A novel mode of action of an ArfGAP, AMAP2/PAG3/Papa lpha, in Arf6 function.
Previously we reported that AMAP2/PAG3/Papalpha/KIAA0400, a GTPase-activating protein (GAP), acts to antagonize Arf6 function when overexpressed, whereas it was shown to exhibit efficient GAP activities for other Arf isoforms in vitro. Here, we found that AMAP2, through its ArfGAP domain, binds to GTP-Arf6 but not to GDP-Arf6 or other Arfs irrespective of nucleotide status. The majority of AMAP2 was localized to intracellular tubulovesicular structures and redistributed to Arf6-enriched membrane areas upon Arf6 activation. In HeLa cells, Arf6 has been shown to be involved in the clathrin-independent endocytosis of Tac, but not the clathrin-dependent endocytosis of transferrin. We found that Arf6 silencing inhibited the internalization of Tac, but not transferrin, in HeLa cells. Internalization of Tac, but not transferrin, was also significantly inhibited by AMAP2 silencing and overexpression. AMAP2 was moreover found to bind to amphiphysin IIm, a component of the endocytic machinery, via its proline-rich domain. We propose that AMAP2 has dual mechanisms for its function; it exhibits efficient catalytic GAP activity for the class I and II Arfs and yet is involved in the cellular function of the class III Arf without immediate GAP activity. These dual mechanisms of AMAP2 may be important for the cellular function of GTP-Arf6.
Hashimoto
Shigeru
S
Department of Molecular Biology, Osaka Bioscience Institute, Osaka 565-0874, Japan.
Hashimoto
Ari
A
Yamada
Atsuko
A
Kojima
Chie
C
Yamamoto
Hiroko
H
Tsutsumi
Tomonari
T
Higashi
Mikito
M
Mizoguchi
Akira
A
Yagi
Ryohei
R
Sabe
Hisataka
H
D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000378:metabolism / Q000502:physiology*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D017931:DNA Primers; D004705:Endocytosis / Q000502:physiology; D020690:GTPase-Activating Proteins / Q000378:metabolism / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000378:metabolism; D011485:Protein Binding
D000089982:ADP-Ribosylation Factor 6; D017931:DNA Primers; D020690:GTPase-Activating Proteins; D009419:Nerve Tissue Proteins; C075637:amphiphysin; C410332:ASAP2 protein, human; D020727:ADP-Ribosylation Factors; C000715348:ARF6 protein, human