Immunocytochemical localization of the neuropeptide-synthesizing enzyme PC1 in AtT-20 cells.
The subtilisin-like enzyme PC1 (also known as PC3) cleaves the neuropeptide precursor proopiomelanocortin at paired basic residues in transfection experiments, thus providing evidence for a critical role in precursor processing. While mRNA for this enzyme is highly enriched in neuroendocrine tissues, little is known about the tissue and subcellular distribution of the PC1 protein. This study used immunocytochemical techniques to investigate the anatomical distribution of PC1, both alone and compared to met-enkephalin (MET-enk), in AtT-20 pituicytes transfected with proenkephalin cDNA. A high density of PC1 immunostaining was 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 were present in the tips, but PC1 was concentrated in a region adjacent to the nucleus while MET-enk punctate staining was dispersed throughout the soma. This codistribution was confirmed in semithin sections of dual-stained cells cut at 1-1.5 microns through the thickness of the cells. PC1 staining resembled that of TGN38, a marker for the trans-Golgi network. When PC1 immunocytochemistry was performed in cells that were pretreated with brefeldin A, a drug that redistributes the proximal Golgi compartments to the endoplasmic reticulum, there was a complete disruption 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 the processed peptide are transported to the tips of the processes.(ABSTRACT TRUNCATED AT 250 WORDS)
Hornby
P J
PJ
Department of Pharmacology, Louisiana State University Medical Center, New Orleans.
Rosenthal
S D
SD
Mathis
J P
JP
Vindrola
O
O
Lindberg
I
I
D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000096:biosynthesis* / Q000276:immunology; D020126:Brefeldin A; D002478:Cells, Cultured; D003517:Cyclopentanes / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004744:Enkephalin, Methionine / Q000096:biosynthesis / Q000276:immunology; D004745:Enkephalins / Q000096:biosynthesis / Q000276:immunology; D006056:Golgi Apparatus / Q000378:metabolism; D007150:Immunohistochemistry; D051379:Mice; D045664:Proprotein Convertase 1; D043484:Proprotein Convertases; D011498:Protein Precursors / Q000096:biosynthesis / Q000276:immunology; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology
D003517:Cyclopentanes; D004745:Enkephalins; D011498:Protein Precursors; D011500:Protein Synthesis Inhibitors; C029992:proenkephalin; D020126:Brefeldin A; D004744:Enkephalin, Methionine; D043484:Proprotein Convertases; C473656:Pcsk1 protein, mouse; D045664:Proprotein Convertase 1; D016282:Aspartic Acid Endopeptidases