g., the slightly reduced pHGluA2 fluorescence decrease seen with DHHC5 transfection; Figure 6D). 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 find more 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, Fernández-Hernando et al., 2006, Greaves

et al., 2008, Ponimaskin et al., 2008 and 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 (Figure 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, Fernández-Hernando www.selleckchem.com/products/cobimetinib-gdc-0973-rg7420.html et al., 2006, Greaves et al., 2008 and 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 and Li et al., 2010). However, to our knowledge, 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 Terminal deoxynucleotidyl transferase 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 (Gratacòs et al., 2009 and 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 data). 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. The following antibodies, from the indicated sources, were used in this study.