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J Mol Biol. 2010 Dec 3;404(3):418-38. doi: 10.1016/j.jmb.2010.09.035. Epub 2010 Oct 1.

A divergent substrate-binding loop within the pro-oncogenic protein anterior gradient-2 forms a docking site for Reptin.

Author information

1
Cancer Research UK p53 Signal Transduction Laboratories, Cell Signalling Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh EX4 2XR, Scotland, UK.

Abstract

Anterior gradient-2 (AGR2) functions in a range of biological systems, including goblet cell formation, limb regeneration, inhibition of p53, and metastasis. There are no well-validated binding proteins for AGR2 protein despite the wealth of data implicating an important cellular function in vertebrates. The yeast two-hybrid system was used to isolate the ATP binding protein Reptin as an AGR2-interacting protein. AGR2 formed a stable complex in human cell lysates with Reptin, thus validating Reptin as an AGR2 binding protein in cells. Reptin was also shown to be overproduced in a panel of primary breast cancer biopsy specimens, relative to normal adjacent tissue from the same patient, suggesting a role in cancer growth in vivo. Mutations were made at the two ATP binding motifs in Reptin to evaluate the effects of ATP on Reptin-AGR2 complex stability. Loss-of-ATP binding mutations at the Walker A motif (K83A) or gain-of-ATP binding mutations at the Walker B motif (D299N) resulted in Reptin mutants with altered oligomerization, thermostability, and AGR2 binding properties. These data indicate that the two ATP binding motifs of Reptin play a role in regulating the stability of the AGR2-Reptin complex. The minimal region of AGR2 interacting with Reptin was localized using overlapping peptide libraries derived from the AGR2 protein sequence. The Reptin docking site was mapped to a divergent octapeptide loop in the AGR2 superfamily between amino acids 104 and 111. Mutations at codon Y104 or F111 in full-length AGR2 destabilized the binding of Reptin. These data highlight the existence of a protein docking motif on AGR2 and an ATP-regulated peptide-binding activity for Reptin. This knowledge has implications for isolating other AGR2-interacting proteins, for developing assays to isolate small molecules that target the Reptin ATP binding site, and for measuring the effects of the Reptin-AGR2 complex in cancer cell growth.

PMID:
20888340
DOI:
10.1016/j.jmb.2010.09.035
[Indexed for MEDLINE]

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