The identification of a plasma membrane 3,3',5-triiodo-L-thyronine binding protein on the cultured Swarm rat chondrosarcoma chondrocyte and the lack of its up-regulation by insulin in vitro.

Primary cultures of Swarm rat chondrosarcoma chondrocytes were examined for the presence of T3 plasma membrane binding proteins and their possible regulation by insulin. Incubation of this tumor cell with [125I]T3 at 4 C yielded saturable and reversible binding of the radioligand. As assessed by LIGAND computer analysis, the binding data in one experiment revealed two classes of [125I]T3 binding sites with association constants of 2.2 X 10(9) M-1 and 4.8 X 10(6) M-1, and binding capacities of 4.9 X 10(3) and 1.9 X 10(6) sites per cell, respectively. Whole cells and a preparation enriched for plasma membrane were affinity labeled with N-bromoacetyl-[125I]T3 (N-BrAc-[125I] T3), and the molecular weights of the radiolabeled proteins were analyzed both by sodium dodecyl sulfate polyacrylamide gel electrophoresis and by Sephadex G-200 gel filtration chromatography. One major chondrosarcoma protein of 55,000 mol wt and two minor proteins of 47,000 and 33,000 mol wt bound N-BrAc-[125I]T3, suggesting that the mol wt of the T3 binding protein was 55,000. As assessed by isoelectric focusing, the 55,000 mol wt protein had an isoelectric point of 5.1. The radiolabeled 55,000 mol wt chondrosarcoma protein was immunoprecipitated with anti-T3 and anti-GH3 plasma membrane T3 antisera. The high degree of homology between this chondrocyte N-BrAc-[125I]T3 binding protein and the protein on rat GH3 cells was demonstrated by a comparison of the peptide maps of Staphylococcus aureus V8 protease and elastase digested radiolabeled binding protein. Although culture of the chondrocytes in medium containing insulin resulted in an approximate 400% increase in plasma membrane [125I]insulin binding, no significant increase in [125I]T3 binding was observed. Thus, expression of the T3 plasma membrane binding protein was similar to that observed previously for the insulin-like growth factor-II receptor on these chondrosarcoma chondrocytes in not being influenced by the concentration of insulin in the culture medium.