QUANTITATIVE AND TEMPORAL ANALYSIS OF THE CELLULAR INTERACTION OF FK-506 AND RAPAMYCIN IN T-LYMPHOCYTES

The structurally related immunosuppressive macrolides FK-506 and rapam ycin (RAP) exert distinct biological effects: inhibition of interleuki n-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 [H-3]-dihydro-FK-506 as a pr obe to examine the binding of these macrolides in both normal mouse sp lenic T-cells and the human Jurkat T-cell lymphoma. These cells were f ound to accumulate the radioligand, predominantly in the cytosol, to a saturable level corresponding to an estimated concentration of 6 to 7 mu M. Half-maximal suppression of T-cell activation was shown to requ ire radioligand occupancy of only 3 to 5% of the pool of available int racellular binding sites (FKBPs). Moreover, the binding and immunosupp ressive effect of the radioligand could not be removed by extensive wa shing and remained stable for at least 6 hr upon incubation of the cel ls at 37 degrees C. However, a molar excess of either FK-506 or RAP wa s found to rapidly displace [H-3]-dihydro-FK-506 from its cellular bin ding sites. Consistently, FK-506 and RAP were able to antagonize mutua lly their immunosuppressive activities even when added several hr afte r each other to T-cell cultures. We took advantage of the reciprocal a ntagonism of FK-506 and RAP to define their apparent affinities for th e functionally relevant cellular receptors by Schild analysis. This in dicated that the drugs compete for a single cellular receptor with sim ilar K(d)s and, therefore, may mediate their immunosuppressive action upon interaction with similar or highly related FKBPs.