Abstract
Many endocrine and neuroendocrine cells contain specialized secretory organelles called dense core secretory granules. These organelles are the repository of proteins and peptides that are secreted in a regulated manner when the cell receives a physiological stimulus. The targeting of proteins to these secretory granules is crucial for the generation of certain peptide hormones, including insulin and ACTH. Although previous work has demonstrated that proteins destined to a variety of cellular locations, including secretory granules, contain targeting sequences, no single consensus sequence for secretory granule-sorting signals has emerged. We have shown previously that alpha-helical domains in the C-terminal tail of the prohormone convertase PC1/3 play an important role in the ability of this region of the protein to direct secretory granule targeting (Jutras, I. Seidah, N. G., and Reudelhuber, T. L. (2000) J. Biol. Chem. 275, 40337-40343). In this study, we show that a variety of alpha-helical domains are capable of directing a heterologous secretory protein to granules. By testing a series of synthetic alpha-helices, we also demonstrate that the presence of charged (either positive or negative) amino acids spatially segregated from a hydrophobic patch in the alpha-helices of secretory proteins likely plays a critical role in the ability of these structures to direct secretory granule sorting.
Highlights
Several protein domains have been implicated in the process of sorting proteins to dense core secretory granules
AtT-20 cells contain dense core secretory granules in which endogenous POMC is processed into adrenocorticotropic hormone (ACTH) by a series of proteolytic cleavages involving PC1/3
We have provided evidence that a variety of ␣-helical domains can direct a linked secretory protein to dense core secretory granules
Summary
Recombinant Plasmid Construction—Naturally occurring peptide fragments to be analyzed for secretory granule sorting were derived from the mouse PC1/3 (GenBankTM accession number NM_013628) and human prorenin (accession number NM_000537) cDNAs. Mammalian Cell Culture, Transfection, and Secretion Analysis—Mouse corticotropic AtT-20 cells were grown in Dulbecco’s minimal essential medium (Invitrogen, Burlington, Ontario, Canada) containing 10% fetal bovine serum in a humidified incubator at 37 °C in 10% CO2. Expression vectors were stably transfected into AtT-20 cells by cotransfection with pSV-Neo (Invitrogen), and selection of stable pools was carried out in Geneticin (G418, Invitrogen). Results were compared by one-way analysis of variance using Dunnett’s post-test and are expressed in Table 1 as the means Ϯ S.E. The forskolin-stimulated secretion of the endogenous granule cargo peptide -endorphin (2.00 Ϯ 0.14, n ϭ 15) was determined by radioimmunoassay in each experiment and was used to ensure that the stimulation of AtT-20 cell granule release was comparable in all experiments. The slides were subsequently washed with Tris-buffered saline and incubated with anti-rabbit IgG antibody conjugated to rhodamine (1:100 dilution; Chemicon, Temecula, Ca) for 1 h at room temperature. Light antifade kit (Invitrogen) and visualized using a Zeiss LSM 510 confocal microscope
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