Phosphorylation of stargazin by protein kinase A regulates its interaction with PSD-95
Phosphorylation of stargazin by protein kinase A regulates its interaction with PSD-95
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DOI：
10.1074/jbc.m200528200
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发表时间：
2002-04-05
影响因子：
4.8
通讯作者：
Kim, E
中科院分区：
文献类型：
--
作者：
Choi, J;Ko, J;Kim, E
Stargazin是已知与AMPA（α-氨基-3-羟基-5-甲基异恶唑-4-丙酸酯）谷氨酸受体（AMPAR）缔合并通过两种不同机制调节其突触靶向的第一种跨膜蛋白，特别是通过将AMPAR递送至表面膜和通过结合PSD-95/SAP-90和相关PDZ蛋白来突触靶向这些受体。然而，尚不清楚这种stargazin介导的AMP的突触靶向是否以及如何调节。Stargazin通过C-末端PDZ结合基序与PSD-95的PDZ结构域相互作用。Stargazin C末端含有一个由cAMP依赖性蛋白激酶A（PKA）磷酸化的共有序列。磷酸化位点特异性stargazin抗体显示stargazin C末端在异源细胞和体内的Thr-321残基处被磷酸化。Stargazin磷酸化被PKA的催化亚基增强。模拟stargazin磷酸化（T321 E和T321 D）的突变导致酵母双杂交相互作用，体外共免疫沉淀，和stargazin和PSD-95之间的共聚类消除。磷酸化stargazin显示了选择性的损失与PSD-95在异源细胞和有限的富集在大鼠脑突触后密度组分的免疫共沉淀。这些结果表明，PKA对stargazin C末端的磷酸化调节其与PSD-95的相互作用和AMPAR的突触靶向。
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 AMPs its 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.