Comparison of the Regulation of Carboxypeptidase E and Prolactin in GH&lt;sub&gt;4&lt;/sub&gt;C&lt;sub&gt;1&lt;/sub&gt; Cells, a Rat Pituitary Cell Line

Fricker, Lloyd D.; Reaves, Barbara J.; Das, Banasree; Dannies, Priscilla S.

doi:10.1159/000125407

Cited by 28 publications

(3 citation statements)

References 15 publications

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“…CPE activity was determined as described (23). Acid phosphatase, a lysosomal marker, and ␣-mannosidase II, a Golgi marker, were assayed as described (24,25). Protein was determined by the Bradford assay.…”

Section: Methodsmentioning

confidence: 99%

The C-terminal Region of Carboxypeptidase E Involved in Membrane Binding Is Distinct from the Region Involved with Intracellular Routing

Varlamov

Fricker

1996

Journal of Biological Chemistry

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Carboxypeptidase E (CPE) is involved in the biosynthesis of numerous peptide hormones and neurotransmitters. Previously, the C-terminal region of CPE has been shown to participate in the binding of the protein to membranes and to also contribute to the sorting of CPE into the regulated pathway. In this study, the role of the C-terminal region of CPE was further examined using several approaches. A series of CPE mutants with C-terminal deletions was expressed in the baculovirus system; constructs with a deletion of 14 or 23 residues were expressed at levels comparable to wild-type CPE. In contrast, deletion of 33 or more residues eliminated CPE activity, and the resulting protein was not secreted from the cells. Even though CPE mutants with a deletion of 14 or 23 residues were expressed normally, the resulting protein was mainly soluble, whereas approximately 55% of wild-type CPE was membrane associated. When expressed in AtT-20 cells, CPE with a deletion of 43 C-terminal amino acids was not secreted, whereas CPE with a deletion of 23 residues was secreted via the regulated pathway. Pulse-chase analysis revealed the protein with a deletion of 43 residues to be degraded in a non-acidic intracellular compartment. To investigate whether the C-terminal region of CPE can confer membrane binding and regulated pathway sorting to another protein, portions of the CPE C-terminal region were attached to the C terminus of albumin and the fusion proteins expressed in AtT-20 cells. Of the constructs examined, only the protein containing 51 amino acids of CPE was sorted to the regulated pathway, although with reduced efficiency compared to endogenous CPE. Although the C-terminal 14 amino acids of CPE are sufficient to target albumin to membranes, this fusion protein is not sorted into the regulated pathway. Taken together, these results indicate that the C-terminal 14 amino acids of CPE are important for membrane binding and that membrane binding and sorting require distinct signals.

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Section: Methodsmentioning

confidence: 99%

The C-terminal Region of Carboxypeptidase E Involved in Membrane Binding Is Distinct from the Region Involved with Intracellular Routing

Varlamov

Fricker

1996

Journal of Biological Chemistry

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“…Thyroidectomy leads in the anterior pituitary to a severalfold increase of PAM (Ouafik et al, 1990) and PCl and PC2 (Day et al, 1992) levels and again only a modest rise in carboxypeptidase H mRNA abundance (Smith et al, 1992). In hormone-treated somatomammotroph pituitary cell lines the PC2 mRNA can be up-regulated to a similar extent as the mRNA of prolactin (Seidah et al, 1990), whereas that of carboxypeptidase H is not altered (Fricker et al, 1990~).…”

Section: A High Dose Ofreserpine Causes a Strong Reflex Stimulation Omentioning

confidence: 99%

Large Dense‐Core Vesicles in Rat Adrenal After Reserpine: Levels of mRNAs of Soluble and Membrane‐Bound: Constituents in Chromaffin and Ganglion Cells Indicate a Biosynthesis of Vesicles with Higher Secretory Quanta

Laslop

Mahata

Wolkersdorfer

et al. 1994

Journal of Neurochemistry

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Rats were injected with a large dose of reserpine known to stimulate the adrenal medulla. Various times after drug treatment the mRNA levels of several constituents of large dense-core vesicles were determined by northern blot analysis and in situ hybridization. The latter method allowed detection of changes in mRNA levels not only in chromaffin cells, but also in the ganglion cells found in adrenal medulla. Levels of the mRNAs of secretory components of large dense-core vesicles (chromogranins A and B, secretogranin II, VGF, and neuropeptide Y) increased in chromaffin cells by 21 5 8 5 7 % after 1-3 days of drug treatment. For partly membrane-bound components (dopamine P-hydroxylase, prohormone convertase 2, carboxypeptidase H, and peptidylglycine a-amidating monooxygenase) the changes ranged from 182 to 31 5%, whereas for glycoprotein 111 and for intrinsic membrane proteins (cytochrome bS6, and vesicle monoamine transporter 2) no change occurred. In ganglion cells the mRNAs that could be detected for VGF, neuropeptide Y, secretogranin II, carboxypeptidase H, and vesicle monoamine transporter 1 showed an analogous pattern of change, with significant increases for the secretory proteins and no change for the membrane components. From these and previous results we suggest the following concept: Longlasting stimulation of chromaffin cells or neurons does not induce the biosynthesis of a larger number of vesicles but rather leads to the formation of vesicles containing higher secretory quanta of chromogranins and neuropeptides.Abbrevialions used: LDV, large dense-core vesicle; NPY, neuropeptide Y; PAM, peptidylglycine tu-amidating monooxygenase; PC1 and PC2, prohormone convertase 1 and 2, respectively; vMATl and vMAT2, vesicle monoamine transporter 1 and 2, respectively.

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“…Acid phosphatase activity was determined as described (Fricker et al, 1990) usingp-nitrophenyl phosphate as substrate. Lactate dehydrogenase activity was determined as described (Schwartz and Bodansky, 1966) using sodium pyruvate as substrate.…”

Section: Acid Phosphatase and Lactate Dehydrogenase Assaysmentioning

confidence: 99%

Regulation of Neuropeptide‐Processing Enzymes by Nitric Oxide in Cultured Astrocytes

Devi

Petanceska

Liu

et al. 1994

Journal of Neurochemistry

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Nitric oxide (NO), a recently discovered neurotransmitter, has been shown to have a cytostatic effect on cultured glia. A NO-generating agent, S-nitroso-N-acetylpenicillamine (SNAP), was used to treat C6 glioma and primary cortical astrocytes. The levels of a monobasic peptide-processing enzyme activity and carboxypeptidase E activity were examined. The cellular levels of these two enzymes are specifically reduced in response to treatment with SNAP. A decrease of -30-50% in these two enzyme activities was seen in both primary astrocytes and C6 glioma cells. This decrease in cellular enzyme activities is not due to increased secretion because the secreted activity is also reduced in response to SNAP treatment in both the glioma cells and the primary astrocytes. Removal of SNAP treatment causes the carboxypeptidase enzyme activity to return to control levels within 3 days. Northern and western blot analyses indicate that the reduced cellular level of carboxypeptidase E is not due to reduced expression of the messenger RNA or protein, suggesting that the SNAP treatment is affecting factors that influence carboxypeptidase E activity. Taken together, these results imply that NO is involved in the regulation of peptide biosynthetic enzymes and this could lead to the antimitogenic action of SNAP on glia.

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Comparison of the Regulation of Carboxypeptidase E and Prolactin in GH<sub>4</sub>C<sub>1</sub> Cells, a Rat Pituitary Cell Line

Cited by 28 publications

References 15 publications

The C-terminal Region of Carboxypeptidase E Involved in Membrane Binding Is Distinct from the Region Involved with Intracellular Routing

The C-terminal Region of Carboxypeptidase E Involved in Membrane Binding Is Distinct from the Region Involved with Intracellular Routing

Large Dense‐Core Vesicles in Rat Adrenal After Reserpine: Levels of mRNAs of Soluble and Membrane‐Bound: Constituents in Chromaffin and Ganglion Cells Indicate a Biosynthesis of Vesicles with Higher Secretory Quanta

Regulation of Neuropeptide‐Processing Enzymes by Nitric Oxide in Cultured Astrocytes

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