Nerve growth factor processing and trafficking events following TrkA-mediated endocytosis.

Abstract

We expressed the high affinity nerve growth factor receptor TrkA in Chinese hamster ovary (CHO) fibroblasts to study nerve growth factor (NGF) trafficking and processing events following receptor-mediated ligand internalization in a nonneuronal and p75 minus cell line. These stable clonal cell lines express approximately 2.5 x 10(5) TrkA receptors and bind 125I-NGF with high affinity (Kd = 4 x 10(-10) M). The TrkA receptors are autophosphorylated on tyrosine residues upon NGF stimulation and are capable of tyrosine phosphorylating downstream signaling molecules. The t1/2 of 125I-NGF internalization is 5 min, and the probability of an occupied TrkA receptor internalizing within 1 min at 37 C is 9.8%. By 2 h following endocytosis, less than 10% of internalized 125I-NGF is degraded, as determined by TCA precipitation. Thirty minutes following ligand endocytosis, endocytosed 125I-NGF is delivered back to the cell surface and released by the cell (retroendocytosis), possibly by remaining associated with recycling TrkA receptors. We measured the effect of acidification on 125I-NGF-TrkA association and found that, at pH 6, 40% of 125I-NGF remains bound. Thus, NGF may remain associated with the TrkA receptor at low pH conditions in the endosome and can thereby be targeted back to the plasma membrane for release by the cell. 1) TrkA, in the absence of p75, is fully capable of mediating 125I-NGF endocytosis; 2) internalized 125I-NGF is slowly and inefficiently degraded; 3) following internalization, 125I-NGF is retroendocytosed; and 4) the ability of 125I-NGF to remain receptor-associated during acidic conditions may provide a mechanism for its retroendocytosis via recycling TrkA vesicles.