyroid Hormone Receptors beta / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D014162:Transfection; D020798:Two-Hybrid System Techniques
D000596:Amino Acids; D003062:Codon; D009687:Nuclear Proteins; D056971:Nuclear Receptor Co-Repressor 1; D018168:Receptors, Retinoic Acid; D011994:Recombinant Proteins; D012097:Repressor Proteins; D047488:Retinoid X Receptors; D037042:Thyroid Hormone Receptors beta; D014157:Transcription Factors; D007930:Leucine