Arfophilin is a common target of both class II and class III ADP-ribosylation factors.
Arfophilin was first identified as a target protein for GTP-ARF5. The N-terminus of ARF5 (amino acids 2-17), which is distinct from that of class I or class III ARFs, is essential for binding to the C-terminus of arfophilin (amino acids 612-756). This study using GST fusion proteins in pulldown experiments in CHO-K1 cell lysates showed that, unexpectedly, ARF6 also bound to full-length arfophilin or the C-terminus of arfophilin (amino acids 612-756) in a GTP-dependent manner. Studies with ARF1/ARF6 chimeras further showed that the amino acid sequence of residues 37-80 of ARF6, which is different from the corresponding sequences in class I and class II ARFs, was essential for binding to arfophilin. Both GTP-ARF5 and GTP-ARF6 bound to arfophilin in CHO-K1 cell lysates, while GTP-ARF1 did not bind. In contrast, all three forms of ARF bound to arfaptin 2, with ARF1 showing the strongest binding. Yeast two-hybrid studies with wild-type, dominant negative, and constitutively active forms of ARF1, -5, and -6 and with ARF1/ARF6 chimeras confirmed these results, except that constitutively active ARF6 was autoactivating. Our findings suggest that both class II and III ARFs may influence the same cellular pathways through arfophilin as a common downstream effector.
Shin
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Howard Hughes Medical Institute and Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
Couvillon
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Exton
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D020823:ADP-Ribosylation Factor 1 / Q000737:chemistry; D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002352:Carrier Proteins / Q000378:metabolism*; D003001:Cloning, Molecular; D006224:Cricetinae; D004926:Escherichia coli; D005982:Glutathione Transferase / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014162:Transfection
D000089982:ADP-Ribosylation Factor 6; D002352:Carrier Proteins; D011993:Recombinant Fusion Proteins; C403131:arfophilin; D006160:Guanosine Triphosphate; D005982:Glutathione Transferase; D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors; C116691:ARF5 protein, human; C000715348:ARF6 protein, human