Pj. Hornby et al., IMMUNOCYTOCHEMICAL LOCALIZATION OF THE NEUROPEPTIDE-SYNTHESIZING ENZYME PC1 IN ATT-20 CELLS, Neuroendocrinology, 58(5), 1993, pp. 555-563
The subtilisin-like enzyme PC1 (also known as PC3) cleaves the neurope
ptide precursor proopiomelanocortin at paired basic residues in transf
ection experiments, thus providing evidence for a critical role in pre
cursor processing. While mRNA for this enzyme is highly enriched in ne
uroendocrine tissues, little is known about the tissue and subcellular
distribution of the PC1 protein. This study used immunocytochemical t
echniques to investigate the anatomical distribution of PC1, both alon
e and compared to met-enkephalin (MET-enk), in AtT-20 pituicytes trans
fected with proenkephalin cDNA. A high density of PC1 immunostaining w
as observed in a small region adjacent to the nucleus and in the tips
of the processes of these cells. Dual-staining immunocytochemistry of
whole cells illustrated that both PC1 and MET-enk immunoreactivity wer
e present in the tips, but PC1 was concentrated in a region adjacent t
o the nucleus while MET-enk punctate staining was dispersed throughout
the soma. This codistribution was confirmed in semithin sections of d
ual-stained cells cut at 1-1.5 mum through the thickness of the cells.
PC1 staining resembled that of TGN38, a marker for the trans-Golgi ne
twork. When PC1 immunocytochemistry was performed in cells that were p
retreated with brefeldin A, a drug that redistributes the proximal Gol
gi compartments to the endoplasmic reticulum, there was a complete dis
ruption of the defined locus of PC1 immunoreactivity. Taken together,
our data indicate that (1) PC1 is concentrated in a region of the cell
body resembling the trans-Golgi network and (2) both the enzyme and t
he processed peptide are transported to the tips of the processes. The
observation of abundant PCI within the Golgi complex supports the not
ion that proteolytic processing of neuropeptide precursors may be init
iated in the Golgi apparatus rather than occurring solely within secre
tory granules.