Inhibition of Clathrin Assembly by High Affinity Binding of Specific Inositol Polyphosphates to the Synapse-specific Clathrin Assembly Protein AP-3 (*)
Inhibition of Clathrin Assembly by High Affinity Binding of Specific Inositol Polyphosphates to the Synapse-specific Clathrin Assembly Protein AP-3 (*)
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DOI：
10.1074/jbc.270.4.1564
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发表时间：
1995-01
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影响因子：
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通讯作者：
Weilan Ye;N. Ali;M. Bembenek;S. Shears;E. Lafer
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文献类型：
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作者：
Weilan Ye;N. Ali;M. Bembenek;S. Shears;E. Lafer
在细菌中表达突触特异性蛋白AP-3(F1-20/AP180/NP185/pp155)，与高亲和力的六磷酸肌醇(InsP6)(K=239 nM)和二磷酸肌醇五磷酸(PP-InsP5)(K=22 nM)结合。结合的[~3H]InsP6的竞争性置换证明了这种配体结合的特异性。IC值如下：PP-InsP5=50 nM，InsP6=240 nM，肌醇-1，2，4，5，6-五磷酸(INS(1，2，4，5，6)P5)=2.2μM，肌醇-1，3，4，5，6-五磷酸(INS(1，3，4，5)P5)=5μM，肌醇-1，3，4，5-四磷酸(INS(1，3，4，5)P4)>；10μM，肌醇-1，4，5-三磷酸(INS(1，4，5)P3)>；10μM。此外，10μ肌醇六硫酸盐(InsS6)仅置换了15%的[~3H]InsP6。这种结合的生理意义在于配体特异性地抑制网状蛋白组装(PP-InsP5>；InsP6>；Ins(1，2，4，5，6)P5)；Ins(1，3，4，5，6)P5和InsS6不抑制网状蛋白组装。我们还观察到InsP6与纯化的牛脑AP-3的高亲和力结合。我们分别表达了33 kDa的氨基末端和58 kDa的羧基末端，前者含有高亲和力的肌醇多聚磷酸结合部位。这些研究表明，特定的肌醇多磷酸可能通过与突触特异的网状蛋白组装蛋白AP-3相互作用而在突触功能的调节中发挥作用。
Bacterially expressed synapse-specific clathrin assembly protein, AP-3 (F1-20/AP180/NP185/pp155), bound with high affinity both inositol hexakisphosphate (InsP6) (K = 239 nM) and diphosphoinositol pentakisphosphate (PP-InsP5) (K = 22 nM). The specificity of this ligand binding was demonstrated by competitive displacement of bound [3H]InsP6. IC values were as follows: PP-InsP5 = 50 nM, InsP6 = 240 nM, inositol-1,2,4,5,6-pentakisphosphate (Ins(1,2,4,5,6)P5) = 2.2 μM, inositol-1,3,4,5,6-pentakisphosphate (Ins(1,3,4,5,6)P5) = 5 μM, inositol-1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) > 10 μM, inositol-1,4,5-trisphosphate (Ins(1,4,5)P3) > 10 μM. Moreover, 10 μM inositol hexasulfate (InsS6) displaced only 15% of [3H]InsP6. The physiological significance of this binding is the ligand-specific inhibition of clathrin assembly (PP-InsP5 > InsP6 > Ins(1,2,4,5,6)P5); Ins(1,3,4,5,6)P5 and InsS6 did not inhibit clathrin assembly. We also observed high affinity binding of InsP6 to purified bovine brain AP-3. We separately expressed the 33-kDa amino terminus and the 58-kDa carboxyl terminus, and it was the former that contained the high affinity inositol polyphosphate binding site. These studies suggest that specific inositol polyphosphates may play a role in the regulation of synaptic function by interacting with the synapse-specific clathrin assembly protein AP-3.