Phosphorylation of the Postsynaptic Density-95 (PSD-95)/Discs Large/Zona Occludens-1 Binding Site of Stargazin Regulates Binding to PSD-95 and Synaptic Targeting of AMPA Receptors

Dynamic regulation of AMPA-type receptors at the synapse is proposed to play a critical role in alterations of the synaptic strength seen in cellular models of learning and memory such as long-term potentiation in the hippocampus. Stargazin, previously identified as an AMPA receptor (AMPAR)-interacting protein, is critical for surface expression and synaptic targeting of AMPARs. Stargazin interacts with postsynaptic density-95/discs large/zona occludens-1 (PDZ) proteins via a C-terminal PDZ binding motif. Interestingly, the C terminal of stargazin also predicts phosphorylation at a threonine residue critical for PDZ protein binding. Because protein phosphorylation regulates synaptic plasticity, we characterized this site and the effects of stargazin phosphorylation on AMPAR function. In vitro peptide phosphorylation assays and Western blot analysis with phospho-stargazin-specific antibodies indicate that the critical threonine within the stargazin PDZ binding site is phosphorylated by protein kinase A. This phosphorylation disrupts stargazin interaction and clustering with postsynaptic density-95 (PSD-95) in transfected COS-7 cells. Furthermore, a stargazin construct with a Thr-to-Glu mutation that mimics phosphorylation fails to cluster at synaptic spines and downregulates synaptic AMPAR function in cultured hippocampal neurons. These data suggest that phosphorylation of the stargazin PDZ ligand can disrupt stargazin interaction with PSD-95 and thereby regulate synaptic AMPAR function.

[1]  R. Nicoll,et al.  Mechanisms underlying long-term potentiation of synaptic transmission. , 1991, Annual review of neuroscience.

[2]  S. Nakanishi.  Molecular diversity of glutamate receptors and implications for brain function. , 1992, Science.

[3]  T. Bliss,et al.  A synaptic model of memory: long-term potentiation in the hippocampus , 1993, Nature.

[4]  P. Seeburg.  The TINS/TiPS Lecture the molecular biology of mammalian glutamate receptor channels , 1993, Trends in Neurosciences.

[5]  S. Heinemann,et al.  Cloned glutamate receptors. , 1994, Annual review of neuroscience.

[6]  R. Huganir,et al.  Characterization of Multiple Phosphorylation Sites on the AMPA Receptor GluR1 Subunit , 1996, Neuron.

[7]  Solomon H. Snyder,et al.  Binding of the Inward Rectifier K+ Channel Kir 2.3 to PSD-95 Is Regulated by Protein Kinase A Phosphorylation , 1996, Neuron.

[8]  John H. Lewis,et al.  Crystal Structures of a Complexed and Peptide-Free Membrane Protein–Binding Domain: Molecular Basis of Peptide Recognition by PDZ , 1996, Cell.

[9]  E. Ziff.  Enlightening the Postsynaptic Density , 1997, Neuron.

[10]  P. Seeburg,et al.  Interaction of ion channels and receptors with PDZ domain proteins , 1997, Current Opinion in Neurobiology.

[11]  D. Bredt,et al.  PDZ Proteins Organize Synaptic Signaling Pathways , 1998, Cell.

[12]  R. Huganir,et al.  Interaction of the N-Ethylmaleimide–Sensitive Factor with AMPA Receptors , 1998, Neuron.

[13]  C. Mahaffey,et al.  The mouse stargazer gene encodes a neuronal Ca2+-channel γ subunit , 1998, Nature Genetics.

[14]  R. Abagyan,et al.  Novel Anchorage of GluR2/3 to the Postsynaptic Density by the AMPA Receptor–Binding Protein ABP , 1998, Neuron.

[15]  D. Bredt,et al.  N-Terminal Palmitoylation of PSD-95 Regulates Association with Cell Membranes and Interaction with K+ Channel Kv1.4 , 1998, Neuron.

[16]  A. Bretscher,et al.  A kinase-regulated PDZ-domain interaction controls endocytic sorting of the β2-adrenergic receptor , 1999, Nature.

[17]  D. Bredt,et al.  Synaptic Targeting of the Postsynaptic Density Protein PSD-95 Mediated by Lipid and Protein Motifs , 1999, Neuron.

[18]  Andreas Lüthi,et al.  Hippocampal LTD Expression Involves a Pool of AMPARs Regulated by the NSF–GluR2 Interaction , 1999, Neuron.

[19]  J. Noebels,et al.  Identification of three novel Ca(2+) channel gamma subunit genes reveals molecular diversification by tandem and chromosome duplication. , 1999, Genome research.

[20]  Mark von Zastrow,et al.  Role of AMPA Receptor Cycling in Synaptic Transmission and Plasticity , 1999, Neuron.

[21]  R. Huganir,et al.  Organization and regulation of proteins at synapses. , 1999, Current opinion in cell biology.

[22]  R. Nicoll,et al.  PSD-95 involvement in maturation of excitatory synapses. , 2000, Science.

[23]  M. Kennedy,et al.  Signal-processing machines at the postsynaptic density. , 2000, Science.

[24]  R. Huganir,et al.  Phosphorylation of the AMPA Receptor Subunit GluR2 Differentially Regulates Its Interaction with PDZ Domain-Containing Proteins , 2000, The Journal of Neuroscience.

[25]  R. Huganir,et al.  Cerebellar Long-Term Depression Requires PKC-Regulated Interactions between GluR2/3 and PDZ Domain–Containing Proteins , 2000, Neuron.

[26]  R. Huganir,et al.  PDZ domains in synapse assembly and signalling. , 2000, Trends in cell biology.

[27]  G. Collingridge,et al.  PDZ Proteins Interacting with C-Terminal GluR2/3 Are Involved in a PKC-Dependent Regulation of AMPA Receptors at Hippocampal Synapses , 2000, Neuron.

[28]  T. Soderling,et al.  CaM-kinases: modulators of synaptic plasticity , 2000, Current Opinion in Neurobiology.

[29]  Dane M. Chetkovich,et al.  Stargazin regulates synaptic targeting of AMPA receptors by two distinct mechanisms , 2000, Nature.

[30]  M. Ehlers,et al.  Reinsertion or Degradation of AMPA Receptors Determined by Activity-Dependent Endocytic Sorting , 2000, Neuron.

[31]  R. Nicoll,et al.  Synaptic plasticity and dynamic modulation of the postsynaptic membrane , 2000, Nature Neuroscience.

[32]  M. Bear,et al.  Regulation of distinct AMPA receptor phosphorylation sites during bidirectional synaptic plasticity , 2000, Nature.

[33]  Yu Tian Wang,et al.  Regulation of AMPA Receptor–Mediated Synaptic Transmission by Clathrin-Dependent Receptor Internalization , 2000, Neuron.

[34]  Roberto Malinow,et al.  LTP mechanisms: from silence to four-lane traffic , 2000, Current Opinion in Neurobiology.

[35]  N. Klugbauer,et al.  A family of gamma-like calcium channel subunits. , 2000, FEBS Letters.

[36]  N. Klugbauer,et al.  A family of γ‐like calcium channel subunits , 2000 .

[37]  R. Nicoll,et al.  PDZ Protein Interactions Regulating Glutamate Receptor Function and Plasticity , 2001, The Journal of cell biology.

[38]  M. Sheng,et al.  PDZ domains and the organization of supramolecular complexes. , 2001, Annual review of neuroscience.

[39]  Eunjoon Kim,et al.  Phosphorylation of Stargazin by Protein Kinase A Regulates Its Interaction with PSD-95* , 2002, The Journal of Biological Chemistry.

[40]  Kevin J. Tracey,et al.  Synaptic activity at calcium-permeable AMPA receptors induces a switch in receptor subtype , 2022 .