Molecular kinetics of nerve growth factor receptor trafficking and activation

Abstract

A growing body of evidence indicates a close relationship between tyrosine kinase receptor trafficking and signaling. Biochemical and molecular analyses of the expression, fate, and kinetics of membrane trafficking of the nerve growth factor (NGF) receptor TrkA were performed in PC12 cells. Pulse-chase experiments indicate that TrkA is synthesized as a 110-kDa N-glycosylated precursor that leads to the mature 140-kDa form of the receptor with a half-life of conversion of ∼24 ± 0.5 min. Neuraminidase digestion shows that modification of the carbohydrate moiety of the receptor by sialylation occurs during maturation. The 140-kDa form is rapidly translocated to the cell surface as assessed by cell surface biotinylation performed on intact PC12 cells. Mature receptor half-life is ∼138 ± 4 min and is shortened to 86 ± 8 min by NGF treatment. Flow cytometric analysis indicates that NGF induces clearing of this receptor from the cell surface within minutes of treatment. The addition of NGF decreases the half-life of cell surface gp140TrkA from 100 to 35 min and leads to enhanced lysosomal degradation of the receptor. The process of NGF-induced TrkA internalization is clearly affected by interfering with ligand binding to p75NTR. An analysis of receptor activation kinetics also shows that receptor signaling primarily takes place from an intracellular location. Together, these data show that the primary effect of NGF treatment is a p75NTR-modulated decrease in TrkA transit time at the cell surface.