title: Analysis of the potential role of GluA4 carboxyl-terminus in PDZ interactions.
abstract: BACKGROUND
  Specific delivery to synapses of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors with long-tailed subunits is believed to be a key event in many forms of activity-dependent changes in synaptic strength. GluA1, the best characterized long-tailed AMPA receptor subunit, contains a C-terminal class I PDZ binding motif, which mediates its interaction with scaffold and trafficking proteins, including synapse-associated protein 97 (SAP97). In GluA4, another long-tailed subunit implicated in synaptic plasticity, the PDZ motif is blocked by a single proline residue. This feature is highly conserved in vertebrates, whereas the closest invertebrate homologs of GluA4 have a canonical class I PDZ binding motif. In this work, we have examined the role of GluA4 in PDZ interactions.
  
  
  RESULTS
  Deletion of the carboxy-terminal proline residue of recombinant GluA4 conferred avid binding to SAP97 in cultured cells as shown by coimmunoprecipitation, whereas wild-type GluA4 did not associate with SAP97. Native GluA4 and SAP97 coimmunoprecipitated from mouse brain independently of the GluA1 subunit, supporting the possibility of in vivo PDZ interaction. To obtain evidence for or against the exposure of the PDZ motif by carboxyterminal processing of native GluA4 receptors, we generated an antibody reagent specific for proline-deleted GluA4 C-terminus. Immunoprecipitation and mass spectrometric analyses indicated that the carboxyl-terminus of native GluA4 AMPA receptors is intact and that the postulated single-residue cleavage does not occur to any significant extent.
  
  
  CONCLUSIONS
  We conclude that native GluA4 receptors are not capable of canonical PDZ interactions and that their association with SAP97 is likely to be indirect.
authors.affiliation: Department of Biosciences, Division of Biochemistry, Viikki Biocenter, University of Helsinki, Helsinki, Finland.
authors.forename: Sarah K
authors.initials: SK
authors.lastname: Coleman
authors.forename: Chunlin
authors.initials: C
authors.lastname: Cai
authors.forename: Nisse
authors.initials: N
authors.lastname: Kalkkinen
authors.forename: Esa R
authors.initials: ER
authors.lastname: Korpi
authors.forename: Kari
authors.initials: K
authors.lastname: Keinänen
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D000074288:Discs Large Homolog 1 Protein; D018698:Glutamic Acid / Q000378:metabolism*; D051528:Guanylate Kinases; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D054731:PDZ Domains; D018091:Receptors, AMPA / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000074288:Discs Large Homolog 1 Protein; C000614189:Dlg1 protein, mouse; D008565:Membrane Proteins; D018091:Receptors, AMPA; D018698:Glutamic Acid; D051528:Guanylate Kinases