Abstract

The structurally related immunosuppressive macrolides FK-506 and rapamycin (RAP) exert distinct biological effects: inhibition of interleukin-2 production and inhibition of interleukin-2-induced proliferation, respectively, through binding to intracellular receptors, termed FKBPs. Although the interaction of these drugs with purified FKBPs in vitro has been well characterized, little is known about their interaction with FKBPs in living cells. Here, we used [3H]-dihydro-FK-506 as a probe to examine the binding of these macrolides in both normal mouse splenic T-cells and the human Jurkat T-cell lymphoma. These cells were found to accumulate the radioligand, predominantly in the cytosol, to a saturable level corresponding to an estimated concentration of 6 to 7 microM. Half-maximal suppression of T-cell activation was shown to require radioligand occupancy of only 3 to 5% of the pool of available intracellular binding sites (FKBPs). Moreover, the binding and immunosuppressive effect of the radioligand could not be removed by extensive washing and remained stable for at least 6 hr upon incubation of the cells at 37 degrees C. However, a molar excess of either FK-506 or RAP was found to rapidly displace [3H]-dihydro-FK-506 from its cellular binding sites. Consistently, FK-506 and RAP were able to antagonize mutually their immunosuppressive activities even when added several hr after each other to T-cell cultures. We took advantage of the reciprocal antagonism of FK-506 and RAP to define their apparent affinities for the functionally relevant cellular receptors by Schild analysis. This indicated that the drugs compete for a single cellular receptor with similar KdS and, therefore, may mediate their immunosuppressive action upon interaction with similar or highly related FKBPs.