Expression and characterization of VIP and two VIP mutants in NIH 3T3 cells.
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.
Wulff
B S
BS
University Department of Clinical Biochemistry, Bispebjerg Hospital, Copenhagen NV, Denmark.
Georg
B
B
Fahrenkrug
J
J
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016133:Polymerase Chain Reaction; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014660:Vasoactive Intestinal Peptide / Q000235:genetics* / Q000378:metabolism
D011994:Recombinant Proteins; D014660:Vasoactive Intestinal Peptide; D004247:DNA