The autocrine motility factor (AMF) and AMF-receptor combination needs sugar chain recognition ability and interaction using the C-terminal region of AMF.
The autocrine motility factor (AMF) promotes cellular locomotion or invasion, and regulates tumor angiogenesis or ascites accumulation. These signals are triggered by binding between AMF and its receptor (AMFR), a glycoprotein on the cell surface. AMF has been identified as phosphohexose isomerase (PHI). Previous reports have suggested that the substrate-recognition of exo-PHI is significant for receptor binding. Crystallographic studies have shown that AMF consists of three domains, and that the substrate or inhibitor of PHI is stored between the large and small domains, corresponding to approximately residues 117-288. Here, site-directed mutagenesis was used to investigate 18 recombinant human AMF point mutants involving critical amino acid residues for substrate or enzyme inhibitor recognition or binding. Mutation of residues that interact with the phosphate group of the PHI substrate significantly reduced the cell motility-stimulating activity. Their binding capacities for AMFR were also lower than wild-type human AMF. Mutants that retained the enzymic activity showed the motility-stimulating effect and receptor binding and had sensitivity to a PHI inhibitor. Mutant AMFR lacking the N-sugar chain was expressed on the cell membrane but did not respond to AMF-stimulation, and N-glycosidase-treated AMFR did not compete with receptor binding of AMF. Furthermore, the AMF domains that contain the substrate storage domain and C-terminal region stimulate cell locomotion. These results suggest that the N-glyco side-chain of AMFR is a trigger and that interaction between the 117-C-terminal part of AMF and the extracellular core protein of AMFR is needed during AMF-AMFR interactions.
Gifu Pharmaceutical University, 5-6-1 Mitahora-Higashi, Gifu 502-8585, Japan. arayo@gifu-pu.ac.jp
Arayo
A
Haga
Nobutada
N
Tanaka
Tatsuyoshi
T
Funasaka
Kazunori
K
Hashimoto
Kazuo T
KT
Nakamura
Hideomi
H
Watanabe
Avraham
A
Raz
Hisamitsu
H
Nagase
D045744:Cell Line, Tumor; D002465:Cell Movement; D005947:Glucose / Q000378:metabolism*; D005956:Glucose-6-Phosphate Isomerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D060285:Receptors, Autocrine Motility Factor; D018121:Receptors, Cytokine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases
D018121:Receptors, Cytokine; D011994:Recombinant Proteins; C555000:AMFR protein, human; D060285:Receptors, Autocrine Motility Factor; D044767:Ubiquitin-Protein Ligases; D005956:Glucose-6-Phosphate Isomerase; D005947:Glucose