Phosphorylation of stargazin by protein kinase A regulates its interaction with PSD-95.
Stargazin is the first transmembrane protein known to associate with AMPA (alpha-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.
Choi
Jeonghoon
J
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Korea.
Ko
Jaewon
J
Park
Eunhye
E
Lee
Jae-Ran
JR
Yoon
Jiyoung
J
Lim
Sangmi
S
Kim
Eunjoon
E
D020816:Amino Acid Motifs; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D019556:COS Cells; D015220:Calcium Channels / Q000737:chemistry* / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D000076122:Disks Large Homolog 4 Protein; D004305:Dose-Response Relationship, Drug; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D008856:Microscopy, Fluorescence; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D015398:Signal Transduction; D013347:Subcellular Fractions / Q000378:metabolism; D013912:Threonine / Q000378:metabolism; D014162:Transfection; D020798:Two-Hybrid System Techniques
C491911:Cacng2 protein, rat; D015220:Calcium Channels; D000076122:Disks Large Homolog 4 Protein; C501449:Dlg4 protein, rat; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; C033652:postsynaptic density proteins; D013912:Threonine; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases