The Effect of Trypsinization on the Plasma Membrane Binding and Action of 3,5,3′-Triiodothyronine in Rat Thymocytes*

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Abstract
We have previously demonstrated that in the rat thymocyte T3 produces a prompt, extranuclear effect to increase cellular cAMP concentration and 2-deoxyglucose (2-DG) uptake. We have also demonstrated the presence of specific receptors for T3 in the rat thymocyte plasma membrane. From these and other observations we have suggested that T3 initiates these actions by binding to its receptors on the surface of this cell. To test this hypothesis further we have now examined whether a decrease in the binding of T3 to the thymocyte plasma membrane, induced by mild trypsinization, attenuates the effect of T3 on thymocyte cAMP concentration and 2-DG uptake. Mild trypsin treatment on rat thymocytes in cell suspension did not cause appreciable nonspecific damage to the cells, since it did not change the ability of the cells to exclude either trypan blue or mannitol, a marker of extracellular water. Further, treatment with trypsin did not alter the ability of thymocytes to bind concanavalin A. It did produce, however, a dose-related decrease in the binding of T3 to its plasma membrane binding sites owing to a decrease in their number, but not their affinity. This was associated with a progressive decrease in the T3-induced increase in both 2-DG uptake and cAMP concentration. Thus, low concentrations of trypsin had no significant effect on 2-DG uptake in the absence of T3, but decreased significantly the increase in sugar uptake induced by T3, whereas a higher concentration of trypin (5 mg/dl) decreased basal 2-DG uptake by only 23%, but abolished the response to T3 altogether. T3, epinephrine, and trypsin alone induced dose-related increases in cellular cAMP concentration, the response to trypsin exceeding that to T3 or epinephrine. Trypsinization also resulted in a progressive decrease in the response of cellular cAMP to both hormones, so that at a trypsin concentration of 5 mg/ml no further increase was produced by either hormone. The possibility that a maximum activation of adenylate cyclase trypsin could explain these results was rendered unlikely to experiments with prostaglandin E1 (PGE1). The increase in cellular cAMP concentration induced by this agent was vastly greater than that produced by trypsin alone. Although the response to PGE1 was greatly inhibited by trypsin, cellular cAMP concentration in thymocytes incubated with trypsin (5 mg/ml) plus PGE1 was much higher than that in cells treated with trypsin alone. This suggests that adenylate cyclase was not maximally activated by trypsin, and that the inhibition of the response to T3 and epinephrine that trypsin induced cannot be ascribed to this mechanism. These findings indicate that trypsin-sensitive proteins are functionally important components of the T3 receptor in the thymocyte plasma membrane, and they support the suggestion that binding of T3 to such receptors is a prerequisite of its effect to increase cAMP concentration and 2-DG uptake in these cells.