Phosphorylation of Stargazin by Protein Kinase A Regulates Its Interaction with PSD-95
2002; Elsevier BV; Volume: 277; Issue: 14 Linguagem: Inglês
10.1074/jbc.m200528200
ISSN1083-351X
AutoresJeonghoon Choi, Jaewon Ko, Eunhye Park, Jae-Ran Lee, Jiyoung Yoon, Sangmi Lim, Eunjoon Kim,
Tópico(s)Photoreceptor and optogenetics research
ResumoStargazin 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 (1217, 1 μg/ml), PSD-95 (SM55, 1 μg/ml), and synaptophysin (Sigma, 1:1000) antibodies. To determine whether the T321 residue of stargazin is the phosphorylation site, we generated phosphorylation site-specific (termed Stg-pT321) antibodies (1218 rabbit and 1229 guinea pig) using the last 10 residues of stargazin with phosphorylated T321 as immunogen. Stg-pT321 antibodies were tested against stargazin expressed in heterologous cells (Fig. 1). Immunoblotting of COS cell lysates transfected with EGFP-tagged stargazin (EGFP-stargazin) with Stg-pT321 antibodies revealed a single protein band of about 66 kDa (38 kDa stargazin + 28 kDa EGFP) (Fig. 1A). In contrast, mutant stargazin (T321A) lacking the hydroxyl group for phosphorylation was not detected by Stg-pT321 antibodies (Fig. 1A). Preincubation of Stg-pT321 antibodies with excess free phosphorylated (but not unphosphorylated) peptide prevented the recognition of EGFP-stargazin (wild-type) (Fig. 1B). Moreover, pretreatment of the membrane with λ-phosphatase abolished recognition of the EGFP-stargazin (wild-type) by Stg-pT321 antibodies (Fig. 1B). These results indicate that stargazin is phosphorylated at T321 in heterologous cells. To examine whether T321 of stargazin is phosphorylated in vivo, we performed immunoblotting analyses on protein samples from rat brain with various stargazin antibodies (Fig. 2). Both Stg-pT321 antibodies (1218 and 1229) recognized a major band of about 38 kDa corresponding to the size of reported 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 crude synaptosomal (P2) and small membrane (S2) fractions. Similarly sized stargazin bands were detected by additional stargazin antibodies raised against the C-terminal peptide (the last 10 residues with unphosphorylated T321, Stg-C-term) and the entire C-terminal cytoplasmic region (aa 203–323; Stg-Cyto). Our data indicate that stargazin is phosphorylated at the T321 residue in vivo. It is possible that Stg-pT321 and Stg-C-term antibodies may recognize closely related proteins such as the γ-3 and γ-4 subunits of voltage-dependent 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 they have similar C-terminal sequences (the last 7 residues are identical) and calculated molecular masses (stargazin, 35.9 kDa; γ-3, 35.6 kDa; γ-4, 36.5 kDa in mice). To determine whether the stargazin C terminus is a substrate of PKA, we examined phosphorylation levels of this region of stargazin in COS cells doubly transfected with stargazin and the PKA catalytic subunit (wild-type and an inactive mutant) (Fig. 3). When COS cells singly transfected with stargazin were immunoblotted with Stg-pT321 antibodies, a relatively small but significant amount of phosphorylated stargazin was detected, suggesting that stargazin is basally phosphorylated in COS cells (Fig. 3). Stargazin phosphorylation was markedly increased upon coexpression with the PKA catalytic subunit but not with an inactive catalytic subunit (25.Maurer R.A. J. Biol. Chem. 1989; 264: 6870-6873Abstract Full Text PDF PubMed Google Scholar) (Fig. 3). These results indicate that PKA phosphorylates the C terminus of stargazin at T321. To investigate whether phosphorylation of T321 of stargazin affects its binding to PSD-95, we generated two mutations mimicking the PKA-phosphorylated state of the protein (RRTDPV and RRTEPV; mutations are underlined). When these stargazin mutations were tested for their interactions with the PDZ domains of PSD-95 and SAP97 in the yeast two-hybrid assay, neither mutant bound PSD-95 or SAP97 (Fig. 4). In control experiments, the wild-type stargazin C terminus specifically interacted with the PDZ domains of PSD-95 and SAP97 but not with unrelated PDZ domains of GRIP1, an AMPAR-interacting multi-PDZ protein (26.Dong H. RJ O.B. Fung E.T. Lanahan A.A. Worley P.F. Huganir R.L. Nature. 1997; 386: 279-284Crossref PubMed Scopus (758) Google Scholar) (Fig. 4). Additional mutant stargazin (RRTTPA) lost the ability to bind both PSD-95 and SAP97, indicating that the C terminus of stargazin specifically interacts with the PDZ domains of the PSD-95 family members. These findings demonstrate that the stargazin mutations mimicking T321 phosphorylation disrupt interactions with members of the PSD-95 family. As an independent test of the effects of stargazin mutations on the stargazin-PSD-95 interaction, we performed coimmunoprecipitation experiments on COS cell lysates doubly transfected with EGFP-stargazin (wild-type and mutants) and PSD-95 (Fig. 5). PSD-95 antibodies pulled down, in addition to their cognate antigen PSD-95, wild-type stargazin (RRTTPV) but none of the mutant stargazins tested (RRTDPV, RRTEPV, and RRTTPA) (Fig. 5). In control immunoprecipitation, singly transfected stargazin (wild-type) was not brought down by PSD-95 antibodies (Fig. 5). These results indicate that stargazin mutations mimicking T321 phosphorylation disrupt the biochemical association between stargazin and PSD-95 in heterologous cells. Coexpression of stargazin and PSD-95 in heterologous cells results in the formation of clusters where the two proteins colocalize (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). We examined the effects of stargazin mutations on the coclustering between stargazin and PSD-95 (Fig. 6). Wild-type EGFP-stargazin when coexpressed with PSD-95 in COS cells formed clusters in which both proteins colocalized (Fig. 6A), consistent with previous 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, none of the stargazin mutants (RRTDPV, RRTEPV and RRTTPA) formed typical clusters on coexpression, and both proteins were diffusely distributed throughout the cells (Fig. 6, B–D). These data suggest that T321 phosphorylation of stargazin regulates the interaction between stargazin and PSD-95, in agreement with the yeast two-hybrid results. To investigate whether phosphorylation of stargazin reduces its interaction with PSD-95, we performed coimmunoprecipitation experiments with COS cells triply transfected with stargazin (wild-type), PSD-95, and the PKA catalytic subunit (Fig. 7). Immunoprecipitation of COS cell lysates with PSD-95 antibodies did not bring down any detectable amount of phosphorylated stargazin, as visualized by Stg-pT321 antibodies (Fig. 7, top panel). In contrast, PSD-95 antibodies brought down a significant amount of total stargazin, as revealed by EGFP antibodies (Fig. 7, middle panel), suggesting that unphosphorylated stargazin retains the ability to coimmunoprecipitate with the PSD-95. These findings further confirm that phosphorylation of stargazin at the C-terminal T321 residue inhibits its interaction with PSD-95. Stargazin is enriched in detergent-insoluble PSD fractions along 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 test whether phosphorylated stargazin shows an altered enrichment in the PSD, we performed immunoblot analysis on PSD fractions of rat brain (Fig. 8). Stg-pT321 antibodies revealed that phosphorylated stargazin is minimally enriched in PSD fractions. In particular, phosphorylated stargazin was not detectable in the PSD III fraction, a core of the PSD extracted with Triton X-100 and sarcosyl detergents. In contrast, Stg-Cyto and Stg-C-term antibodies showed a significant enrichment of total stargazin in all PSD fractions. These results indicate that phosphorylated stargazin is less tightly associated with the PSD. Our results demonstrate that phosphorylation of stargazin at T321 by PKA inhibits its interaction with PSD-95. The C-terminal sequence of stargazin (NRRTTPV) is identical or similar to those in other closely related γ-subunits of voltage-dependent calcium channels (NRRTTPV in γ-3 and γ-4 and NRKTTPV in γ-8) that are expressed 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 similar interactions with PSD-95 and phosphorylation by PKA. Conversely, stargazin interacts with all known members of the PSD-95 family (Fig. 4) (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 display diverse distribution patterns in the brain (16.Sheng M. Sala C. Annu. Rev. Neurosci. 2001; 24: 1-29Crossref PubMed Scopus (1049) Google Scholar). These results suggest that the PKA-dependent phosphorylation of stargazin at T321 regulates interactions between various members of the stargazin/γ and PSD-95 families. To our knowledge, this is the second report thus far showing that phosphorylation of the C-terminal PDZ-binding motif regulates PDZ interactions. We propose three possible roles of stargazin phosphorylation. First, stargazin phosphorylation may regulate the synaptic targeting of AMPARs by modulating interactions between the stargazin C terminus and the PDZ domains of PSD-95 and related PDZ proteins. Consistently, 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). In cultured hippocampal neurons, stargazinΔC is diffusely distributed and down-regulates 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). Second, phosphorylation may regulate the stability of stargazin on the synaptic surface membrane. Phosphorylated stargazin may lose its ability to interact with synaptic PDZ anchors and consequently diffuse away laterally from the synaptic surface or become internalized. Consistently, PSD-95 markedly suppresses internalization of its binding partners including Kv1.4 potassium channel (27.Jugloff D.G. Khanna R. Schlichter L.C. Jones O.T. J. Biol. Chem. 2000; 275: 1357-1364Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar) and β1-adrenergic receptor (28.Hu L.A. Tang Y. Miller W.E. Cong M. Lau A.G. Lefkowitz R.J. Hall R.A. J. Biol. Chem. 2000; 275: 38659-38666Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Stargazin phosphorylation may not be the only factor that determines its synaptic stability, if stargazin delivered to synaptic sites still remains associated with AMPARs, which are known to interact with their own anchors such as GRIP/ABP (26.Dong H. RJ O.B. Fung E.T. Lanahan A.A. Worley P.F. Huganir R.L. Nature. 1997; 386: 279-284Crossref PubMed Scopus (758) Google Scholar, 29.Srivastava S. Osten P. Vilim F.S. Khatri L. Inman G. States B. Daly C. DeSouza S. Abagyan R. Valtschanoff J.G. Weinberg R.J. Ziff E.B. Neuron. 1998; 21: 581-591Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar, 30.Wyszynski M. Kim E. Yang F.C. Sheng M. Neuropharmacology. 1998; 37: 1335-1344Crossref PubMed Scopus (65) Google Scholar). However, a mutant GluR2 subunit of AMPARs lacking GRIP binding loses its stability at the synaptic surface (31.Osten P. Khatri L. Perez J.L. Kohr G. Giese G. Daly C. Schulz T.W. Wensky A. Lee L.M. Ziff E.B. Neuron. 2000; 27: 313-325Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar), suggesting that stargazin may dissociate from GluR2 after initial synaptic targeting. Third, stargazin phosphorylation may regulate translocation of AMPARs to the cell surface. Stargazin phosphorylation may release the stargazin-AMPAR complex from cytosolic PDZ anchors and facilitate its delivery to the surface membrane. Interestingly, phosphorylation of the Ser-845 residue of GluR1, a subunit of AMPARs that binds 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), correlates well with its surface association in cultured neurons (32.Ehlers M.D. Neuron. 2000; 28: 511-525Abstract Full Text Full Text PDF PubMed Scopus (903) Google Scholar). In addition, PKA activation reduces N-methyl-d-aspartic acid-induced GluR1 endocytosis, and PKA inhibition reduces GluR1 reinsertion following NMDA treatment (32.Ehlers M.D. Neuron. 2000; 28: 511-525Abstract Full Text Full Text PDF PubMed Scopus (903) Google Scholar). It is known that Ser-845 of GluR1 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, 33.Roche K.W. O'Brien R.J. Mammen A.L. Bernhardt J. Huganir R.L. Neuron. 1996; 16: 1179-1188Abstract Full Text Full Text PDF PubMed Scopus (666) Google Scholar) and that this phosphorylation increases the peak open probability of AMPARs (34.Banke T.G. Bowie D. Lee H. Huganir R.L. Schousboe A. Traynelis S.F. J. Neurosci. 2000; 20: 89-102Crossref PubMed Google Scholar). However, little is known about its involvement in GluR1 trafficking. Stargazin phosphorylation by PKA may be a molecular mechanism that explains the effects of PKA on AMPAR surface expression. We thank Dr. Kaang of Seoul National University for generously providing cDNA of wild-type and mutant PKA catalytic subunits.
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