Abstract
Nerve growth factor (NGF) binds to two cell surface receptors, p140trk and p75NGFR, which are both expressed in responsive sensory, sympathetic, and basal forebrain cholinergic neurons. While p140trk belongs to the family of receptor tyrosine kinases, p75NGFR is a member of the TNF/Fas/CD40/CD30 family of receptors. Current views of neurotrophin receptor function have tended to interpret p140trk as the high affinity NGF-binding site. To assess if the binding of NGF to p140trk was distinguishable from binding to high affinity sites on neuronal cells, PC12 cell sublines were generated which expressed p140trk alone, or coexpressed both p140trk and p75NGFR. Kinetic analysis of 125I-NGF binding indicates that it has an unusually slow rate of association with p140trk (k + 1 = 8 x 10(5) M-1 s-1). When both p140trk and p75NGFR receptors are coexpressed, the rate of association of NGF is increased 25-fold to produce a higher affinity binding site. An increase in the rate of internalization was also observed. Since high affinity binding and internalization are believed to be prerequisite for the biological activities of NGF, these results suggest that the biological effects by NGF are derived from a novel kinetic binding site that requires the expression of both receptors. The implications of these results with respect to multisubunit polypeptide receptors are discussed.
Highlights
From the QHematology/Oncology Division, $Department of Cell Biology and Anatomy, Cornel1 University Medical College, New York, New York 10021
Cells were harvested by trituration in PBS- 2 m EDTA, washed twice in PBS-EDTA, and resuspended at a concentration of0.5-1.0 x lo6 celldml in PBS containing 1 mg/mlBSA and 1 mg/mlglucose.Cells were incubated with increasing concentrationsof lZ5I-Nerve growth factor (NGF)(0.001-5 nM) in the presence or absence of an excess of unlabeled NGF (0.8PM) for 2 h
To confrm that high and low affinity sites could be dis- off-rate of lZ5I-NGF fromits receptors, intact cells were incutinguished at 0.4 "C,steady state binding measurements were bated with radiolabeled ligand for 2 h at 0.4 "C to assure that repeated using cells expressing either p75 or p140trk,or cells equilibrium binding had been reached
Summary
From the QHematology/Oncology Division, $Department of Cell Biology and Anatomy, Cornel1 University Medical College, New York, New York 10021. The inset in D represents an independent binding experiment, performed with higher concentration of ligand, to assess low affinity sites in trk-PC12 cells at 1 x lo6 cellstml.
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