Presence of Functional Domains in NADPH-dependent Cytosolic 3,5,3'-Triiodo-L-thyronine (T₃)-binding Protein (p38CTBP) Molecule: Analyses with Deletion Mutants

Abstract

Functional domains required for NADPH-binding, T₃-binding, protein dimerization and cytosolic retention were analyzed in NADPH-dependent cytosolic 3,5,3′-triiodothyronine (T₃)-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 T₃-binding activity was not observed in all mutant p38CTBPs studied, although wild-type p38CTBP showed high-affinity T₃-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 T₃-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.

Crystallin - Homodimer - Thyroid hormone-binding protein

References

• 1 Larsen P R, Davis T F, Hay I D. The thyroid gland. In: Wilson JD, Foster DW, Kronenberg HM, Larsen PR (eds) Williams Textbook of Endocrinology,. Philadelphia; Saunders WB 1998: 389-515
• 2 Hashizume K, Kobayashi M, Miyamoto T. Active and inactive forms of 3,5,3’-triiodo-L-thyronine (T₃)-binding protein in rat kidney cytosol: possible role of nicotinamide adenine dinucleotide phosphate in activation of T₃ binding.  Endocrinology. 1986;  119 710-719
• 3 Hashizume K, Miyamoto T, Ichikawa K, Yamauchi K, Kobayashi M, Sakurai A, Ohtsuka H, Nishii Y, Yamada T. Purification and characterization of NADPH-dependent cytosolic 3,5,3’-triiodo-L-thyronine binding protein in rat kidney.  J Biol Chem. 1989;  264 4857-4863
• 4 Suzuki S, Hashizume K, Ichikawa K, Takeda T. Ontogenesis of the high affinity NADPH-dependent cytosolic 3,5,3’-triiodo-L-thyronine-binding protein in rat.  Endocrinology. 1991;  129 2571-2574
• 5 Kobayashi M, Hashizume K, Suzuki S, Ichikawa K, Takeda T. A novel NADPH-dependent cytosolic 3,5,3’-triiodo-L-thyronine-binding protein (CTBP;5.1S) in rat liver: a comparison with 4.7S NADPH-dependent CTBP.  Endocrinology. 1991;  129 1701-1708
• 6 Vie M P, Evrard C, Osty J, Breton-Gilet A, Blanchet P, Pomerance M, Rouget P, Francon J, Blondeau J P. Purification, molecular cloning, and functional expression of the human nicodinamide-adenine dinucleotide phosphate-regulated thyroid hormone-binding protein.  Mol Endocrinol. 1997;  11 1728-1736
• 7 Wistow G, Kim H. Lens protein expression in mammals: Taxon-specificity and the recruitment of crystallins.  J Mol Evol. 1991;  32 262-269
• 8 Kim R Y, Gasser R, Wistow G J. µ-crystallin is a mammalian homologue of Agrobacterium ornithine cyclodeaminase and is expressed in human retina.  Proc Natl Acad Sci USA. 1992;  89 9292-9296
• 9 Mori J, Suzuki S, Kobayashi M, Inagaki T, Komatsu A, Takeda T, Miyamoto T, Ichikawa K, Hashizume K. Nicotinamide adenine dinucleotide phosphate-dependent cytosolic T₃ binding protein as a regulator for T₃-mediated transactivation.  Endocrinology. 2002;  143 1538-1544
• 10 Suzuki S, Mori J-I, Kobayashi M, Inagaki T, Inaba H, Komatsu A, Yamashita K, Takeda T, Miyamoto T, Ichikawa K, Hashizume K. Cell specific expression of NADPH-dependent cytosolic 3,5,3’-triiodo-L-thyronine (T₃) binding protein (p38CTBP).  Eur J Endocrinol. 2003;  148 259-268
• 11 Abe S, Katagiri T, Saito-Hisaminato A, Usami S, Inoue Y, Tsunoda T, Nakamura Y. Identification of CRYM as a candidate responsible for nonsyndromic deafness, through cDNA microarray analysis of human cochlear and vestibular tissues.  Am J Hum Genet. 2003;  72 73-82
• 12 Studer F W, Moffatt B A. Use of bacteriophage T₇ RNA polymerase to direct selective high-level expression of cloned genes.  J Mol Biol. 1986;  189 113-130
• 13 Chou P Y, Fasman G D. Conformational parameters for amino acids in helical, beta-sheet, and random coil regions calculated from proteins.  Biochemistry. 1974;  13 211-222
• 14 Garnier J, Osquthorpe D J, Robson B. Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins.  J Mol Biol. 1978;  120 97-120
• 15 Scatchard G. The attractions of proteins for small molecules and ions.  Ann NY Acad Sci. 1949;  51 660-672
• 16 Bradford M M. A rapid and sensitive method for the quantitation of micogram quantities of protein utilizing the principle of protein-dye binding.  Anal Biochem. 1976;  72 248-254
• 17 Kakizawa T, Miyamoto T, Ichikawa K, Kaneko A, Suzuki S, Hara M, Nagasawa T, Takeda T, Mori J, Kumagai M, Hashizume K. Functional interaction between Oct-1 and retinoid X receptor. J. Biol.  Chem.. 1999;  274 19 103-19 108
• 18 Sambrook J, Fritsch E F, Maniatis T. A laboratory manual, Second edition. New York; Cold Spring Harbor Laboratory Press 1989: 16.33-16.36
• 19 Hashizume K, Miyamoto T, Ichikawa K, Yamauchi K, Sakurai A, Ohtsuka H, Kobayashi M, Nishii Y, Yamada T. Evidence for the presence of two active forms of cytosolic 3,5,3’-triiodo-L-thyronine(T₃)-binding protein (CTBP) in rat kidney. Specialized functions of two CTBPs in intracellular T₃ translocation.  J Biol Chem. 1989;  264 4864-4871
• 20 Yen P M, Darling D S, Carter R L, Forgione M, Umeda P K, Chin W W. Triiodothyronine (T₃) decreases binding to DNA by T₃-receptor homodimers but not receptor-auxiliary protein heterodimers.  J Biol Chem. 1992;  267 3565-3568
• 21 Miyamoto T, Suzuki S, DeGroot L J. High affinity and specificity of dimeric binding of thyroid hormone receptors to DNA and their ligand-dependent dissociation.  Mol Endocrinol. 1993;  7 224-231
• 22 Lazar M A. Thyroid hormone receptors: Multiple forms, multiple possibilities.  Endocr Rev. 1993;  14 184-193