Mutational analysis of the C-terminus in ion transport peptide (ITP) expressed in Drosophila Kc1 cells.
Ion transport peptide (ITP) stimulates Cl(-) transport (measured as short-circuit current, I(sc)) and fluid reabsorption in Schistocerca gregaria ilea. We report that Drosophila Kc1 cells transfected with preproITP cDNA secrete a peptide (KcITP(75)) that, while cleaved correctly at the N-terminus, had reduced (10-fold) stimulatory activity on ileal I(sc) compared to both native ITP (ScgITP) and synthetic ITP (synITP). We provide evidence that the reduced activity of KcITP(75) is due to incomplete processing of the C-terminal sequence LGKK (KcITP(75)) to L-amide. In support of this, in vitro amidation of glycine extended ITP (i.e., KcITP(73) ending in LG) but not KcITP(75) (ending in LGKK) significantly increased specific activity in the bioassay. Further evidence for C-terminus involvement includes complete loss of stimulation by truncated mutants (e.g., KcITP(71) which lacks LGKK) and a mutant in which alanine is substituted for the terminal glycine in KcITP(73). Moreover a natural homologue (KcITP-L, which differs only in the C-terminal sequence) expressed by Kc1 cells does not stimulate ileal I(sc). Rather KcITP-L acts as a weak ITP antagonist, as does the truncated mutant KcITP(71). KcITP(70) has no antagonistic effect. A short synthetic peptide fragment of the C-terminus (VEIL-amide) does not stimulate ileal I(sc), indicating that other regions of ITP are also essential to biological activity. Arch.
Wang
Y J
YJ
Department of Zoology, University of British Columbia, Vancouver, British Columbia, Canada.
Zhao
Y
Y
Meredith
J
J
Phillips
J E
JE
Theilmann
D A
DA
Brock
H W
HW
D000818:Animals; D001681:Biological Assay; D001692:Biological Transport; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D002460:Cell Line; D018118:Chloride Channels / Q000502:physiology; D004252:DNA Mutational Analysis; D004305:Dose-Response Relationship, Drug; D004330:Drosophila; D006110:Grasshoppers / Q000235:genetics / Q000378:metabolism; D007082:Ileum / Q000187:drug effects / Q000378:metabolism; D019476:Insect Proteins; D016297:Mutagenesis, Site-Directed; D009479:Neuropeptides / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000494:pharmacology; D017422:Sequence Analysis, DNA; D014162:Transfection
D002352:Carrier Proteins; D018118:Chloride Channels; D019476:Insect Proteins; D009479:Neuropeptides; D010446:Peptide Fragments; C079361:ion-transport peptide, Schistocerca gregaria