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 vitro coimmunoprecipitation, 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. 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 vitro coimmunoprecipitation, 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. Stargazin is a transmembrane protein that interacts with AMPARs 1The abbreviations used are: AMPARAMPA (α-amino-3-hydroxy-5-methylisoxazole-4-propionate) glutamate receptoraaamino acid(s)PKAprotein kinase APSDpostsynaptic densityEGFPenhanced green fluorescent protein and regulates their synaptic targeting (1.Tomita S. Nicoll R.A. Bredt D.S. J. Cell Biol. 2001; 153: F19-F24Crossref PubMed Google Scholar, 2.Nakagawa T. Sheng M. Science. 2000; 290: 2270-2271PubMed Google Scholar). The stargazer, a spontaneous mutant mouse (3.Noebels J.L. Qiao X. Bronson R.T. Spencer C. Davisson M.T. Epilepsy Res. 1990; 7: 129-135Crossref PubMed Scopus (177) Google Scholar) with defects in the stargazin gene (Cacng2) (4.Letts V.A. Felix R. Biddlecome G.H. Arikkath J. Mahaffey C.L. Valenzuela A. Bartlett 2nd, F.S. Mori Y. Campbell K.P. Frankel W.N. Nat. Genet. 1998; 19: 340-347Crossref PubMed Scopus (489) Google Scholar), 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.Noebels J.L. Qiao X. Bronson R.T. Spencer C. Davisson M.T. Epilepsy Res. 1990; 7: 129-135Crossref PubMed Scopus (177) Google Scholar). In addition, stargazer mice develop an ataxic gait (3.Noebels J.L. Qiao X. Bronson R.T. Spencer C. Davisson M.T. Epilepsy Res. 1990; 7: 129-135Crossref PubMed Scopus (177) Google Scholar) and severe impairment in classical eye-blink conditioning (5.Qiao X. Chen L. Gao H. Bao S. Hefti F. Thompson R.F. Knusel B. J. Neurosci. 1998; 18: 6990-6999Crossref PubMed Google Scholar), 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.Qiao X. Chen L. Gao H. Bao S. Hefti F. Thompson R.F. Knusel B. J. Neurosci. 1998; 18: 6990-6999Crossref PubMed Google Scholar, 6.Qiao X. Hefti F. Knusel B. Noebels J.L. J. Neurosci. 1996; 16: 640-648Crossref PubMed Google Scholar). 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 γ subunits of voltage-gated calcium channels (7.Klugbauer N. Dai S. Specht V. Lacinova L. Marais E. Bohn G. Hofmann F. FEBS Lett. 2000; 470: 189-197Crossref PubMed Scopus (153) Google Scholar, 8.Burgess D.L. Davis C.F. Gefrides L.A. Noebels J.L. Genome Res. 1999; 9: 1204-1213Crossref PubMed Scopus (56) Google Scholar, 9.Chu P.J. Robertson H.M. Best P.M. Gene. 2001; 280: 37-48Crossref PubMed Scopus (81) Google Scholar, 10.Burgess D.L. Gefrides L.A. Foreman P.J. Noebels J.L. Genomics. 2001; 71: 339-350Crossref PubMed Scopus (85) Google Scholar). Stargazin (or γ-2) associates with neuronal calcium channel subunits in vivo (11.Kang M.G. Chen C.C. Felix R. Letts V.A. Frankel W.N. Mori Y. Campbell K.P. J. Biol. Chem. 2001; 276: 32917-32924Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar) and inhibits calcium channel activity by increasing steady-state inactivation (4.Letts V.A. Felix R. Biddlecome G.H. Arikkath J. Mahaffey C.L. Valenzuela A. Bartlett 2nd, F.S. Mori Y. Campbell K.P. Frankel W.N. Nat. Genet. 1998; 19: 340-347Crossref PubMed Scopus (489) Google Scholar, 7.Klugbauer N. Dai S. Specht V. Lacinova L. Marais E. Bohn G. Hofmann F. FEBS Lett. 2000; 470: 189-197Crossref PubMed Scopus (153) Google Scholar, 11.Kang M.G. Chen C.C. Felix R. Letts V.A. Frankel W.N. Mori Y. Campbell K.P. J. Biol. Chem. 2001; 276: 32917-32924Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 12.Puranam R.S. McNamara J.O. Nat. Genet. 1998; 19: 313-314Crossref PubMed Scopus (4) Google Scholar). AMPA (α-amino-3-hydroxy-5-methylisoxazole-4-propionate) glutamate receptor amino acid(s) protein kinase A postsynaptic density enhanced green fluorescent protein 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.Hashimoto K. Fukaya M. Qiao X. Sakimura K. Watanabe M. Kano M. J. Neurosci. 1999; 19: 6027-6036Crossref PubMed Google Scholar). A subsequent study revealed that stargazin mediates synaptic targeting of AMPARs by two distinct mechanisms (14.Chen L. Chetkovich D.M. Petralia R.S. Sweeney N.T. Kawasaki Y. Wenthold R.J. Bredt D.S. Nicoll R.A. Nature. 2000; 408: 936-943Crossref PubMed Scopus (9) Google Scholar). 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 (stargazinΔC) rescues extrasynaptic but not synaptic AMPAR currents in cerebellar granule cells of stargazer mice (14.Chen L. Chetkovich D.M. Petralia R.S. Sweeney N.T. Kawasaki Y. Wenthold R.J. Bredt D.S. Nicoll R.A. Nature. 2000; 408: 936-943Crossref PubMed Scopus (9) Google Scholar). 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 RRTTPV, 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.Doyle D.A. Lee A. Lewis J. Kim E. Sheng M. MacKinnon R. Cell. 1996; 85: 1067-1076Abstract Full Text Full Text PDF PubMed Scopus (976) Google Scholar, 16.Sheng M. Sala C. Annu. Rev. Neurosci. 2001; 24: 1-29Crossref PubMed Scopus (1049) Google Scholar, 17.Songyang Z. Fanning A.S. Fu C. Xu J. Marfatia S.M. Chishti A.H. Crompton A. Chan A.C. Anderson J.M. Cantley L.C. Science. 1997; 275: 73-77Crossref PubMed Scopus (1224) Google Scholar). 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 −2 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.Niethammer M. Valtschanoff J.G. Kapoor T.M. Allison D.W. Weinberg T.M. Craig A.M. Sheng M. Neuron. 1998; 20: 693-707Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar), reveals that the Thr residue at the −2 position interacts with His-372 of PDZ3 (15.Doyle D.A. Lee A. Lewis J. Kim E. Sheng M. MacKinnon R. Cell. 1996; 85: 1067-1076Abstract Full Text Full Text PDF PubMed Scopus (976) Google Scholar). 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 −2 position of Kir2.3 (an inward rectifier potassium channel) by PKA disrupts interactions with PSD-95 (19.Cohen N.A. Brenman J.E. Snyder S.H. Bredt D.S. Neuron. 1996; 17: 759-767Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). Here we report that PKA phosphorylates the stargazin C terminus and consequently regulates its interaction with PSD-95. In view of the hypothesis that stargazin regulates synaptic targeting of AMPARs by binding to PSD-95 and related PDZ proteins, our results suggest that phosphorylation of the C terminus of stargazin regulates AMPAR synaptic targeting. Anti-fusion protein stargazin antibody (Stg-Cyto 1222, guinea pig polyclonal) was generated using an H6 fusion protein containing two copies of the C-terminal cytoplasmic region of stargazin (aa 203–323) as immunogen. Anti-peptide stargazin antibody (Stg-C-term 1217, rabbit polyclonal) was generated using a synthetic peptide mimicking the last 10 aa residues of stargazin (CNTANRRTTPV). The underlined cysteine residue was added for coupling to keyhole limpet hemocyanin or the SulfoLink column (Pierce). Affinity purification of antibodies was performed using the SulfoLink column coupled with the peptides. To generate phosphorylation site-specific stargazin antibodies (Stg-pT321 1218 rabbit and 1229 guinea pig polyclonal), the same synthetic stargazin C-terminal peptide with phosphorylated T321 was employed as immunogen. For affinity purification of the Stg-pT321 antibody, antisera were passed through the SulfoLink column coupled with the phophorylated peptide followed by a column coupled with unphosphorylated C-terminal peptide as described previously (20.Mammen A.L. Kameyama K. Roche K.W. Huganir R.L. J. Biol. Chem. 1997; 272: 32528-32533Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar). Rabbit polyclonal EGFP (1167) and PSD-95 (SM55) antibodies were generated using H6-EGFP (aa 1–240) and H6-PSD-95 (aa 77–299) (21.Kim E. Niethammer M. Rothschild A. Jan Y.N. Sheng M. Nature. 1995; 378: 85-88Crossref PubMed Scopus (900) Google Scholar) as immunogens. The PSD-95 (HM319) antibody is described in the literature (21.Kim E. Niethammer M. Rothschild A. Jan Y.N. Sheng M. Nature. 1995; 378: 85-88Crossref PubMed Scopus (900) Google Scholar). The yeast two-hybrid assay was performed as described earlier (21.Kim E. Niethammer M. Rothschild A. Jan Y.N. Sheng M. Nature. 1995; 378: 85-88Crossref PubMed Scopus (900) Google Scholar). The L40 yeast strain harboring reporter genes HIS3 and LacZ, under control of the upstream LexA DNA-binding domain was used in the assay. For pBHA (a bait vector containing the LexA DNA-binding domain) constructs, the last 121 aa residues (aa 203–323) of stargazin were amplified by PCR and subcloned in-frame into pBHA. Mutant pBHA stargazin constructs (RRTDPV, RRTEPV, and RRTTPA) were generated using the QuikChange site-directed mutagenesis kit (Stratagene). For pGAD10 constructs, the following PDZ domains were subcloned in-frame into pGAD10 (a prey vector, CLONTECH): PDZ1 (aa 224–311), PDZ2 (aa 318–404), PDZ3 (aa 465–545), and PDZ1–2 (aa 224–404) of SAP97; PDZ 4–6 (aa 463–761) of GRIP1. pGAD10 constructs containing the PDZ domains of PSD-95 are described (21.Kim E. Niethammer M. Rothschild A. Jan Y.N. Sheng M. Nature. 1995; 378: 85-88Crossref PubMed Scopus (900) Google Scholar). Stargazin-PDZ interactions were measured by semiquantitative yeast two-hybrid assays using his3 and lacZ as reporter genes. The entire open reading frame and 75 bp of the 5′ untranslated region of stargazin were amplified by RT-PCR using mouse brain total RNA, digested with HindIII and EcoRI, and subcloned into GW1 (British Biotechnology). For EGFP tagging of stargazin (EGFP-stargazin), a fragment containing EGFP (aa 1–240) was amplified by PCR and subcloned in-frame into the BglII site at the C-terminal cytoplasmic region of stargazin, thus generating a construct containing EGFP between aa 269 and 270 of the protein. Mutant stargazin expression constructs with RRTDPV, RRTEPV and RRTTPA at the C-terminal end were generated using the QuikChange kit (Stratagene) with GW1 EGFP-stargazin as template. GW1 PSD-95 is described in the literature (21.Kim E. Niethammer M. Rothschild A. Jan Y.N. Sheng M. Nature. 1995; 378: 85-88Crossref PubMed Scopus (900) Google Scholar). For coimmunoprecipitation experiments, COS cells were transfected with combinations of PSD-95, EGFP-stargazin (wild-type and mutants) and the PKA catalytic subunit using LipofectAMINE (Invitrogen), extracted with phosphate-buffered saline containing 1% Triton X-100, incubated with PSD-95 (HM319) antibodies (4 μg/ml), and precipitated with protein A-Sepharose (Amersham Biosciences, Inc.), followed by immunoblotting with EGFP (1 μg/ml), Stg-pT321 (1218, 1 μg/ml), or PSD-95 (SM55, 1 μg/ml) antibodies. The coclustering assay was performed as described earlier (22.Kim E. Naisbitt S. Hsueh Y.P. Rao A. Rothschild A. Craig A.M. Sheng M. J. Cell Biol. 1997; 136: 669-678Crossref PubMed Scopus (434) Google Scholar). Briefly, COS-7 cells doubly transfected with GW1 PSD-95 and stargazin (wild-type and mutants) were fixed with 2% paraformaldehyde, permeabilized with 0.1% Triton X-100, and stained with EGFP (1167) and PSD-95 (HM319) antibodies followed by double immunofluorescence staining with fluorescein isothiocyanate- and Cy3-conjugated secondary antibodies (Jackson ImmunoResearch). Images were captured by confocal laser scanning microscopy (LSM510, Zeiss). PSD fractions were purified as described (23.Carlin R.K. Grab D.J. Cohen R.S. Siekevitz P. J. Cell Biol. 1980; 86: 831-845Crossref PubMed Scopus (606) Google Scholar, 24.Cho K.O. Hunt C.A. Kennedy M.B. Neuron. 1992; 9: 929-942Abstract Full Text PDF PubMed Scopus (1008) Google Scholar). To obtain PSD fractions, the synaptosomal fraction was extracted with detergents, once with Triton X-100 (PSD I), twice with Triton X-100 (PSD II), once with Triton X-100, and once with sarcosyl (PSD III). PSD fractions were immunoblotted with Stg-pT321 (1229, 1 μg/ml), Stg-Cyto (1222, 1 μg/ml), Stg-C-term 1 μg/ml), PSD-95 (SM55, 1 μg/ml), and antibodies. To whether the T321 residue of stargazin is the phosphorylation we generated phosphorylation site-specific antibodies rabbit and 1229 guinea using the last 10 residues of stargazin with phosphorylated T321 as immunogen. Stg-pT321 antibodies were stargazin in heterologous cells of COS transfected with stargazin with Stg-pT321 antibodies revealed a protein of about stargazin In mutant stargazin lacking the hydroxyl for phosphorylation was not by Stg-pT321 antibodies of Stg-pT321 antibodies with phosphorylated not peptide the of EGFP-stargazin of the membrane with of the EGFP-stargazin by Stg-pT321 antibodies These results that stargazin is phosphorylated at T321 in heterologous To whether T321 of stargazin is phosphorylated in we performed immunoblotting protein rat brain with stargazin antibodies Both Stg-pT321 antibodies and a of about to the of stargazin (4.Letts V.A. Felix R. Biddlecome G.H. Arikkath J. Mahaffey C.L. Valenzuela A. Bartlett 2nd, F.S. Mori Y. Campbell K.P. Frankel W.N. Nat. Genet. 1998; 19: 340-347Crossref PubMed Scopus (489) Google Scholar) in synaptosomal and membrane stargazin were by stargazin antibodies the C-terminal peptide last 10 residues with unphosphorylated and the entire C-terminal cytoplasmic region (aa data that stargazin is phosphorylated at the T321 residue in vivo. is that Stg-pT321 and Stg-C-term antibodies may closely related as the and subunits of calcium channels (7.Klugbauer N. Dai S. Specht V. Lacinova L. Marais E. Bohn G. Hofmann F. FEBS Lett. 2000; 470: 189-197Crossref PubMed Scopus (153) Google Scholar, 8.Burgess D.L. Davis C.F. Gefrides L.A. Noebels J.L. Genome Res. 1999; 9: 1204-1213Crossref PubMed Scopus (56) Google Scholar, 10.Burgess D.L. Gefrides L.A. Foreman P.J. Noebels J.L. Genomics. 2001; 71: 339-350Crossref PubMed Scopus (85) Google Scholar), because C-terminal last residues are and calculated molecular in To whether the stargazin C terminus is a of PKA, we phosphorylation levels of this region of stargazin in COS cells doubly transfected with stargazin and the PKA catalytic subunit (wild-type and an COS cells transfected with stargazin were immunoblotted with Stg-pT321 a but of phosphorylated stargazin was suggesting that stargazin is phosphorylated in COS cells Stargazin phosphorylation was with the PKA catalytic subunit but not with an catalytic subunit R.A. J. Biol. Chem. Full Text PDF PubMed Google Scholar) These results that PKA phosphorylates the C terminus of stargazin at To whether phosphorylation of T321 of stargazin its binding to PSD-95, we generated two mimicking the of the protein and are these stargazin were for their interactions with the PDZ domains of PSD-95 and in the yeast two-hybrid mutant PSD-95 or In control experiments, the stargazin C terminus specifically with the PDZ domains of PSD-95 and but not with PDZ domains of an protein H. Huganir R.L. Nature. 1997; PubMed Scopus Google Scholar) mutant stargazin the to PSD-95 and that the C terminus of stargazin specifically interacts with the PDZ domains of the PSD-95 These demonstrate that the stargazin mimicking T321 phosphorylation interactions with of the PSD-95 an of the of stargazin the we performed coimmunoprecipitation COS doubly transfected with EGFP-stargazin (wild-type and mutants) and PSD-95 PSD-95 antibodies in to their PSD-95, stargazin but of the mutant (RRTDPV, RRTEPV, and RRTTPA) In control transfected stargazin was not by PSD-95 antibodies These results that stargazin mimicking T321 phosphorylation the association between stargazin and PSD-95 in heterologous of stargazin and PSD-95 in heterologous cells results in the of the two (14.Chen L. Chetkovich D.M. Petralia R.S. Sweeney N.T. Kawasaki Y. Wenthold R.J. Bredt D.S. Nicoll R.A. Nature. 2000; 408: 936-943Crossref PubMed Scopus (9) Google Scholar). the of stargazin the coclustering between stargazin and PSD-95 EGFP-stargazin with PSD-95 in COS cells in which with results (14.Chen L. Chetkovich D.M. Petralia R.S. Sweeney N.T. Kawasaki Y. Wenthold R.J. Bredt D.S. Nicoll R.A. Nature. 2000; 408: 936-943Crossref PubMed Scopus (9) Google Scholar). However, of the stargazin (RRTDPV, RRTEPV and RRTTPA) and were the cells These data suggest that T321 phosphorylation of stargazin regulates the interaction between stargazin and PSD-95, in with the yeast two-hybrid To whether phosphorylation of stargazin its interaction with PSD-95, we performed coimmunoprecipitation with COS cells transfected with stargazin PSD-95, and the PKA catalytic subunit of COS with PSD-95 antibodies not any of phosphorylated stargazin, as by Stg-pT321 antibodies In PSD-95 antibodies a of total stargazin, as revealed by EGFP antibodies suggesting that unphosphorylated stargazin the to with the PSD-95. These that phosphorylation of stargazin at the C-terminal T321 residue inhibits its interaction with PSD-95. Stargazin is in PSD fractions with AMPAR subunits and PSD-95 (14.Chen L. Chetkovich D.M. Petralia R.S. Sweeney N.T. Kawasaki Y. Wenthold R.J. Bredt D.S. Nicoll R.A. Nature. 2000; 408: 936-943Crossref PubMed Scopus (9) Google Scholar). To whether phosphorylated stargazin shows an enrichment in the we performed PSD fractions of rat brain Stg-pT321 antibodies revealed that phosphorylated stargazin is in PSD In phosphorylated stargazin was not in the PSD a of the PSD extracted with Triton X-100 and sarcosyl In Stg-Cyto and Stg-C-term antibodies a enrichment of total stargazin in PSD These results that phosphorylated stargazin is with the results demonstrate that phosphorylation of stargazin at T321 by PKA inhibits its interaction with PSD-95. The C-terminal sequence of stargazin is or to in closely related of calcium channels in and and in that are in the brain (7.Klugbauer N. Dai S. Specht V. Lacinova L. Marais E. Bohn G. Hofmann F. FEBS Lett. 2000; 470: 189-197Crossref PubMed Scopus (153) Google Scholar, 8.Burgess D.L. Davis C.F. Gefrides L.A. Noebels J.L. Genome Res. 1999; 9: 1204-1213Crossref PubMed Scopus (56) Google Scholar, 10.Burgess D.L. Gefrides L.A. Foreman P.J. Noebels J.L. Genomics. 2001; 71: 339-350Crossref PubMed Scopus (85) Google Scholar), suggesting interactions with PSD-95 and phosphorylation by PKA. stargazin interacts with known of the PSD-95 (14.Chen L. Chetkovich D.M. Petralia R.S. Sweeney N.T. Kawasaki Y. Wenthold R.J. Bredt D.S. Nicoll R.A. Nature. 2000; 408: 936-943Crossref PubMed Scopus (9) Google Scholar), which in the brain M. Sala C. Annu. Rev. Neurosci. 2001; 24: 1-29Crossref PubMed Scopus (1049) Google Scholar). These results suggest that the phosphorylation of stargazin at T321 regulates interactions between of the and PSD-95 To our this is the report thus that phosphorylation of the C-terminal PDZ-binding regulates PDZ of stargazin stargazin phosphorylation may regulate the synaptic targeting of AMPARs by interactions between the stargazin C terminus and the PDZ domains of PSD-95 and related PDZ proteins. Consistently, rescues extrasynaptic but not synaptic AMPAR currents in cerebellar granule cells of stargazer mice (14.Chen L. Chetkovich D.M. Petralia R.S. Sweeney N.T. Kawasaki Y. Wenthold R.J. Bredt D.S. Nicoll R.A. Nature. 2000; 408: 936-943Crossref PubMed Scopus (9) Google Scholar). In is and synaptic AMPAR currents (14.Chen L. Chetkovich D.M. Petralia R.S. Sweeney N.T. Kawasaki Y. Wenthold R.J. Bredt D.S. Nicoll R.A. Nature. 2000; 408: 936-943Crossref PubMed Scopus (9) Google Scholar). phosphorylation may regulate the of stargazin the synaptic surface membrane. Phosphorylated stargazin may its to with synaptic PDZ and consequently the synaptic surface or Consistently, PSD-95 of its binding potassium channel R. L.C. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar) and receptor L.A. Y. M. R.J. R.A. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). Stargazin phosphorylation may not the factor that its synaptic if stargazin to synaptic sites with which are known to with their as H. Huganir R.L. Nature. 1997; PubMed Scopus Google Scholar, S. P. F.S. L. G. B. C. S. R. Valtschanoff J.G. Weinberg R.J. Neuron. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, M. Kim E. Sheng M. 1998; PubMed Scopus Google Scholar). However, a mutant subunit of AMPARs lacking binding its at the synaptic surface P. L. J.L. G. G. C. A. Lee Neuron. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar), suggesting that stargazin may synaptic targeting. stargazin phosphorylation may regulate translocation of AMPARs to the Stargazin phosphorylation may the complex cytosolic PDZ and its delivery to the surface membrane. Interestingly, phosphorylation of the residue of a subunit of AMPARs that to stargazin (14.Chen L. Chetkovich D.M. Petralia R.S. Sweeney N.T. Kawasaki Y. Wenthold R.J. Bredt D.S. Nicoll R.A. Nature. 2000; 408: 936-943Crossref PubMed Scopus (9) Google Scholar), with its surface association in Neuron. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). In addition, PKA and PKA following Neuron. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). is known that of is phosphorylated by PKA (20.Mammen A.L. Kameyama K. Roche K.W. Huganir R.L. J. Biol. Chem. 1997; 272: 32528-32533Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar, K.W. R.J. A.L. J. Huganir R.L. Neuron. 1996; 16: Full Text Full Text PDF PubMed Scopus Google Scholar) and that this phosphorylation the open of AMPARs Lee H. Huganir R.L. A. J. Neurosci. 2000; 20: PubMed Google Scholar). However, little is known about its in Stargazin phosphorylation by PKA may a molecular that the of PKA AMPAR surface of for of and mutant PKA catalytic