GRIP1 regulates AMPA receptor targeting to dendrites and the recycling of AMPA receptors to the plasma membrane following NMDA receptor (NMDAR) activation (Setou et al., 2002 and Mao et al., 2010). We, therefore, hypothesized that GRIP1b palmitoylation might in turn affect GRIP1b’s ability to regulate AMPA receptor recycling. To address this possibility, we transfected hippocampal neurons with wild-type, nonpalmitoylatable, or constitutively membrane-targeted forms of GRIP1b, together with a pHluorin-tagged GluA2 AMPA receptor, to which GRIP1 directly binds (Dong et al., 1997 and Mao et al., 2010). The pHluorin tag fluoresces

brightly at neutral pH, as when the receptor Selleck PFT�� is present on the plasma membrane. Brief treatment with NMDA drives internalization STI571 mw of pHGluA2 to recycling endosomes, whose acidity (pH <6.6) dramatically quenches pHGluA2 fluorescence, while NMDA washout induces pHGluA2 recycling to the plasma membrane and fluorescence recovery (Ashby et al., 2004, Lin and Huganir, 2007, Thomas et al., 2008 and Mao et al., 2010; Figure 6A). Fluorescence of pHGluA2,

therefore, acts as a readout of receptor distribution and can be used to determine rates and degrees of internalization and recycling. In particular the T1/2 of fluorescence recovery time, derived from a single exponential fit of the recycling phase, provides a quantitative measure of recycling rate. In neurons transfected with GRIP1bwt or GRIP1bC11S, rates of pHGluA2 internalization and recycling were highly similar to neurons transfected with vector alone (Figure 6B). However, pHGluA2 PAK6 recycling was markedly accelerated in neurons transfected with Myr-GRIP1b (Figure 6C). This accelerated recycling was also seen in neurons transfected with

DHHC5, which is predicted to increase palmitoylation of endogenous GRIP1b (Figures 6D and Figures S5A). Both Myr-GRIP1b and DHHC5 caused accelerated recycling of both somatic and dendritic pHGluA2 (Figures 6 and Figures S5B–S5E). The effect of transfected DHHC5 is likely due to direct palmitoylation of GRIP1b, as although GluA2 is a known palmitoylated protein (Hayashi et al., 2005), it is not detectably palmitoylated by DHHC5 (Figure S5B). AMPA receptor recycling is, therefore, significantly accelerated under conditions where GRIP1b membrane attachment is enhanced (Figures 6E and Figures S5E). Myr-GRIP1b, which is targeted to trafficking vesicles, also colocalized extensively with pH-GluA2 in dendritic puncta in fixed neurons (Figure S5C), suggesting that effects on trafficking were likely due to a direct GRIP1b-pHGluA2 interaction. Here, we report that two PATs use a novel PDZ domain recognition mechanism to palmitoylate and control the distribution and trafficking of GRIP1b.