Loss of α2‐macroglobulin and epidermal growth factor surface binding induced by phenothiazines and naphthalene sulfonamides

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Abstract
We have found that certain naphthalenesulfonamides [e.g., N-6(-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7)] and phenothiazines [e.g., trifluoperazine (TFP)] induce a loss of cell-surface receptors for α2-macroglobulin and epidermal growthfactor (EGF) in fibroblasts. The loss of α2-macroglobulin receptors is independent of receptor occupancy and is rapidly reversed upon removal of these agents from the culture medium. The extent of EGF receptor loss is less than for α2-macroglobulin, and the EGF receptors do not reappear at the surface when W-7 isremoved. Receptor loss was measured as a change in the capacity for binding iodinated ligands;nochange in affinity of binding was observed. This receptor loss could reflect inactivation of receptors or internalization. W-7 did not induce a loss of cell surface β2-microglobulin, a membrane protein which is excluded from coated pits and which isnot internalized, indicating that the effect of W-7 was specific for membrane receptorsandnot a result of bulk depletion of plasma membrane. The loss of α2-macroglobulin and EGF receptors occurs at concentrations which do not cause an increase in the pH of endocytic vesicles or the cytoplasm, indicating that these agents act by a mechanism distinct from the effect of other weak bases. Since both TFP and W-7 are potent inhibitors of calmodulin, we investigated the possibility that inhibition of calmodulin was responsiblefor the loss of receptors. Three lines of evidence suggest that calmodulin inhibition is not responsible for the inhibition of binding and endocytosis: (1) Promethazine, a phenothiazine that is a poor inhibitor of calmodulin, is nearly as effective as TFP at inhibiting endocytosis; calmidazolium, a potent inhibitor of several calmodulin functions, did not cause a loss of binding; (2) the microinjection of calmodulin into cells did not reverse the effects of W-7; using pressure microinjection, we introduced up to a 100-fold excess of calmodulin over native levels into individual gerbil fibroma cells; using rho-damine-labeled α2-macroglobulln, we saw that the W-7 induced inhibition of receptor-mediated endocytosis was the same in injected and uninjected cells; (3) we injected calcineurin, a calmodulin-binding protein, into cells (1-3 pg/ cell) and observed no effect on the receptor-mediated endocytosis of rhoda-mine-labeled α2-macroglobulin. These data indicated that cell surface receptor numbers can be regulated by a cellular component that is not cytoplasmic calmodulin but that shares some drug sensitivities with calmodulin.