Nerve growth factor receptors on PC12 cells: Evidence for two receptor classes with differing cytoskeletal association

Abstract

PC12, an NGF responsive cell line, exhibits two classes of NGF receptors which we designate “Fast” and “Slow.” Fast receptors, accounting for 75% of specific NGF binding, are distinguished by their rapid rates for association and dissociation of 125I-NGF. At 37°C, binding of 125I-NGF to Fast receptors is 5-fold more rapid than to Slow receptors and dissociation of 125I-NGF from Fast receptors is 40-fold more rapid than from Slow receptors. No evidence was obtained for a ligand-induced conversion of receptors from Fast to Slow characteristics. Scatchard analysis of binding experiments indicates that PC12 cells possess 60,000 specific receptors for NGF of which 15,000 are of the Slow class. Despite having very different kinetic constants, Slow and Fast receptors have similar equilibrium binding constants (about 2 × 10 −10 M) due to cancelling effects of differing association and dissociation rates. Brief digestion of PC12 cells with trypsin before addition of NGF inactivates essentially all Fast receptors without significantly affecting Slow receptors. Therefore Fast and Slow classes of receptors must exist prior to addition of NGF, and the observed receptor heterogeneity is not due to ligand-induced changes. 125I-NGF bound to Slow receptors is preferentially associated with preparations of Triton X-100 insoluble cytoskeletons, while 125I-NGF bound to Fast receptors is solubilized by this procedure. Cytoskeletally associated NGF is almost exclusively associated with the extranuclear cytoskeletal matrix rather than with the nucleus itself. Preparation of nuclei by various methods suggests that the presence of contaminating cytoskeletal elements should be considered in evaluating the existence of translocation and binding of NGF to the nucleus. Inhibition of endocytotic internalization of NGF either by lowering of temperature to O°C or by preincubation of cells with sodium azide in medium lacking glucose does not reduce the slowly released component of bound NGF, nor alter its cytoskeletal association. The possible functional roles of Slow and cytoskeletal receptors are discussed.