Mutational analysis of the C-terminus in ion transport peptide (ITP) expressed in Drosophila Kc1 cells

Ton transport peptide (ITP) stimulates Cl- transport (measured as short-cir cuit current, I-sc) and fluid reabsorption in Schistocerca gregaria ilea, W e report that Drosophila Kcl 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,, compared to both native ITP (ScgITP) and synthetic ITP (synITP), We provide evidence that the redu ced activity of KcITP(75) is due to incomplete processing of the C-terminal sequence LGKK (KcITP75) 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) t ending in LGKK) significantly increased specific activity in the bioassay, Further evidence for C-terminus involvement includes complete loss of stimu lation 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). Mor eover a natural homologue (KcITP-L, which differs only in the C-terminal se quence) expressed by Kcl cells does not stimulate ileal I-sc. Rather KcITP- L acts as a weak ITP antagonist, as does the truncated mutant KcITP(71). Kc ITP(70) has no antagonistic effect, A short synthetic peptide fragment of t he C-terminus (VEIL-amide) does not stimulate ileal I-sc, indicating that o ther regions of ITP are also essential to biological activity. (C) 2001 Wil ey-Liss, Inc.