Quantitation of rat tissue thyroid hormone binding receptor isoforms by immunoprecipitation of nuclear triiodothyronine binding capacity.
A panel of anti-thyroid hormone receptor (TR) antisera were generated to allow direct assay of the concentrations of the alpha 1 and beta 1 receptor isoforms in nuclear extracts from adult rat liver, kidney, brain and heart, and fetal brain. An antiserum, immunoglobulin G (IgG)-beta 1, raised against amino acid sequence 62-92 of the rat TR-beta 1 specifically precipitated only TR-beta 1 in vitro translation products. A second antiserum, IgG-alpha 1/beta, generated against a sequence that is identical in the ligand binding region of rat TR-alpha 1 and TR-beta isoforms immunoprecipitated both TR-alpha 1 and -beta 1 translation products. These IgG preparations were used to specifically immunoprecipitate thyroid hormone receptor binding activity from nuclear extracts. IgG-beta 1 cleared almost 80%, and the IgG-alpha 1/beta immunoprecipitated nearly all binding from hepatic nuclear extracts. This distribution of TR protein, 80% beta 1 and 20% alpha 1, is the same as previously reported for their respective mRNAs in liver. In heart, kidney, and brain IgG-beta 1 cleared 45, 43, and 28% of total binding, respectively, and IgG-alpha 1/beta cleared all T3 binding activity from these tissues. In agreement with an earlier study, marked variations in specific protein/mRNA ratios were noted among these tissues. Consistent with our earlier report of the presence of only very low levels of TR-beta 1 mRNA in fetal brain, IgG-beta 1 cleared just 5% of binding in this tissue. Studies using an antiserum (IgG-ch) generated against homologous segments of the hinge region in both TR-alpha 1 and -beta 1 yielded results which contrasted sharply with those of IgG-alpha 1/beta. Whereas IgG-ch could also immunoprecipitate virtually all binding from hepatic extracts it cleared only 40-50% of binding from the other tissues, including fetal brain in which TR-alpha 1 accounts for greater than 90% of binding protein. The data suggest the presence of posttranslational modification of the TR-alpha 1 protein in the hinge region, consistent with the presence in this segment of potential phosphorylation sites.
Schwartz
H L
HL
Department of Medicine, University of Minnesota, Minneapolis 55455.
Strait
K A
KA
Ling
N C
NC
Oppenheimer
J H
JH
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D001921:Brain / Q000196:embryology / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism*; D005260:Female; D007106:Immune Sera; D007668:Kidney / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D009206:Myocardium / Q000378:metabolism; D010455:Peptides / Q000276:immunology; D011233:Precipitin Tests; D014176:Protein Biosynthesis; D051381:Rats; D011919:Rats, Inbred Strains; D011988:Receptors, Thyroid Hormone / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D014284:Triiodothyronine / Q000378:metabolism*
D000906:Antibodies; D007106:Immune Sera; D010455:Peptides; D011988:Receptors, Thyroid Hormone; D014284:Triiodothyronine