Tyrosine dephosphorylation regulates AMPAR internalisation in mGluR-LTD
Introduction
Cellular and molecular mechanisms underlying synaptic plasticity in the central nervous system (CNS) are thought to be involved in memory acquisition. Synaptic plasticity is categorised into two main types, long-term depression (LTD) and long-term potentiation (LTP) involving either a long-lasting decrease or increase in synaptic efficacy, respectively. There are two main forms of coexisting LTD at hippocampal CA1 synapses which are dependent either on synaptic NMDAR activation (NMDAR-LTD; Dudek and Bear, 1992, Mulkey and Malenka, 1992) or on mGluR activation (mGluR-LTD; Bashir et al., 1993, Bolshakov and Siegelbaum, 1994). Bath application of NMDA and the group I selective mGluR agonist (RS)-3,5-dihydroxyphenylglycine (DHPG) can be used to induce robust NMDAR-LTD (NMDA-LTD; Lee et al., 1998) and mGluR-LTD (DHPG-LTD; Palmer et al., 1997, Fitzjohn et al., 1999), respectively. DHPG has been widely used to investigate the induction and expression mechanisms underlying mGluR-LTD in hippocampal neurons (e.g., Palmer et al., 1997, Fitzjohn et al., 1999, Fitzjohn et al., 2001, Schnabel et al., 1999, Huber et al., 2000, Rouach and Nicoll, 2003, Tan et al., 2003, Gallagher et al., 2004, Huang et al., 2004, Moult et al., 2006). A major advantage of DHPG and NMDA induced LTD is that synchronised changes at a large number of synapses facilitate the monitoring of biochemical events.
Characteristically NMDAR-LTD includes the following: (1) increased postsynaptic calcium concentration [Ca2+]i (Mulkey and Malenka, 1992), (2) activation of a serine/threonine phosphatase cascade (Mulkey et al., 1993, Mulkey et al., 1994), (3) activation of GSK-3β (Peineau et al., 2007) and (4) internalisation of AMPARs via an NSF/AP2/clathrin-dependent mechanism (Nishimune et al., 1998, Lüscher et al., 1999, Luthi et al., 1999, Noel et al., 1999, Beattie et al., 2000, Man et al., 2000, Carroll et al., 2001, Lee et al., 2002, Rammes et al., 2003, Ashby et al., 2004).
In contrast to NMDAR-LTD, DHPG-induced mGluR-LTD is Ca2+ independent (Fitzjohn et al., 2001) and does not involve serine/threonine phosphatases (Schnabel et al., 2001). However, mGluR-LTD is dependent on G-protein activation (Kleppisch et al., 2001, Watabe et al., 2002, Huang et al., 2004), protein tyrosine phosphatases (PTPs; Moult et al., 2002, Huang and Hsu, 2006, Moult et al., 2006), MAPK cascades (Rush et al., 2002, Gallagher et al., 2004, Huang et al., 2004, Li et al., 2007b, Moult et al., 2008), arachidonic acid (AA; Feinmark et al., 2003), phosphoinositide-3-kinase (PI3K)-Akt-mammalian target of the rapamycin (mTOR) signalling pathways (Hou and Klann, 2004), cyclinD1–cyclin-dependent kinase 4 (CDK4) complex expression (Li et al., 2007a) and postsynaptic protein synthesis (Huber et al., 2000, Huber et al., 2001, Hou and Klann, 2004).
Phosphorylation is a key post-translational modification that facilitates the precise regulation of synaptic proteins necessary for modulation of synaptic transmission. Dynamic changes in phosphorylation can alter electrophysiological characteristics, protein–protein interactions and synaptic delivery or internalisation of AMPARs (reviewed in Molnár, 2008). These changes underlie the major molecular mechanisms that affect many forms of synaptic plasticity. Compared to roles of serine/threonine phosphorylation/dephosphorylation of AMPAR subunits, much less is known about the role of tyrosine phosphorylation/dephosphorylation in synaptic plasticity. While previous studies demonstrated that DHPG-LTD induction involves tyrosine dephosphorylation of AMPARs (Huang and Hsu, 2006, Moult et al., 2006), details of the underlying molecular and cellular mechanisms have not been established.
In the present study we have investigated: (1) the involvement of individual AMPAR subunits in DHPG-LTD induction, (2) differential changes in endogenous AMPAR subunit phosphorylation in DHPG-LTD and NMDA-LTD and (3) the role of tyrosine dephosphorylation in AMPAR endocytosis in DHPG-LTD. We show that tyrosine dephosphorylation is a specific process that is associated with DHPG-LTD. Thus, while both GluR2 and GluR3 AMPAR subunits are tyrosine phosphorylated at the basal state, only GluR2 is tyrosine dephosphorylated in DHPG-LTD. Furthermore, we find no evidence for tyrosine dephosphorylation in NMDA-LTD. To address how tyrosine dephosphorylation is involved in DHPG-LTD we have used surface biotinylation assays. We find that DHPG-LTD is associated with a temperature-dependent internalisation of AMPARs and that changes in tyrosine phosphorylation are observed only on the cell surface. This suggests that tyrosine dephosphorylation is a trigger for the internalisation of AMPARs during DHPG-LTD.
Section snippets
Analysis of changes in GluR2 and GluR3 tyrosine phosphorylation in DHPG-LTD
While it was previously shown that DHPG-LTD entails tyrosine dephosphorylation of the GluR2 subunit and that GluR1 has very low basal tyrosine phosphorylation levels (Moult et al., 2006), the involvement of the functionally important and structurally highly similar GluR3 AMPAR subunit has not been established. Therefore, our first aim was to determine if GluR3 is phosphorylated under basal conditions and whether changes in GluR3 tyrosine phosphorylation contribute to mGluR-dependent LTD
Discussion
Recent studies revealed that DHPG-LTD is dependent on activation of postsynaptic PTPs which tyrosine dephosphorylate AMPARs (Huang and Hsu, 2006, Moult et al., 2006, Zhang et al., 2008). Here we established that: (1) both GluR2 and GluR3 AMPAR subunits are tyrosine phosphorylated, but only GluR2 is dephosphorylated in DHPG-LTD. (2) Tyrosine dephosphorylation of GluR2 is a specific feature of DHPG-LTD in that it does not occur in NMDA-LTD. (3) PTP-mediated dephosphorylation of cell surface
Materials
(RS)-3,5-DHPG, NMDA, the NMDAR antagonist L-689 560 (trans-2-carboxy-5,7-dichloro-4-phenylaminocarbonylanmino-1,2,3,4-tetrahydroquinoline), the GABAA receptor antagonist picrotoxin (PTX) were all obtained from Tocris Bioscience (Bristol, UK). The PTP inhibitor orthovanadate and all other chemicals were obtained from Sigma-Aldrich Company Ltd. (Gillingham, UK) unless otherwise stated. The following antibodies were used: immunoaffinity purified rabbit polyclonal C-terminal anti-GluR1 (Upstate
Acknowledgments
We are grateful to the Medical Research Council (MRC) UK, the Wellcome Trust, the Biotechnology and Biological Sciences Research Council (BBSRC) UK, the Ontario Mental Health Foundation and the Canadian Institutes of Health Research (CIHR; MOP-42396) for financial support. CMG was an MRC funded PhD student. We would like to thank Dr. Stephen Fitzjohn for helpful advice given during the course of this study.
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