Characterization of Binding and Uptake of 3,3′,5-Triido-L-Thyronine in Cultured Mouse Fibroblasts

Abstract

The binding and internalization of 3,3′-[¹²⁵I]5- triiodo-L-thyronine ([¹²⁵I]T₃) was studied in cultured Swiss 3T3- 4 mouse fibroblasts. At 0 C, the binding of T₃ to cells is saturable, reversible, and stereospecific. These results together with those of earlier fluorescence studies using rhodamine-labeled T₃ demonstrate the presence of specific plasma membrane T₃ receptors. At 37 C, the uptake of T₃ reached a steady state after 1 h, and approximately 57 fmol T₃ were specifically taken up by 10⁶ cells. In other cell lines, 7,19, and 201 fmol T₃ were specifically taken up by Chinese hamster ovary cells (subclone 10001), Kirsten sarcoma virus-transformed NIH 3T3 mouse fibroblasts, and nontransformed NIH 3T3 mouse fibroblasts, respectively. Incorporation of T₃ into nuclei followed similar kinetics and accounted for approximately 9% of the total cellular uptake. Equilibrium binding studies of T₃ to isolated nuclei showed one class of binding sites with an apparent association constant of 5 × 10⁹ M^-1 and a binding capacity of 16 fmol/100 μg DNA. At 37 C, the internalization of T₃ was nearly totally blocked by antimycin A or rotenone, inhibitors of oxidative phosphorylation. These results indicate that the uptake of T₃ is an energy-dependent process. In the presence of bacitracin or monodansylcadaverine, substances that inhibit the receptor-mediated endocytosis of α₂- macroglobulin, the cellular uptake of T₃ as well as the nuclear incorporation of T₃ were inhibited in a concentration-dependent manner. The half-maximal inhibitory concentrations for the cellular uptake of T₃ were 90 and 660 μM for monodansylcadaverine and bacitracin, respectively; for nuclear incorporation, they were 70 and 350 μM for monodansylcadaverine and bacitracin, respectively. These results indicate that receptor-mediated endocytotic uptake of T₃ is a physiologically significant pathway.