Mechanism of Action of Secreted Newt Anterior Gradient Protein.
Anterior gradient (AG) proteins have a thioredoxin fold and are targeted to the secretory pathway where they may act in the ER, as well as after secretion into the extracellular space. A newt member of the family (nAG) was previously identified as interacting with the GPI-anchored salamander-specific three-finger protein called Prod1. Expression of nAG has been implicated in the nerve dependence of limb regeneration in salamanders, and nAG acted as a growth factor for cultured newt limb blastemal (progenitor) cells, but the mechanism of action was not understood. Here we show that addition of a peptide antibody to Prod1 specifically inhibit the proliferation of blastema cells, suggesting that Prod1 acts as a cell surface receptor for secreted nAG, leading to S phase entry. Mutation of the single cysteine residue in the canonical active site of nAG to alanine or serine leads to protein degradation, but addition of residues at the C terminus stabilises the secreted protein. The mutation of the cysteine residue led to no detectable activity on S phase entry in cultured newt limb blastemal cells. In addition, our phylogenetic analyses have identified a new Caudata AG protein called AG4. A comparison of the AG proteins in a cell culture assay indicates that nAG secretion is significantly higher than AGR2 or AG4, suggesting that this property may vary in different members of the family.
Institute of Structural and Molecular Biology, Division of Biosciences, University College London, London, United Kingdom.
Kathrin S
KS
Grassme
The Francis Crick Institute, Mill Hill Laboratory, London, United Kingdom.
Acely
A
Garza-Garcia
Institute of Structural and Molecular Biology, Division of Biosciences, University College London, London, United Kingdom.
Jean-Paul
JP
Delgado
Institute of Structural and Molecular Biology, Division of Biosciences, University College London, London, United Kingdom.
James W
JW
Godwin
Institute of Structural and Molecular Biology, Division of Biosciences, University College London, London, United Kingdom.
Anoop
A
Kumar
Institute of Structural and Molecular Biology, Division of Biosciences, University College London, London, United Kingdom.
Phillip B
PB
Gates
The Francis Crick Institute, Mill Hill Laboratory, London, United Kingdom.
Paul C
0000-0002-4124-6950
PC
Driscoll
Institute of Structural and Molecular Biology, Division of Biosciences, University College London, London, United Kingdom.
Jeremy P
JP
Brockes
D000595:Amino Acid Sequence; D029845:Amphibian Proteins / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D049109:Cell Proliferation / Q000235:genetics / Q000502:physiology*; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000378:metabolism*; D009154:Mutation; D010802:Phylogeny; D011485:Protein Binding; D016196:S Phase / Q000235:genetics / Q000502:physiology; D012455:Salamandridae / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D029845:Amphibian Proteins; D002352:Carrier Proteins; D003545:Cysteine