Structural Similarities between the Plasma Membrane Binding Sites for L-Thyroxine and 3,3′,5-Triiodo-L- Thyronine in Cultured Cells

Abstract

Using ¹²⁵I-labeled L-thyroxine (T₄), the binding of [¹²⁵I]T4 to GH₃ rat pituitary tumor cells was studied. At 15^±C, the binding of [¹²⁵I]T₄ to cells is saturable and specific. Least squares analysis of binding data showed two classes of binding sites with apparent dissociation constants of 4.3 ± 0.3 nM and 350 ± 30nM and binding capacities of (3.8 ± 0.5) × 10⁴ and (9.1 ± 0.35) × 10⁶ sites/cell, respectively. Affinity labeling of cells or purified plasma membranes with N-bromoacetyl-[¹²⁵I]T₄ (BrAc[¹²⁵I]T₄ showed ±a major specifically labeled protein band with an apparent molecular mass of 55 kilodaltons (kDal). Digestion of the 55-kDal protein from cells and plasma membrane by Staphylococcus aureus V8 protease or elastase gave similar peptide fragments. Thus, the 55-kDal protein labeled from intact cells is the same protein as that from purified plasma membranes. Peptide mapping was further used to compare the 55-kDal protein specifically labeled by either N-bromoacetyl-3, [¹²⁵I]3′,5-triiodo-L-thyronine (BrAc[¹²⁵I]T₃) or BrAc[¹²⁵I]T₄ in intact cells and highly purified plasma membranes. Very similar patterns were obtained. These results indicate that plasma membrane T₃ and T₄ binding sites have similar hormone binding domains. In addition the plasma membrane T₃ and T₄ binding sites of Swiss 3T3-4 mouse fibroblasts and A431 human epithelioid carcinoma cells are structurally similar to the T₃ and T₄ binding sites of GH₃ cells.