ESTIMATION OF THE PROTEIN-CONTENT OF THYROID-HORMONE RECEPTOR ALPHA-1 AND BETA-1 IN RAT-TISSUES BY WESTERN BLOTTING

Recent studies of the expression of c-erbA/thyroid hormone receptor (TR) mRNAs have revealed a dissociation between T3-binding activity and the behavior of the mRNAs that code the functional TRs in some tissues. Compared with T3-binding activity, TR(alpha1 + beta1) mRNA is disproportionally high in the brain and low in the liver. Using anti-TR antiserum, 4BII, which recognizes TRalpha1 and beta1, but not the alpha2-variant, we measured TR protein content in rat tissues by Western blotting. Two protein bands of 47 and 55 kilodaltons (kDa) were specifically identified as TR proteins. The positions of the in vitro transcription/translation products of c-erbA/TRalpha1 and beta1 cDNA on the gel were consistent with those of the 47- and 55-kDa bands, respectively. The 47- and 55-kDa proteins in nuclear proteins extracted with 0.4 m KCl from rat tissues were analyzed by Western blotting, and the intensity of TR protein bands in each tissue was measured by a densitometer. The relative TR protein concentration was highest in liver, followed by brain, kidney, and testis. We compared the TR protein level measured by Western blotting with the maximal T3-binding capacity (C(max)) in the same aliquot of samples from liver and brain. Both the TR protein level and the C(max) in the brain were about 40% of those in the liver, suggesting that the C(max) per receptor molecule is constant in these two tissues, and an abundant amount of functional TR proteins exists in the liver, corresponding to the high level of T3-binding activity.