Photoaffinity labeling of thyroid hormone nuclear receptors. Influence of n-butyrate and analysis of the half-lives of the 57,000 and 47,000 molecular weight receptor forms.
The thyroid hormone receptor is a nuclear-associated protein which appears to mediate the actions of 3,5,3'-triiodo-L-thyronine (L-T3) and 3,5,3',5'-tetraiodo-L-thyronine (L-T4) in mammalian cells. In a previous study we reported that N-2-diazo-3,3,3-trifluoropropionyl-3,5,3'-triiodo-L-thyronine (L-T3-PAL) serves as an effective photoaffinity label probe of the receptor in GH1 cells, a growth hormone producting rat pituitary cell line. Irradiation of cells at 254 nm covalently cross-links L-[125I]T3-PAL to two molecular weight (Mr) nuclear receptor forms, an abundant 47,000 Mr component and a less abundant 57,000 Mr species (Pascual, A., Casanova, J., and Samuels, H. H. (1982) J. Biol. Chem. 257, 9640-9647). In this study we have explored a number of possible interrelationships of the different Mr receptor forms. Denaturing gel electrophoresis and autoradiography indicates that the 57,000 Mr form is a doublet species which differ in Mr by 1,000 to 2,000. The various receptor forms are not an artifact of the L-[125I]T3-PAL probe, and identical forms can be labeled at 310 nm using underivatized L-[125I]T4 with a 15-fold lower coupling efficiency. The 57,000 and 47,000 Mr receptor forms are not generated by indiscriminate proteolysis, UV peptide cleavage, or zero length protein-protein cross-linking by irradiation at 254 nm. Micrococcal nuclease excises both the 57,000 and 47,000 Mr forms, and receptor is not identified in the residual nuclear matrix fraction. Receptor is also not detected in the cytoplasmic fraction. By coupling dense amino acid labeling and photoaffinity labeling of receptor we determined a half-life of 2.4 h for the 57,000 Mr species and 5.6 h for the 47,000 Mr form while both species have similar relative synthetic rates. n-Butyrate has been previously shown to decrease receptor levels in GH1 cells. We demonstrate that n-butyrate decreases receptor levels primarily by shortening the half-life of the 47,000 Mr form.
D000345:Affinity Labels / Q000378:metabolism; D000818:Animals; D002087:Butyrates / Q000494:pharmacology*; D020148:Butyric Acid; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D003979:Diazonium Compounds / Q000378:metabolism; D013006:Growth Hormone / Q000494:pharmacology; D006207:Half-Life; D008836:Micrococcal Nuclease / Q000378:metabolism; D008970:Molecular Weight; D010777:Photochemistry; D010911:Pituitary Neoplasms / Q000032:analysis; D051381:Rats; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011988:Receptors, Thyroid Hormone; D014284:Triiodothyronine / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology
D000345:Affinity Labels; D002087:Butyrates; D003979:Diazonium Compounds; D011956:Receptors, Cell Surface; D011988:Receptors, Thyroid Hormone; D014284:Triiodothyronine; D020148:Butyric Acid; C035514:N-2-diazo-3,3,3-trifluoropropionyl-3,5,3'-triiodothyronine; D013006:Growth Hormone; D008836:Micrococcal Nuclease