TY - JOUR
T1 - Processing of pro-CGRP in a rat medullary thyroid carcinoma cell line transfected with protease inhibitors
AU - Johansen, Teit Eliot
AU - Schifter, S
AU - Vogel, Charlotte Katrine
AU - Tolstoy, Susanne
AU - Schwartz, Thue W.
N1 - Keywords: Animals; Calcitonin; Cloning, Molecular; Mutagenesis, Site-Directed; Neuropeptide Y; Plasmids; Protease Inhibitors; Protein Precursors; Protein Processing, Post-Translational; Rats; Thyroid Gland; Transfection; Tumor Cells, Cultured
PY - 1991
Y1 - 1991
N2 - A rat medullary thyroid carcinoma cell line, CA77, was used to study the effect of a series of biosynthesized protease inhibitors on the proteolytic cleavage of the endogenously synthesized pro-CGRP. This cell line efficiently converted the pro-CGRP to mature CGRP as assessed by chromatography of cell extracts followed by radioimmunoassay for CGRP. CA77 cells were transfected with expression vectors encoding protease inhibitors: the Arg-serpins, alpha 1-antitrypsin Pittsburgh (358 Met----Arg) and plasminogen activator inhibitor 1, the Kazal type serine protease inhibitor, pancreatic secretory trypsin inhibitor, and the general thiol protease inhibitor, cystatin C. Only the chromatography of cell extracts from CA77 cells transfected with a plasmid encoding cystatin C showed an apparent higher content of unprocessed pro-CGRP as compared to non-transfected cells. No effect on pro-CGRP processing could be measured in the CA77 cells transfected with plasmids encoding the three serine protease inhibitors. CA77 cells were also transfected with two constructs encoding chimeric proteins consisting of cystatin C and the precursor for neuropeptide Y. Release experiments using 8-bromo cAMP as the secretagogue showed that the chimer was co-released with CGRP. However, no effect of this chimer upon pro-CGRP processing could be detected. It is concluded that the processing of pro-CGRP in the CA77 cell line was very efficient and that four different protease inhibitors and two cystatin C/NPY chimers synthesized by this neuroendocrine cell line had only minimal effect upon the processing of CGRP.
AB - A rat medullary thyroid carcinoma cell line, CA77, was used to study the effect of a series of biosynthesized protease inhibitors on the proteolytic cleavage of the endogenously synthesized pro-CGRP. This cell line efficiently converted the pro-CGRP to mature CGRP as assessed by chromatography of cell extracts followed by radioimmunoassay for CGRP. CA77 cells were transfected with expression vectors encoding protease inhibitors: the Arg-serpins, alpha 1-antitrypsin Pittsburgh (358 Met----Arg) and plasminogen activator inhibitor 1, the Kazal type serine protease inhibitor, pancreatic secretory trypsin inhibitor, and the general thiol protease inhibitor, cystatin C. Only the chromatography of cell extracts from CA77 cells transfected with a plasmid encoding cystatin C showed an apparent higher content of unprocessed pro-CGRP as compared to non-transfected cells. No effect on pro-CGRP processing could be measured in the CA77 cells transfected with plasmids encoding the three serine protease inhibitors. CA77 cells were also transfected with two constructs encoding chimeric proteins consisting of cystatin C and the precursor for neuropeptide Y. Release experiments using 8-bromo cAMP as the secretagogue showed that the chimer was co-released with CGRP. However, no effect of this chimer upon pro-CGRP processing could be detected. It is concluded that the processing of pro-CGRP in the CA77 cell line was very efficient and that four different protease inhibitors and two cystatin C/NPY chimers synthesized by this neuroendocrine cell line had only minimal effect upon the processing of CGRP.
U2 - 10.1016/0303-7207(91)90008-G
DO - 10.1016/0303-7207(91)90008-G
M3 - Journal article
C2 - 1761166
SN - 0303-7207
VL - 82
SP - 51
EP - 60
JO - Molecular and Cellular Endocrinology
JF - Molecular and Cellular Endocrinology
IS - 1
ER -