Abstract
The processing of two homologous precursors, pro-neuropeptide Y (pro-NPY) and pro-pancreatic poly-peptide (pro-PP), was studied in four neuroendocrine cell lines after transfection: CA-77 medullary thyroid carcinoma cells, AtT-20 corticotrope pituitary cells, RIN2A-19 pancreatic endocrine cells, and NB1 neuroblastoma cells. Northern blot analysis indicated that the AtT-20 cells only expressed precursor convertase 3; in contrast, NB1 cells only expressed precursor convertase 2, whereas the RIN2A-19 and CA-77 cells expressed both enzymes. Despite these differences in expression pattern of precursor convertases the four cell lines were, surprisingly, indistinguishable in respect to their processing of pro-PP and pro-NPY. In all four cell lines, pro-NPY was almost completely converted to NPY, and, in all four cell lines, only around 50% of the PP precursor was converted to PP. The relatively poor processing efficiency of pro-PP was rather similar to the processing efficiency of the endogenously produced precursors in the respective cell lines, pro-calcitonin (CA-77), proopiomelanocortin (AtT-20), proinsulin (RIN2A-19), and pro-vasoactive intestinal polypeptide (NB1). At least in the CA-77 cells, NPY and PP were apparently sorted to the regulated secretory pathway, as upon stimulation with secretagogue the release of the transfected peptides increased in parallel with the endogenously expressed peptide, calcitonin gene-related peptide. Mutagenesis studies showed that on the N-terminal side of the di-basic processing site, the otherwise important difference in structure between PP and NPY, a proline for glutamine in position 34, was not responsible for the difference in processing efficiency. On the C-terminal side of the processing site, the efficient processing of pro-NPY could not be transferred to pro-PP by exchanging the whole C-terminal domains of the precursors. It is concluded that pro-NPY is processed more efficiently than pro-PP in all neuroendocrine cell lines tested independent on their expression of the two main precursor convertases and that mutagenesis data indicate that the structural element responsible for the efficient processing of pro-NPY is not located on the N-terminal side of the dibasic processing site.
Highlights
From the $Laboratory of Molecular Endocrinology, University Department of Clinical Chemistry, Rigshospitalet6321, Blegdamsuej 9, DK-2100 Copenhagen, Denmark and )INovo Nordisk AIS, DK-2880 Bagsumrd, Denmark
Expression of Precursor Conuertases-As shown in Fig. 2, mRNA for precursor convertase 3 (PC3)could be detected in RNA isolated from AtT-20 cells, RIN2A-19 cells and to a smaller degree in CA-77 cells but not in the RNA isolated from the NB1 cells
Processing of [ P r ~ ~ ~, p r o -CP-pPeptidelpro-NPY and [pro- In the present investigation, we find that four endocrine
Summary
The neuroendocrine cell lines used were: 1)AtT-20, murine corticotrope cells, producing various peptides from the proopiomelanocortin precursor, obtained from Steven Brand (Gastrointestinal Unit, the structures of PP-fold peptides in solution are similar to Massachusetts General Hospital, Harvard Medical School, Boston, the crystal structure of avian PP [14, 15].The processing of MA); 2) RIN2A-19, rat insulinoma cells, producing insulin, obtained pro-PP has previously been characterized in detail in primary from Ole Madsen (Hagedorn Research Laboratory, Gentofte, Dencell cultures, for example by metabolic labeling and pulsemark); 3) a subclone of the CA-77cell line, from a rat medullary thyroid carcinoma, producing mainly CGRP andonly a small amount chase experiments [16, 17]. It was found that after removal of calcitonin, obtained from Bernard Ross (Department of Medicine, of the signal peptide, the dibasic processing site inthe middle V.A. University Hospital of Washington, Seattle, WA); and 4) NB1, of pro-PP is cleaved first, followed by a more gradual cleavage a human neuroblastoma cell line that synthesizes VIP and pro-VIP at the monobasic site in the C-peptide part. The AtT-20 cells were cultured in Dulbecco's modified Eagle's medium containing10% horse serum and 5% fetal calf serum in an atmosphere of 10% COZ.RIN2A-19 and NB1 cells weregrown in RPMI 1640 containing 10%fetal calf serum and in 5% COP, and the transfection [10, 20]. CA-77 cells weregrown in Dulbecco's modified Eagle's medium/ Ham's F-10 medium (1:l)containing 5 pg/ml transferrin, 10 pg/ml insulin, and 30 mM selenous acid and in 10% CO,.All cell culture media and fetal calf serum were obtained from GIBCO/BRL. EXPERIMENTALPROCEDURES commenced 2 days after transfection in media containing 0.4 mg/ml
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