Insoluble State by Anti Nerve Growth Factor An tibodied

Two populations of nerve growth factor (NGF) receptors can be distinguished on PC12 cells and have been termed “fast” and “slow” receptors on the basis of their respective rates of dissociation. Since a slowly dissociating state of the NGF receptor could be produced by receptor clustering, we sought to examine the effects of artificially clustering NGF-receptor complexes by using anti-NGF antibodies. At an IZSI-NGF concentration of 500 pM, slow receptors account for 25% of the total NGF binding. However, if lz5I-NGF is first bound to the cell surface and an anti-NGF immunoglobulin G (IgG) is added subsequently, >90% of the total binding becomes slowly dissociating. Anti-NGF IgG also changes the proportion of Triton X-100 insoluble NGF binding from 10% to 50% possibly reflecting an association of the NGF receptor with the cytoskeleton. The effects on NGF binding of low concentrations of anti-NGF IgG could be enhanced by the addition of goat anti-rabbit IgG. Neither Fab S i n c e its discovery over 25 years ago, nerve growth factor (a 26 000-dalton polypeptide) has remained the only purified agent whose importance in neuronal development has been clearly demonstrated (Thoenen & Barde, 1980). The actions of nerve growth factor (NGF) on the pheochromocytoma clonal cell line PC 12 have been extensively studied in recent years as a model system for understanding neuronal differentiation (Greene & Trischler, 1976). NGF causes this cell line of neural crest origin to extend neurites and become electrically excitable. Two distinct populations of cell-surface receptors for NGF have been identified on PC12 cells on the basis of their dissociation kinetics (Landreth & Shooter, 1980; Schechter & Bothwell, 1981). Upon the addition of excess unlabeled ligand, lZ51-NGF is released rapidly from one receptor subtype ( t ! ,2 of dissociation = 30 s) and slowly from another (t l12 of dissociation = 30 min). The two receptor species have been termed “fast” and “slow” by Schechter & Bothwell (1981) in reference to their dissociation properties. Slow receptors can be further distinguished from fast receptors by their resistance to degradation by trypsin (Landreth & Shooter, 1980; Schechter & Bothwell, 1981) and by their relative insolubility after Triton X-100 extraction (Schechter & Bothwell, 198 1). Landreth & Shooter (1 980) originally proposed that slow receptors may arise from fast receptors by a ligand-induced conversion process. Schechter & Bothwell (1981), on the other hand, suggested that fast and slow receptors are separate and independent entities. In an attempt to distinguish between such From the Department of Neurobiology, Stanford University Medical Center, Stanford, California 94305. Received May 2. 2983. This work was supported by grants from the National Insthutes of Health (NS 04270) and the American Cancer Society (BC 325). R.D.V. is a trainee in the Medical Scientist Training Program supported by a grant from the NIH (GM 07365). 0006-2960183 10422-5022%01 .SO10 fragments nor an anti-NGF monoclonal antibody affects NGF binding, indicating that the cross-linking capacity of anti-NGF IgG is required for its activity. Wheat germ agglutinin (WGA), a multivalent lectin, also converts NGF binding into a predominantly slowly dissociating, Triton X100 insoluble state. Both WGA and anti-NGF IgG produce their effects on NGF binding at 37 and 4 OC and protect the 1251-NGFreceptor complex from protease digestion. WGA and antiNGF IgG act synergistically to convert NGF binding into a Triton X-100 insoluble form and affect NGF binding to the melanoma A875 cell line in a similar fashion as to PC12 cells. Since WGA and anti-NGF IgG share the potential capability of cross-linking NGF-receptor complexes, these results suggest that receptor clustering may play a role in the formation of a slowly dissociating, Triton X-100 insoluble state of the NGF receptor on PC12 and A875 cells. models, Vale & Shooter (1982) found that fast receptors could be converted into slow receptors by the lectin wheat germ agglutinin (WGA), indicating that the two receptors may be interrelated. The WGA-induced receptor conversion occurred within seconds, was largely independent of temperature, and was accompanied by an association of the NGF receptor with the Triton X100 insoluble cytoskeleton, findings which have been independently observed by Grob and Bothwell (unpublished results). More recently, Buxser et al. (1983) have extended these observations to show that WGA also induces a receptor conversion in detergent-soluble membrane extracts and membrane extracts reconstituted into phospholipid vesicles. Since WGA is a multivalent lectin, its ability to cross-link receptors may be important in producing its effects on the binding properties and Triton X-100 solubility of the NGF receptor. Since it has not been possible to produce a functionally monovalent derivative of WGA, as has been achieved with concanavalin A (Gunther et al., 1973), we decided to investigate how other clustering agents affect the binding properties of the NGF receptor. Schechter et al. (1979a) previously demonstrated that anti-insulin immunoglobulin G (IgG) increased the specific binding of 1251-insulin to liver membranes by apparently converting a class of low-affinity receptors to high-affinity sites. The clustering of insulin receptors was important for this process, since monovalent Fab fragments did not produce this effect. In the present study, we have investigated the effects of anti-NGF IgG on the binding properties of the NGF-receptor complex. Similar to the results previously reported with WGA (Vale & Shooter, 1982), anti-NGF IgG converter receptor-bound ‘251-NGF into a slowly dissociating and protease-resistant state. These antibodies also increased by severalfold the proportion of 1251NGF binding which is insoluble after Triton X100 extraction. Conversion of NGF bound to fast receptors into a slowly