Nerve growth factor receptors on cultured rat Schwann cells

Abstract

Neonatal rat Schwann cells were grown in tissue culture and assayed for NGF receptors with time in culture. NGF receptor levels on freshly prepared Schwann cells (day 0) were low but increased dramatically during the first week in culture. Characterization of 125I-NGF binding to resuspended cells grown for 4 d in culture revealed that binding was not saturable at high ligand concentrations (50-70 nM) and that a high-capacity, low-affinity NGF binding component existed on these cells as compared to PC12 cells. The monoclonal antibody, 192-IgG, which recognizes the rat NGF receptor, was used as an immunohistochemical tool to verify the presence of NGF receptors on the cultured rat Schwann cells. In contrast to radiolabeled NGF, 125I-192-IgG demonstrated saturable binding to Schwann cells in suspension, with Kd and Bmax values of 4 nM and 115 fmol/10(6) cells, respectively. Schwann cells showed no evidence of slow dissociation or internalization of NGF binding at any of several NGF concentrations. 192-IgG was used to immunoprecipitate 125I-NGF chemically crosslinked to cell membranes. SDS-PAGE and subsequent autoradiography of the immunoprecipitated NGF receptors revealed that 2 species of NGF receptors were precipitated from Schwann cells and PC12 cells. In PC12 cells, 2 bands with molecular weights of 90 and 210 kDa were identified. The Schwann cell NGF receptor species migrated slower on the gels, with apparent molecular weights of 95 and 220 kDa. Further analysis of glial cell NGF receptors showed that Schwann cells isolated from the vagus nerve of neonatal rats also expressed NGF receptors in culture; however, astrocytes cultured from neonatal rat cerebral cortex, cultured under conditions reported here, were devoid of detectable NGF receptors. These results show that NGF receptor levels on Schwann cells increase with time in culture, and this resembles what is observed in Schwann cells in vivo when adult peripheral nerve is injured. The data are discussed in terms of a supportive role for the Schwann cell in facilitating peripheral nerve development and regeneration.