Processing and Secretion of Nerve Growth Factor: Expression in Mammalian Cells with a Vaccinia Virus Vector

To study posttranslational mechanisms for the control of nerve growth factor (NGF), we used a recombinant vaccinia virus vector to independently express the two major NGF transcripts in a variety of mammalian cell lines. The two major transcripts contain NGF (12.5 kilodaltons [kDa]) at the C-terminus and differ by alternative splicing of an N-terminal exon, so that the large precursor (34 kDa) had 67 amino acids upstream of an internal signal peptide and the smaller precursor (27 kDa) had this signal peptide at its N-terminus. In L929 cells, expression of either NGF transcript with the vaccinia virus vector gave rise to an apparently identical intracellular 35-kDa glycosylated precursor formed by cleavage of the primary gene product after the signal peptide. These cells also secreted biologically active NGF. To determine whether NGF processing is restricted by cell type, we infected a variety of mammalian cell lines with both recombinant viruses; all accumulated the same 35-kDa precursor and secreted NGF. Thus, many types of cells have the machinery to process and secrete NGF. However, NGF accumulated intracellularly (presumably in secretory granules) in cells with a regulated pathway of secretion (e.g., AtT-20 and HIT cells). In these cells, a membrane-permeable cyclic AMP analog, 8-bromo-cyclic AMP, stimulated NGF secretion. This suggests a mechanism for the regulation of NGF levels in which specific secretagogues, e.g., neurotransmitters, control NGF secretion.