Phosphorylation of Stargazin by Protein Kinase A Regulates Its Interaction with PSD-95*

Stargazin is the first transmembrane protein known to associate with AMPA (α-amino-3-hydroxy-5-methylisoxazole-4-propionate) glutamate receptors (AMPARs) and regulate their synaptic targeting by two distinct mechanisms, specifically via delivery of AMPARs to the surface membrane and synaptic targeting of these receptors by binding to PSD-95/SAP-90 and related PDZ proteins. However, it is not known whether and how this stargazin-mediated synaptic targeting of AMPARs is regulated. Stargazin interacts with the PDZ domains of PSD-95 through the C-terminal PDZ-binding motif. The stargazin C terminus contains a consensus sequence for phosphorylation by cAMP-dependent protein kinase A (PKA). Phosphorylation site-specific stargazin antibodies reveal that the stargazin C terminus is phosphorylated at the Thr-321 residue in heterologous cells and in vivo. Stargazin phosphorylation is enhanced by the catalytic subunit of PKA. Mutations mimicking stargazin phosphorylation (T321E and T321D) lead to elimination of yeast two-hybrid interactions, in vitrocoimmunoprecipitation, and coclustering between stargazin and PSD-95. Phosphorylated stargazin shows a selective loss of coimmunoprecipitation with PSD-95 in heterologous cells and limited enrichment in postsynaptic density fractions of rat brain. These results suggest that phosphorylation of the stargazin C terminus by PKA regulates its interaction with PSD-95 and synaptic targeting of AMPARs.

[1]  H. Robertson,et al.  Calcium channel γ subunits provide insights into the evolution of this gene family , 2001 .

[2]  W. Frankel,et al.  Biochemical and Biophysical Evidence for γ2 Subunit Association with Neuronal Voltage-activated Ca2+Channels* , 2001, The Journal of Biological Chemistry.

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

[4]  J. Noebels,et al.  A Cluster of Three Novel Ca2+ Channel γ Subunit Genes on Chromosome 19q13.4: Evolution and Expression Profile of the γ Subunit Gene Family ☆ , 2001 .

[5]  T. Nakagawa,et al.  A Stargazer Foretells the Way to the Synapse , 2000, Science.

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

[7]  Liaoyuan A. Hu,et al.  β1-Adrenergic Receptor Association with PSD-95 , 2000, The Journal of Biological Chemistry.

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

[9]  P. Osten,et al.  Mutagenesis Reveals a Role for ABP/GRIP Binding to GluR2 in Synaptic Surface Accumulation of the AMPA Receptor , 2000, Neuron.

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

[11]  O. Jones,et al.  Internalization of the Kv1.4 Potassium Channel Is Suppressed by Clustering Interactions with PSD-95* , 2000, The Journal of Biological Chemistry.

[12]  R. Huganir,et al.  Control of GluR1 AMPA Receptor Function by cAMP-Dependent Protein Kinase , 2000, The Journal of Neuroscience.

[13]  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.

[14]  Masahiko Watanabe,et al.  Impairment of AMPA Receptor Function in Cerebellar Granule Cells of Ataxic Mutant Mouse Stargazer , 1999, The Journal of Neuroscience.

[15]  M. Sheng,et al.  Biochemical and immunocytochemical characterization of GRIP, a putative AMPA receptor anchoring protein, in rat brain , 1998, Neuropharmacology.

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

[17]  Richard F. Thompson,et al.  Cerebellar Brain-Derived Neurotrophic Factor–TrkB Defect Associated with Impairment of Eyeblink Conditioning in StargazerMutant Mice , 1998, The Journal of Neuroscience.

[18]  J. McNamara,et al.  Stargazing nets new calcium channel subunit , 1998, Nature Genetics.

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

[20]  Ann Marie Craig,et al.  CRIPT, a Novel Postsynaptic Protein that Binds to the Third PDZ Domain of PSD-95/SAP90 , 1998, Neuron.

[21]  R. Huganir,et al.  Phosphorylation of the α-Amino-3-hydroxy-5-methylisoxazole4-propionic Acid Receptor GluR1 Subunit by Calcium/ Calmodulin-dependent Kinase II* , 1997, The Journal of Biological Chemistry.

[22]  Richard L. Huganir,et al.  GRIP: a synaptic PDZ domain-containing protein that interacts with AMPA receptors , 1997, Nature.

[23]  M. Sheng,et al.  GKAP, a Novel Synaptic Protein That Interacts with the Guanylate Kinase-like Domain of the PSD-95/SAP90 Family of Channel Clustering Molecules , 1997, The Journal of cell biology.

[24]  L. Cantley,et al.  Recognition of Unique Carboxyl-Terminal Motifs by Distinct PDZ Domains , 1997, Science.

[25]  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.

[26]  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.

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

[28]  F. Hefti,et al.  Selective failure of brain-derived neurotrophic factor mRNA expression in the cerebellum of stargazer, a mutant mouse with ataxia , 1996, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[29]  Y. Jan,et al.  Clustering of Shaker-type K+ channels by interaction with a family of membrane-associated guanylate kinases , 1995, Nature.

[30]  M. Kennedy,et al.  The rat brain postsynaptic density fraction contains a homolog of the drosophila discs-large tumor suppressor protein , 1992, Neuron.

[31]  M. T. Davisson,et al.  Stargazer: a new neurological mutant on chromosome 15 in the mouse with prolonged cortical seizures , 1990, Epilepsy Research.

[32]  R. Maurer.  Both isoforms of the cAMP-dependent protein kinase catalytic subunit can activate transcription of the prolactin gene. , 1989, The Journal of biological chemistry.

[33]  P Siekevitz,et al.  Isolation and characterization of postsynaptic densities from various brain regions: enrichment of different types of postsynaptic densities , 1980, The Journal of cell biology.

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