Here we report that two PATs use a novel PDZ domain recognition mechanism to palmitoylate and control the distribution and trafficking of GRIP1b. The role of GRIP1b palmitoylation is distinct from that observed for many palmitoyl-proteins: palmitoylation targets GRIP1b to motile trafficking vesicles in neuronal dendrites, and drives accelerated recycling of AMPA-type glutamate receptors. These findings are consistent with both the dendritic localization of the major GRIP1 PAT, DHHC5, and the known role of GRIP1 in the dendritic trafficking of its interacting partners, most notably AMPA-type glutamate receptors (Setou et al., 2002
; Mao et al., 2010
). Why, though, is palmitoylated GRIP1b not detected at the plasma membrane, as observed for several other palmitoylated proteins? A likely explanation is that the GRIP1b N-terminus lacks a second membrane-targeting signal, such as an additional lipid modification site or a polybasic sequence (Sigal et al., 1994
; Dunphy and Linder, 1998; Resh, 2006
). 'Two signal' modification of this type is essential for plasma membrane targeting of GFP, while GFP modified with only a single lipid and lacking a polybasic sequence localizes to intracellular vesicles that are most likely endosomes (McCabe and Berthiaume, 2001
). The `single signal' present in GRIP1b would therefore be predicted to direct targeting to vesicles. Querying databases for conserved N-terminal Cysteines surrounded by non-basic residues may well reveal further proteins that are targeted to vesicles by palmitoylation.
Several lines of evidence support the conclusion that palmitoylated GRIP1b is targeted to dendritic endosomes; endogenous GRIP1b, which is highly palmitoylated, shows a dendritic distribution very similar to the palmitoylation mimic Myr-GRIP1b. Moreover, DHHC5 targets GRIP1bwt, but not the palmitoylation mutant GRIP1b-C11S, to similar dendritic puncta. Notably, though, the endosomal targeting of palmitoylated GRIP1b is distinct from the synaptic targeting described for the closely related palmitoylated GRIP2b (DeSouza et al., 2002
; Misra et al., 2010
). Consistent with these reports, we also observed prominent GRIP2b targeting to dendritic spines, which did not require DHHC5 or DHHC8 co-expression (not shown). Although GRIP1 and GRIP2 can compensate for one another in cerebellar Purkinje neurons (Takamiya et al., 2008
), two related issues likely underlie the distinct regulation of these two proteins in forebrain. First, plasma membrane / synaptic targeting of GRIP2b is consistent with the additional basic residues that surround the palmitoylated Cysteine at the GRIP2b N-terminus, compared to GRIP1b. Second, while the PDZ domains of GRIP1 and GRIP2 are highly homologous, the KIF5-binding region of GRIP1 (between PDZ6 and PDZ7; Setou et al., 2002
) is poorly conserved in GRIP2, suggesting that GRIP1 is unique in its ability to interact with motor proteins that control vesicular cargoes. In further support of their distinct regulation, we also observed no increase in GRIP2b palmitoylation by either DHHC5 or DHHC8 in transfected heterologous cells (not shown), and a much slower GRIP2 palmitoylation turnover rate in neurons, compared to GRIP1b (not shown). Together, these findings suggest that palmitoylated GRIP1b plays a unique role in endosomal trafficking and coupling to kinesin motor proteins.
Our finding that GRIP1b palmitoylation specifically affects activity-dependent AMPA-R recycling would appear to differ from a recent report (Hanley and Henley, 2010), which implicates GRIP1b in NMDA-induced AMPA-R internalization. However, we suspect that experimental differences likely underlie this discrepancy and that our findings more accurately reflect the physiological role of GRIP1b. In particular, Hanley and Henley used Sindbis virus infection to express GRIP1b, a system that has two key issues when used to study intracellular trafficking. First, host cell protein synthesis is shut down, complicating the analysis of intracellular trafficking phenotypes. Second, GRIP1b is over-expressed at high levels, leading to intracellular aggregation (visible in some images from this report (Hanley and Henley, 2010). Moreover, the authors used a large, N-terminal (YFP) tag close to the site of GRIP1b palmitoylation, which may well affect regulation of GRIP1b palmitoylation and/or functional downstream effects that depend on this modification.
We recently developed a more physiological genetic manipulation approach (Mao et al., 2010
) to circumvent many issues associated with GRIP1 overexpression. This approach allowed us to reveal a specific role for GRIP1 in activity-dependent recycling of both endogenous and exogenous (pHluorin-tagged) AMPA-Rs. In contrast, we observed no role for GRIP1 on basal or activity-induced AMPA-R internalization. The findings reported here are highly consistent with the report by (Mao et al., 2010
) and with recent work from our collaborators (Mejias et al., 2011), again observing a role for GRIP1 in activity-dependent AMPA-R recycling. Moreover, in this study we also deliberately transfected only small amounts of plasmid DNA, expressing GRIP1 from a weak promoter (see Methods) to avoid GRIP1b aggregation. Our GRIP1b constructs carried only a small C-terminal myc tag far from the site of palmitoylation, which is unlikely to interfere with GRIP1b function. Evidence from multiple readouts, using both endogenous and exogenous AMPA-Rs, therefore suggests that the predominant physiological role of GRIP1 is to control activity-dependent AMPA-R recycling, and that palmitoylated GRIP1b enhances this process.
We note that, in addition to GRIP1b described here, their prominent dendritic distribution suggests that DHHC5/8 are well placed to palmitoylate other proteins at or near glutamatergic synapses. Although DHHC5 does not palmitoylate GluA2 (Fig S6B
), its targets may include other AMPA-R subunits (Hayashi et al., 2005
), NMDA-Rs (Hayashi et al., 2009
) or other PDZ domain adaptor proteins (Fukata and Fukata, 2010
), all of which are known to be palmitoylated. Indeed, other DHHC5 substrates may underlie effects that cannot be fully attributed to GRIP1b palmitoylation (e.g. the slightly reduced pHGluA2 fluorescence decrease seen with DHHC5 transfection; ). In addition, the presence of transfected DHHC5 in long, aspiny neurites that are likely axons, suggests that DHHC5 may palmitoylate additional axonal / presynaptic substrates in addition to its dendritic regulation of GRIP1b described here.
The identification of additional DHHC5/8 substrates remains an exciting area for future investigation. We note with interest that other PATs cannot compensate for loss of DHHC5/8 to palmitoylate GRIP1 in neurons, and in transfected cells even PATs that display broad substrate specificity (DHHC3, DHHC7; Fukata et al., 2004
; Fernandez-Hernando et al., 2006
; Greaves et al., 2008
; Ponimaskin et al., 2008
; Tsutsumi et al., 2009
) or preferentially palmitoylate cysteines located close to the N-termini of their substrates (DHHC20; Draper and Smith, 2010
) do not palmitoylate GRIP1b (Fig S1
). These findings suggest that GRIP1b palmitoylation by DHHC5/8 has distinct requirements, namely that the PDZ domain interaction unique to DHHC5 and DHHC8 is essential to render GRIP1b accessible as a substrate. DHHC5, in particular, is a major GRIP1b PAT in neurons but cannot palmitoylate several other palmitoyl-proteins (Fukata et al., 2004
; Fernandez-Hernando et al. 2008; Greaves et al., 2008
; Tsutsumi et al., 2009
), suggesting that PDZ domain-dependent recognition is a key determinant of DHHC5 substrate specificity.
Multiple studies link DHHC5 and DHHC8 to both normal higher brain function and neuropsychiatric disease (Mukai et al., 2004
; Mukai et al., 2008
; Li et al., 2010
). However, no neuronal substrates have been identified for DHHC5, and although PSD-95 palmitoylation is reduced in DHHC8 knockout mice (Mukai et al., 2008
), other PATs are also reported to directly palmitoylate PSD-95 in neurons (Noritake et al., 2009
), raising the possibility that this may be an indirect effect. Thus, our identification of GRIP1b as the first bona fide neuronal substrate for DHHC5/8 has broad implications, since GRIP1 is also genetically linked to neuropsychiatric conditions and to autism (Gratacos et al., 2006; Mejias et al., 2011). This raises the possibility that abnormal dendritic and/or synaptic palmitoylation of PDZ domain proteins such as GRIP1 contributes to the pathogenesis of these conditions. Indeed, another PDZ domain protein linked to neuropsychiatric disease is also palmitoylated by DHHC5 and DHHC8 in a PDZ ligand manner (G.M.T., T.H and R.L.H., unpublished). These findings raise the hope that therapeutic targeting of specific PATs and/or their interactions with specific substrates may provide a new approach to better therapeutic treatments for these diseases.