Expression and characterization of VIP and two VIP mutants in NIH 3T3 cells

University Department of Clinical Biochemistry, Bispebjerg Hospital, Copenhagen NV, Denmark.
FEBS Letters (Impact Factor: 3.17). 04/1994; 341(1):43-8. DOI: 10.1016/0014-5793(94)80237-8
Source: PubMed


Prepro-vasoactive intestinal peptide (prepro VIP) was expressed in NIH 3T3 cells, and the prepro VIP-derived peptides produced by the cells were analyzed by chromatography combined with sequence-specific radio-immunoanalysis. In accordance with what has previously been reported on processing in non-endocrine cell lines, the VIP precursor was processed poorly in these non-endocrine cells. Mainly an extended form of VIP could be detected in the media from the cells, and no immunoreactivity specific for amidated VIP was found. However, by changing the dibasic cleavage site positioned N-terminal to the VIP sequence in the precursor into the consensus sequence (Arg, X,Lys/Arg,Arg) for the ubiquitous processing enzyme furin, thought to process, e.g. insulin receptors, factor VII, and by deleting residues 156-170 in the VIP precursor, expression of amidated VIP was obtained in this fibroblast cell line. Peptides from the wild-type VIP precursor as well as peptides from the mutated VIP precursor were found to be able to stimulate the adenylate cyclase in cells expressing the VIP receptor.

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Available from: Birgitte Wulff, Oct 02, 2015
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    ABSTRACT: The precursor for rat vasoactive intestinal polypeptide (preproVIP) is processed by proteolytic cleavage into a signal peptide and five further functional domains: preproVIP 22-79, peptide histidine isoleucine (PHI), preproVIP 111-122, VIP, and preproVIP 156-170. To investigate the biosynthetic processing of preproVIP in peripheral parasympathetic neurons, the sphenopalatine ganglion and one of its projection areas, the nasal mucosa, were used. By immunohistochemistry it was shown that in the sphenopalatine ganglion, preproVIP-derived peptides are localized mainly in neuronal cell bodies, whereas in the nasal mucosa immunoreactivity was found only in nerve fibers and terminals. The peptides were quantified and characterized by radioimmunoassay, HPLC, and gel chromatography using antisera specific for the different precursor products. In the rat sphenopalatine ganglion, the different peptides were found in approximately equimolar amounts, with the exception of PHI and its C-terminally extended variant, PHV, which were present at considerably lower concentrations. However, in the nasal mucosa there was a preferential accumulation of VIP to at least three times the concentration of any of the other peptides. Our results suggest that all preproVIP-derived peptides are present and processed in the sphenopalatine ganglion but that there is a selective accumulation of VIP in the nerve terminals. This indicates that VIP is physiologically the most important transmitter among the preproVIP-derived peptides in parasympathetic nerves originating in the sphenopalatine ganglion.
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    ABSTRACT: The structural requirements of vasoactive intestinal polypeptide (VIP) for receptor binding and cAMP production were studied in a cell line stable transfected with the cDNA for rat VIP receptor 1 (rVIPR 1). Using a number of chimeric constructs of VIP and the homologue peptide secretin, it was found that the N-terminal half of VIP (1-11) can be exchanged with the corresponding sequences in secretin with only modest influence on binding and activation, whereas the opposite chimeras with N-terminal VIP and C-terminal secretin were unable to bind to the VIP receptor. The data suggest that the C-terminal region of VIP is important for receptor binding and activation.
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