We have demonstrated previously the presence of plasma membrane receptors for 3,3'5-triidodo-L-thyronine (T3) in cultured rat GH3 and human epithelioid carcinoma A431 cells. To further characterize the plasma membrane T3 receptors in GH3 cells, binding of [125I]T3 to the CHAPS solubilized receptors was evaluated. One class of binding sites was detected with a Kd of 7nM and Bmax of 0.84 pmoles/50yg protein. Analogs specificity, salt, pH and temperature dependency of binding were also examined. The intracellular routing of T3 in GH3 cell during internalization was examined by using centrifugation in colloidal silica gel gradients. At 4 degrees C,T3 is associated with a membrane fraction enriched in adenylate cyclase activity indicative of plasma membranes. At 37 degrees c, T3 is internalized ans associated with vesicular structures enriched with galactosyl transferase activity. Concomitantly, T3 is incorporated into nuclei in a time-dependent manner. Thus, the association of T3 with the nuclear fraction probably occurs by a receptor-mediated transfer of the hormone through vesicular structures rather than by a diffusion process. Plasma membrane T3 receptors in A431 cells were purified to homogenity by gel filtration ion exchange chromatography and preparative eletrophoresis. The partial amino acid sequence was determined by micro sequencing technique. Polyclonal and monoclonal antibodies against the purified receptors were raised. Using these reagents, the function and regulation of plasma membrane T3 receptors are being studied.
