Processing Of Nerve Growth Factor Research Articles

Processing of nerve growth factor: The role of basic amino acid clusters in the pro-region

Neurotrophins are synthesized first as precursors called pro-neurotrophins, and their propeptides are then proteolytically removed to form mature neurotrophins. However, a significant proportion of total neurotrophins has been shown to be secreted as pro-neurotrophins. Furthermore, pro- and mature neurotrophins have been shown to elicit opposite effects on cell survival. Thus, the processing step of neurotrophins is very important. In order to understand the mechanism of neurotrophin processing, we focused on the two basic amino acid clusters in the pro-region of nerve growth factor (NGF). Various NGFs mutated at basic amino acids in the pro-region were introduced in COS7 and PC12 cells. The results indicated that these basic amino acid clusters were actually cleaved in the cells by furin, but that their cleavage contributed little to the production of mature NGF. However, one of the two sites was considered to contribute to mature NGF production depending on conditions used.

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

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.

Nerve Growth Factor Processing and Trafficking Events Following TrkA-Mediated Endocytosis

Nerve Growth Factor Processing and Trafficking Events Following TrkA-Mediated Endocytosis

The unprocessed C-terminal dipeptide of recombinant beta-nerve growth factor determines three stable forms with distinct biological activities.

The processing of polypeptide neurotrophins in the nervous system is poorly understood. In this paper, we provide information on the effects of C-terminal processing of nerve growth factor. Three forms of recombinant mouse beta-nerve growth factor (rNGF) were produced and isolated from insect cells infected with a recombinant baculovirus. The three purified forms of rNGF exhibited distinct biological activities and differed in their abilities to compete with high affinity binding of mouse beta-nerve growth factor (mNGF). However, they were chemically and structurally indistinguishable from each other. All three forms of rNGF differed from mature mNGF from mouse submaxillary gland in that the C-terminal Arg-Gly dipeptide had not been proteolytically removed. Removal of the C-terminal dipeptide by gamma-NGF peptidase treatment converted the three forms into a single form identical with mature mNGF. The above results demonstrate that a single polypeptide of rNGF, due to the presence of a C-terminal dipeptide, exhibits three stable dimeric protein conformations with distinct biological activities. The apparent lack of gamma-NGF peptidase in the nervous system raises the possibility that the biologically significant form of NGF may differ from mature mNGF; such a difference may be of physiological relevance.