Presence of functional domains in NADPH-dependent cytosolic 3,5,3'-Triiodo-L-thyronine (T3)-binding protein (p38CTBP) molecule: analyses with deletion mutants.
Functional domains required for NADPH-binding, T(3)-binding, protein dimerization and cytosolic retention were analyzed in NADPH-dependent cytosolic 3,5,3'-triiodothyronine (T(3))-binding protein (p38CTBP) by using the deletion mutants. Wild-type p38CTBP (amino acids; 1-314) and a series of deletion mutants (amino acids; 1-79, 1-128, 1-146, 1-216, 37-314, and 1-145 with 270-314) were bacterially induced. NADPH-dependent T(3)-binding activity was not observed in all mutant p38CTBPs studied, although wild-type p38CTBP showed high-affinity T(3)-binding activity. Wild-type p38CTBP was able to bind a CL-6B column, none of the mutant p38CTBPs showed any binding activity. Pull-down analyses demonstrated that two regions between amino acid 128 and 146, and between 216 and 270, both of which possess helical structures, were required for homodimeric p38CTBP binding. In fluoroscopic studies, GFP-tagged p38CTBP was preferentially observed in cytoplasm. However, C-terminal region-deleted p38CTBP(1-216) was not only observed in cytoplasm, but also in nucleus. These results suggest that 1) multiple regions in p38CTBP molecule are required for T(3)-binding and NADPH binding, 2) two helical structures in p38CTBP molecule may be important in the homodimer formation, and 3) C-terminal region of p38CTBP contains the function to preserve the protein in cytoplasm.
Suzuki
S
S
Department of Aging Medicine and Geriatrics, Institute on Aging and Adaptation, Shinshu University Graduate School, Matsumoto, Nagano, Japan. soutaro@hsp.md.shinshu-u.ac.jp
Mori
J-I
JI
Kobayashi
M
M
Inagaki
T
T
Komatsu
A
A
Yamashita
K
K
Takeda
T
T
Miyamoto
T
T
Ichikawa
K
K
Hashizume
K
K
D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D016466:CHO Cells; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D006224:Cricetinae; D003600:Cytosol / Q000737:chemistry; D019281:Dimerization; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D016296:Mutagenesis; D009249:NADP / Q000378:metabolism; D011993:Recombinant Fusion Proteins; D013329:Structure-Activity Relationship; D013963:Thyroid Hormones / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D014284:Triiodothyronine / Q000378:metabolism; D000097810:Thyroid Hormone-Binding Proteins
D002352:Carrier Proteins; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D008565:Membrane Proteins; D009249:NADP; D011993:Recombinant Fusion Proteins; D013963:Thyroid Hormones; D014284:Triiodothyronine; D000097810:Thyroid Hormone-Binding Proteins