Nerve growth factor receptors. Characterization of two distinct classes of binding sites on chick embryo sensory ganglia cells.

A Sutter,R J Riopelle,R M Harris-Warrick,E M Shooter

doi:10.1016/s0021-9258(18)50507-x

Abstract

Steady state and kinetic studies on the binding of 125I-beta nerve growth factor (NGF) to single cells from sensory ganglia of 8-day-old chick embryos show two distinct, saturable binding sites with dissociation constants of Kd(I) = 2.3 X 10(-11) M and Kd(II) = 1.7 X 10(-9) M. The difference in the affinities is due to different rate constants of dissociation (k-1(I) = 10(-3) s-1, k-1(II) = 2 X 10(-1 s-1). The association to both sites is apparently diffusion controlled (k+1(I) = 4.8 X 10(7) M-1s-1, k+2(II) = 10(7) to 10(8) M-1s-1). The binding of betaNGF to both sites is specific, since none of a number of hormones or proteins tested compete for the binding of 125I-betaNGF to either of those two sites. The heterogeneity of the binding of 125I-betaNGF is not due to heterogeneity of the 125I-betaNGF preparation nor to a negatively cooperative binding. In experiments where the dissociation of 125I-betaNGF is induced by the addition of saturating amounts of unlabeled betaNGF, the ratio of the 125I-betaNGF released with either of the two dissociation rate constants is solely dependent on the occupancy of the two sites before dissociation is started and is independent of the total occupancy of the sites during dissociation. The rate of dissociation of 125I-betaNGF from the higher affinity binding site I is accelerated by unlabeled betaNGF under conditions where the occupancy is both increased and decreased. Although the dissociation characteristics of 125I-beta NGF change with increasing times of exposure of the cells to the ligand, and 125I-beta NGF is degraded after it binds to the cells, these secondary processes do not interfere with the analysis of the binding data. At the lowest concentration of 125I-beta NGF used for the analysis less than 10% of the 125I-beta NGF is degraded. Both kinetic and steady state binding data reveal the two NGF binding sites at 2 degrees C as well as at 37 degrees C.

Highlights

P nerve growth factor (NGF) to single cells from sensory ganglia of g-day-old chick embryos show two distinct, saturable binding sites with dissociation constants of &(I) = 2.3 X 10-l’ M and &(II) = 1.7 X lo-’ M
Key observations in this regard are the appearance of two separate linear segments in the Scatchard plot of the binding data (Figs. 3b and 8) and the agreement of steady state and kinetic binding data observed at 37°C and 2°C when the dissociation kinetics were studied under chase conditions (Figs. 6 and 11)
Under most circumstances different afflnities of labeled and unlabeled ligand will produce linear Scatchard plots if the ligand concentration is altered by varying the concentration of the labeled ligand alone [28], which is the procedure used in the present work

Summary

Introduction

For the three concentrations chosen in the experiment depicted, the percentage of “‘I-PNGF bound to the higher affinity Site I calculated from the steady state binding data was 90% (0.6 X lO-‘i M), 58% (0.16 X lo-” M), and 24%

Results

Conclusion