Cannabinoid Receptor Interacting Protein 1a Competition with β-Arrestin for CB1 Receptor Binding Sites
Cannabinoid Receptor Interacting Protein 1a Competition with β-Arrestin for CB1 Receptor Binding Sites
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DOI：
10.1124/mol.116.104638
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发表时间：
2017-02-01
影响因子：
3.6
通讯作者：
Howlett, Allyn C.
中科院分区：
文献类型：
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作者：
Blume, Lawrence C.;Patten, Theresa;Howlett, Allyn C.
大麻素受体相互作用蛋白 1a (CRIP1a) 是一种 CB1 受体 (CB1R) 远端 C 末端相关蛋白，可改变 CB1R 与 G 蛋白的相互作用。我们测试了这样的假设：CRIP1a 也能够改变 CB1R 与 β-抑制蛋白的相互作用，而β-抑制蛋白与 C 末端的 CB1R 相互作用。免疫共沉淀研究表明，CB1R 与 CRIP1a 或 b-arrestin 形成复合物，但 CRIP1a 和 b-arrestin 不能相互共免疫沉淀。这表明 CRIP1a 和 b-arrestin 与 CB1R 的结合存在竞争，我们假设这可能会减弱 b-arrestin 介导 CB1R 内化的作用。我们确定激动剂介导的细胞表面内源表达的CB(1)Rs密度的降低是网格蛋白和动力依赖性的，并且可以建模为激动剂诱导的瞬时表达的GFP-CB1R的聚集。 CRIP1a 过表达减弱了 CP55940 介导的 GFP-CB1R 以及内源性 β-抑制蛋白对点的重新分布，相反，CRIP1a 敲低增强了 β-抑制蛋白对点的重新分布。模拟 CB1R C 末端的肽可以与细胞提取物中的 CRIP1a 以及纯化的重组 CRIP1a 结合。亲和力下拉研究表明，CB1R 远端 C 末端 14 聚体肽苏氨酸 468 处的磷酸化可减少 CB1R-CRIP1a 关联。 CB1R 蛋白复合物的免疫共沉淀表明，中央或远端 C 端肽与 CRIP1a 竞争 CB1R 关联，但磷酸化中央 C 端肽竞争与 b-arrestin 1 关联，磷酸化中央或远端 C 端肽竞争与 β-arrestin 2 关联。因此，CRIP1a 可以与 β-arrestins 竞争与 C 端 CB1R 结构域的相互作用，从而影响激动剂驱动、βb-抑制蛋白介导的 CB1R 内化。
Cannabinoid receptor interacting protein 1a (CRIP1a) is a CB1 receptor (CB1R) distal C-terminal-associated protein that alters CB1R interactions with G-proteins. We tested the hypothesis that CRIP1a is capable of also altering CB1R interactions with beta-arrestin proteins that interact with the CB1R at the C-terminus. Coimmunoprecipitation studies indicated that CB1R associates in complexes with either CRIP1a or b-arrestin, but CRIP1a and b-arrestin fail to coimmunoprecipitate with each other. This suggests a competition for CRIP1a and b-arrestin binding to the CB1R, which we hypothesized could attenuate the action of b-arrestin to mediate CB1R internalization. We determined that agonist-mediated reduction of the density of cell surface endogenously expressed CB(1)Rs was clathrin and dynamin dependent and could be modeled as agonist-induced aggregation of transiently expressed GFP-CB1R. CRIP1a overexpression attenuated CP55940-mediated GFP-CB1R as well as endogenous beta-arrestin redistribution to punctae, and conversely, CRIP1a knockdown augmented beta-arrestin redistribution to punctae. Peptides mimicking the CB1R C-terminus could bind to both CRIP1a in cell extracts as well as purified recombinant CRIP1a. Affinity pull-down studies revealed that phosphorylation at threonine-468 of a CB1R distal C-terminus 14-mer peptide reduced CB1R-CRIP1a association. Coimmunoprecipitation of CB1R protein complexes demonstrated that central or distal C-terminal peptides competed for the CB1R association with CRIP1a, but that a phosphorylated central C-terminal peptide competed for association with b-arrestin 1, and phosphorylated central or distal C-terminal peptides competed for association with beta-arrestin 2. Thus, CRIP1a can compete with beta-arrestins for interaction with C-terminal CB1R domains that could affect agonist-driven, beta b-arrestin-mediated internalization of the CB1R.