As reported previously (Beattie et al , 2000, Carroll et al , 199

As reported previously (Beattie et al., 2000, Carroll et al., 1999 and Ehlers, 2000), the surface HA-GluA2, which is mainly localized to the somatodendritic domain (Matsuda et al., 2008), rapidly decreased following NMDA stimulation (Figures 4A and 4E). When the GFP-tagged C-terminal fragment of PIP5Kγ661 (PIP5K-CT-WT) was expressed, the NMDA-induced reduction of surface AMPA receptors was blocked (Figures 4B and 4E). Nutlin-3a cost Similarly, the expression of the GFP-tagged dephosphomimetic

mutant of the PIP5Kγ661 C-terminal fragment (PIP5K-CT-S645A), in which Ala replaced Ser 645, blocked NMDA-induced AMPA receptor endocytosis (Figures 4C and 4E). In contrast, expression of GFP-tagged phosphomimetic mutant of the PIP5Kγ661 C-terminal fragment (PIP5K-CT-S645E) failed to do so (Figures 4D and 4E). GST pull-down assays confirmed that wild-type and PIP5K-CT-S645A, but not PIP5K-CT-S645E, bound to β2 adaptin (Figure S5A). These results suggest that overexpression of the wild-type and dephosphomimetic C-terminal PS-341 cost fragment of PIP5Kγ661 interferes with the interaction between endogenous PIP5Kγ661 and AP-2, thereby inhibiting the NMDA-induced AMPA receptor endocytosis. The C

terminus of PIP5Kγ661 is also reported to bind to the μ2 adaptin of AP-2 (Bairstow et al., 2006) and talin (Di Paolo et al., 2002 and Ling et al., 2002). In addition, β2 adaptin binds to clathrin through its ear and hinge domains (Thieman et al., 2009) and to hippocalcin, a protein that translocates to the plasma membrane in the presence of Ca2+ during hippocampal LTD (Palmer et al., 2005). Nevertheless, none of these interactions are reported to be affected by the dephosphorylation of the C-terminal domain of PIP5Kγ661. To examine the specificity of the dephosphorylation-dependent binding of PIP5Kγ661 Org 27569 to β2 adaptin, we performed

GST pull-down assays. Although β2 adaptin bound to GST-CT-WT and GST-CT-S645A with significantly higher affinity than to GST-CT-S645E, μ2 adaptin equally interacted with all GST-fused C-terminal fragments of PIP5Kγ661 (Figure S5A). In addition, although PIP5Kγ661 binding to β2 adaptin was specifically inhibited by the dephophomimetic C-terminal peptide of PIP5Kγ661 (pep-S645A), the clathrin binding to β2 adaptin was not affected by either phophomimetic (pep-S645E) or dephosphomimetic (pep-S645A) peptide (Figure S5B). Furthermore, binding of CT-WT to talin was inhibited by pep-S645A and pep-S645E to the same degree (Figure S5C). The interaction between β2 adaptin and hippocalcin in the presence of Ca2+ was also similarly inhibited by dephosphomimetic or phosphomimetic PIP5Kγ661 C-terminal peptides and fragments (Figures S5D). These results indicate that the dephosphorylated form-specific effect of PIP5Kγ661 (Figure 4) is likely mediated by its specific interaction with β2 adaptin and that this specific interaction is required for NMDA-induced AMPA receptor endocytosis.

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