A divergent substrate-binding loop within the pro-oncogenic protein anterior gradient-2 forms a docking site for Reptin.
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.
Maslon
Magdalena M
MM
Cancer Research UK p53 Signal Transduction Laboratories, Cell Signalling Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh EX4 2XR, Scotland, UK.
Hrstka
Roman
R
Vojtesek
Borek
B
Hupp
Ted R
TR
D000074183:ATPases Associated with Diverse Cellular Activities; D000255:Adenosine Triphosphate / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D001665:Binding Sites; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D004265:DNA Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D005260:Female; D015870:Gene Expression; D006801:Humans; D066298:In Vitro Techniques; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009088:Mucoproteins; D046912:Multiprotein Complexes; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015513:Oncogene Proteins; D054730:Protein Interaction Domains and Motifs; D055550:Protein Stability; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D020798:Two-Hybrid System Techniques
C502296:AGR2 protein, human; D002352:Carrier Proteins; D017931:DNA Primers; D009088:Mucoproteins; D046912:Multiprotein Complexes; D050505:Mutant Proteins; D015513:Oncogene Proteins; D011506:Proteins; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D000074183:ATPases Associated with Diverse Cellular Activities; D004265:DNA Helicases; C122079:RUVBL2 protein, human