Nerve growth factor receptors in human neuroblastoma cells.
Receptors for the nerve growth factor protein (NGFR) present in the human neuroblastoma cell line LAN-1 were characterized. LAN-1 cells display high-affinity (type I, with KD value of 5.9 X 10(-11) M) and low-affinity (type II, with KD value of 9.2 X 10(-9) M) binding to NGF. NGFR were fractionated by preparative isoelectric focusing in a granulated gel (PEGG). High-affinity binding was found in the 5.9-6.2 pH region of the PEGG, and low-affinity binding in the 4.6-4.8 and 8.8-9.3 pH ranges. After further analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) we observed both 92.5- and 200-kDa molecular species associated with NGF binding activity. The 200-kDa protein was found in fractions displaying high-affinity NGF binding and the 92.5-kDa protein in fractions displaying low-affinity NGF binding. Equilibrium binding analysis of NGF in PEGG fractions confirmed the presence of two specific saturable binding sites with KD values similar to those observed for whole dissociated cells. When NGFR II activity from the acidic region of the PEGG chromatogram was incubated with NGFR II from the basic region of the PEGG chromatogram, there was no change in NGF binding or in the number of apparent NGF receptors. However, incubation of these same fractions with a fraction having only NGFR I showed an apparent increase in high-affinity NGF binding and a decrease in low-affinity NGF binding. Immunoprecipitation of this "mixed" fraction and analysis on SDS-PAGE under reduced and nonreduced conditions showed 200-kDa and 92.5-kDa proteins under nonreduced conditions and a 92.5-kDa protein under reduced conditions. Our findings are consistent with the hypothesis that there are two distinct NGF receptors in NGF-responsive cells. The interconvertibility of low- and high-affinity receptors and the possible existence of a modulator type protein or of "silent" type receptors are also in agreement with our findings.
D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D009414:Nerve Growth Factors / Q000378:metabolism*; D009447:Neuroblastoma / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000302:isolation & purification / Q000378:metabolism*; D017475:Receptors, Nerve Growth Factor; D012995:Solubility
D009414:Nerve Growth Factors; D011956:Receptors, Cell Surface; D017475:Receptors, Nerve Growth Factor