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ARF6 stimulates clathrin/AP-2 recruitment to synaptic membranes by activating phosphatidylinositol phosphate kinase type Igamma.

Krauss M, Kinuta M, Wenk MR, De Camilli P, Takei K, Haucke V - J. Cell Biol. (2003)

Bottom Line: Phosphoinositides have been implicated in nucleating coat assembly by directly binding to several endocytotic proteins including AP-2 and AP180.Here, we show that the stimulatory effect of ATP and GTPgammaS on clathrin coat recruitment is mediated at least in part by increased levels of PIP2.These data suggest a model according to which activation of PIPKIgamma by ARF6-GTP facilitates clathrin-coated pit assembly at the synapse.

View Article: PubMed Central - PubMed

Affiliation: Zentrum für Biochemie und Molekulare Zellbiologie, Dept. of Biochemistry II, University of Göttingen, Humboldtallee 23, Göttingen D-37073, Germany.

ABSTRACT
Clathrin-mediated endocytosis of synaptic vesicle membranes involves the recruitment of clathrin and AP-2 adaptor complexes to the presynaptic plasma membrane. Phosphoinositides have been implicated in nucleating coat assembly by directly binding to several endocytotic proteins including AP-2 and AP180. Here, we show that the stimulatory effect of ATP and GTPgammaS on clathrin coat recruitment is mediated at least in part by increased levels of PIP2. We also provide evidence for a role of ADP-ribosylation factor 6 (ARF6) via direct stimulation of a synaptically enriched phosphatidylinositol 4-phosphate 5-kinase type Igamma (PIPKIgamma), in this effect. These data suggest a model according to which activation of PIPKIgamma by ARF6-GTP facilitates clathrin-coated pit assembly at the synapse.

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PIP2 degradation inhibits receptor-mediated internalization of EGF and transferrin. Overexpression of membrane-targeted HA-tagged inositol phosphatase domain of synaptojanin 1 (HA-IPP1-CAAX) inhibits the internalization of epidermal growth factor (A; EGF) or transferrin (B; Tf). Cos7 cells expressing HA-IPP1-CAAX were allowed to internalize Texas red–labeled epidermal growth factor (A) or Alexa® 488-transferrin (B) for 10 min at 37°C. Cells were fixed and analyzed by immunofluorescence microscopy. Bar, 20 μm.
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fig9: PIP2 degradation inhibits receptor-mediated internalization of EGF and transferrin. Overexpression of membrane-targeted HA-tagged inositol phosphatase domain of synaptojanin 1 (HA-IPP1-CAAX) inhibits the internalization of epidermal growth factor (A; EGF) or transferrin (B; Tf). Cos7 cells expressing HA-IPP1-CAAX were allowed to internalize Texas red–labeled epidermal growth factor (A) or Alexa® 488-transferrin (B) for 10 min at 37°C. Cells were fixed and analyzed by immunofluorescence microscopy. Bar, 20 μm.

Mentions: Consistent with its effects on clathrin/AP-2–coated pit formation and with previous observations (Malecz et al., 2000), cells expressing HA-IPP1-CAAX displayed a strongly reduced ability to internalize Texas red epidermal growth factor (EGF; Fig. 9Figure 9.


ARF6 stimulates clathrin/AP-2 recruitment to synaptic membranes by activating phosphatidylinositol phosphate kinase type Igamma.

Krauss M, Kinuta M, Wenk MR, De Camilli P, Takei K, Haucke V - J. Cell Biol. (2003)

PIP2 degradation inhibits receptor-mediated internalization of EGF and transferrin. Overexpression of membrane-targeted HA-tagged inositol phosphatase domain of synaptojanin 1 (HA-IPP1-CAAX) inhibits the internalization of epidermal growth factor (A; EGF) or transferrin (B; Tf). Cos7 cells expressing HA-IPP1-CAAX were allowed to internalize Texas red–labeled epidermal growth factor (A) or Alexa® 488-transferrin (B) for 10 min at 37°C. Cells were fixed and analyzed by immunofluorescence microscopy. Bar, 20 μm.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172713&req=5

fig9: PIP2 degradation inhibits receptor-mediated internalization of EGF and transferrin. Overexpression of membrane-targeted HA-tagged inositol phosphatase domain of synaptojanin 1 (HA-IPP1-CAAX) inhibits the internalization of epidermal growth factor (A; EGF) or transferrin (B; Tf). Cos7 cells expressing HA-IPP1-CAAX were allowed to internalize Texas red–labeled epidermal growth factor (A) or Alexa® 488-transferrin (B) for 10 min at 37°C. Cells were fixed and analyzed by immunofluorescence microscopy. Bar, 20 μm.
Mentions: Consistent with its effects on clathrin/AP-2–coated pit formation and with previous observations (Malecz et al., 2000), cells expressing HA-IPP1-CAAX displayed a strongly reduced ability to internalize Texas red epidermal growth factor (EGF; Fig. 9Figure 9.

Bottom Line: Phosphoinositides have been implicated in nucleating coat assembly by directly binding to several endocytotic proteins including AP-2 and AP180.Here, we show that the stimulatory effect of ATP and GTPgammaS on clathrin coat recruitment is mediated at least in part by increased levels of PIP2.These data suggest a model according to which activation of PIPKIgamma by ARF6-GTP facilitates clathrin-coated pit assembly at the synapse.

View Article: PubMed Central - PubMed

Affiliation: Zentrum für Biochemie und Molekulare Zellbiologie, Dept. of Biochemistry II, University of Göttingen, Humboldtallee 23, Göttingen D-37073, Germany.

ABSTRACT
Clathrin-mediated endocytosis of synaptic vesicle membranes involves the recruitment of clathrin and AP-2 adaptor complexes to the presynaptic plasma membrane. Phosphoinositides have been implicated in nucleating coat assembly by directly binding to several endocytotic proteins including AP-2 and AP180. Here, we show that the stimulatory effect of ATP and GTPgammaS on clathrin coat recruitment is mediated at least in part by increased levels of PIP2. We also provide evidence for a role of ADP-ribosylation factor 6 (ARF6) via direct stimulation of a synaptically enriched phosphatidylinositol 4-phosphate 5-kinase type Igamma (PIPKIgamma), in this effect. These data suggest a model according to which activation of PIPKIgamma by ARF6-GTP facilitates clathrin-coated pit assembly at the synapse.

Show MeSH
Related in: MedlinePlus