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

Abstract

Stargazin is a transmembrane protein that interacts with AMPARs 1 and regulates their synaptic targeting (1, 2). The stargazer, a spontaneous mutant mouse (3) with defects in the stargazin gene (Cacng2) (4), displays an absence seizure (also known as petit-mal or spike-wave) and, as the name implies, a head-tossing movement, probably because of a defect in the vestibular system (3). In addition, stargazer mice develop an ataxic gait (3) and severe impairment in classical eye-blink conditioning (5), probably because of a cerebellar malfunction. Both mRNA and protein levels of brain-derived neurotrophic factor are selectively reduced in cerebellar granule cells of stargazer mice (5, 6). Stargazin, a protein with a calculated molecular mass of 36 kDa, contains four putative transmembrane domains and a cytosolic C terminus, and its primary structure is closely related to that of the ␥ subunits of voltagegated calcium channels (7-10). Stargazin (or ␥-2) associates with neuronal calcium channel subunits in vivo (11) and inhibits calcium channel activity by increasing steady-state inactivation (4,7,11,12).The functional association between stargazin and AMPAR was initially ascertained by the observation that postsynaptic AMPAR currents are selectively impaired in cerebellar granule cells of stargazer mice (13). A subsequent study revealed that stargazin mediates synaptic targeting of AMPARs by two distinct mechanisms (14). Stargazin initially interacts with AMPARs and assists their translocation to the extrasynaptic surface membrane. Next, the AMPAR-stargazin complex is targeted to synaptic sites by binding to PSD-95 and related PDZ proteins. In support of this hypothesis, a stargazin mutant lacking the last four residues (stargazin⌬C) rescues extrasynaptic but not synaptic AMPAR currents in cerebellar granule cells of stargazer mice (14). However, little is known about whether the stargazin-mediated synaptic targeting of AMPARs is regulated and, if so, what these regulatory mechanisms involve.The C terminus of stargazin contains the end sequence RRT-TPV, which belongs to the class I PDZ-binding motif, (S/T)XV (S/T, Ser or Thr; X, any aa residue; V, hydrophobic residue) (15-17). Interestingly, the RRTT sequence of the C terminus additionally corresponds to the consensus sequence for phosphorylation by PKA, (R/K)(R/X)X(S/T), suggesting that Thr at the Ϫ2 position (RRTTPV, designated T321) is phosphorylated by PKA. The crystal structure of the PDZ3 domain of PSD-95 (class I) complexed with the C terminus of CRIPT, a PSD-95-binding protein that ends with the QTSV sequence (18), reveals that the Thr residue at the Ϫ2 position interacts with His-372 of PDZ3 (15). Specifically, the hydroxyl oxygen of the Thr forms a hydrogen bond with the N-3 nitrogen of His-372. Therefore, phosphorylation of T321 at the stargazin C terminus may weaken the interaction between stargazin and the PDZ domains of PSD-95. Consistently, earlier data demonstrate that phosphorylation of the Ser residue at the Ϫ2 position of Kir2.3 (an inward rectifie...

The PDZ-binding domain is essential for the dendritic targeting of 5-HT2A serotonin receptors in cortical pyramidal neurons in vitro

“…1G-I), confirming that this major synaptic protein of the excitatory post-synaptic density was appropriately sorted to the plasma membrane of cortical pyramidal neurons in culture. Furthermore, PSD-95 is known to associate directly with NMDA ionotropic glutamate receptors (NMDA-R) and indirectly with AMPA ionotropic glutamate receptors (AMPA-R) via stargazin (Kornau et al, 1995;Chen et al, 2000;El-Husseini et al, 2000;Chetkovich et al, 2002;Choi et al, 2002;Schnell et al, 2002), and has been shown to directly interact with the 5-HT 2A receptor (Xia et al, 2003). As expected, PSD-95 in cultured neurons co-localized with NMDA receptors (data not shown) and 5-HT 2A receptors (Fig.…”

The Shank Family of Postsynaptic Density Proteins Interacts with and Promotes Synaptic Accumulation of the βPIX Guanine Nucleotide Exchange Factor for Rac1 and Cdc42

“…Specific ␤PIX antibodies were affinity-purified after immobilizing the protein to a polyvinylidene difluoride membrane. The following antibodies have been described: EGFP (1167) (35), PSD-95 (SM55) (35), Shank (3856, pan-Shank) (10), Shank (1123, pan-Shank) (9), and GIT1 (du139) (36). The following antibodies were obtained from commercial sources: HA rabbit polyclonal (Santa Cruz Biotechnology), FLAG M2 monoclonal (Sigma), synaptophysin SVP38 (Sigma), MAP2 (Sigma), phospho-neurofilament H N52 (Sigma), PAK N-20 (Santa Cruz Biotechnology), vinculin hVIN-1 (Sigma), and p130Cas (Transduction Laboratories).…”

Stargazin and Other Transmembrane AMPA Receptor Regulating Proteins Interact with Synaptic Scaffolding Protein MAGI-2 in Brain

“…For example, PKA phosphorylation of a threonine residue (T321) in the -TTPV motif of ␥2 disrupts binding by PSD-95 (Chetkovich et al, 2002;Choi et al, 2002). Thus, phosphorylation by PKA or other kinases is a reasonable candidate for regulating the synaptic targeting of ␥2 and associated AMPARs by other PDZ proteins in vivo, including MAGI-2.…”