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Mutational analysis of the C‐terminus in ion transport peptide (ITP) expressed in Drosophila Kc1 cells
Author(s) -
Wang YJ.,
Zhao Y.,
Meredith J.,
Phillips J.E.,
Theilmann D.A.,
Brock H.W.
Publication year - 2000
Publication title -
archives of insect biochemistry and physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.576
H-Index - 66
eISSN - 1520-6327
pISSN - 0739-4462
DOI - 10.1002/1520-6327(200011)45:3<129::aid-arch4>3.0.co;2-l
Subject(s) - mutant , peptide , biology , peptide sequence , glycine , transfection , c terminus , alanine , complementary dna , biochemistry , in vitro , amino acid , stimulation , microbiology and biotechnology , n terminus , gene , endocrinology
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. Insect Biochem. Physiol. 45:129–138, 2000. © 2001 Wiley‐Liss, Inc.