Secretory protein traffic. Chromogranin A contains a dominant targeting signal for the regulated pathway.
Secretory proteins are targeted into either constitutive (secreted upon synthesis) or regulated (stored in vesicles and released in response to a secretagogue) pathways. To investigate mechanisms of protein targeting into catecholamine storage vesicles (CSV), we stably expressed human chromogranin A (CgA), the major soluble protein in human CSV, in the rat pheochromocytoma PC-12 cell line. Chromaffin cell secretagogues (0.1 mM nicotinic cholinergic agonist, 55 mM K+, or 2 mM Ba++) caused cosecretion of human CgA and catecholamines from human CgA-expressing cells. Sucrose gradients colocalized human CgA and catecholamines to subcellular particles of the same buoyant density. Chimeric proteins, in which human CgA (either full-length [457 amino acids] or truncated [amino-terminal 226 amino acids]) was fused in-frame to the ordinarily nonsecreted protein chloramphenicol acetyltransferase (CAT), were expressed transiently in PC-12 cells. Both constructs directed CAT activity into regulated secretory vesicles, as judged by secretagogue-stimulated release. These data demonstrate that human CgA expressed in PC-12 cells is targeted to regulated secretory vesicles. In addition, human CgA can divert an ordinarily non-secreted protein into the regulated secretory pathway, consistent with the operation of a dominant targeting signal for the regulated pathway within the peptide sequence of CgA.
Parmer
R J
RJ
Department of Medicine, University of California, San Diego, La Jolla 92161.
Xi
X P
XP
Wu
H J
HJ
Helman
L J
LJ
Petz
L N
LN
D000595:Amino Acid Sequence; D000818:Animals; D001358:Avian Sarcoma Viruses / Q000235:genetics; D001483:Base Sequence; D002217:Carbachol / Q000494:pharmacology; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000378:metabolism; D053379:Chromogranin A; D002864:Chromogranins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D003594:Cytoplasmic Granules / Q000378:metabolism* / Q000648:ultrastructure; D005822:Genetic Vectors; D006801:Humans; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009638:Norepinephrine / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides; D016716:PC12 Cells; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection
C506150:CHGA protein, human; D053379:Chromogranin A; D002864:Chromogranins; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; C506153:chromogranin A, rat; D002217:Carbachol; D015500:Chloramphenicol O-Acetyltransferase; D009638:Norepinephrine