The scaffold protein gravin (cAMP-dependent protein kinase-anchoring protein 250) binds the β2-adrenergic receptor via the receptor cytoplasmic Arg-329 to Leu-413 domain and provides a mobile scaffold during desensitization
The scaffold protein gravin (cAMP-dependent protein kinase-anchoring protein 250) binds the β2-adrenergic receptor via the receptor cytoplasmic Arg-329 to Leu-413 domain and provides a mobile scaffold during desensitization
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DOI：
10.1074/jbc.m011199200
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发表时间：
2001-06-29
影响因子：
4.8
通讯作者：
Malbon, CC
中科院分区：
文献类型：
--
作者：
Fan, GF;Shumay, E;Malbon, CC
环状 AMP 依赖性激酶锚定蛋白 (AKAP) 充当多种蛋白质-蛋白质相互作用的支架。被称为 gravin 的 250 kDa AKAP 在组织 G 蛋白偶联受体与蛋白激酶和磷酸酶的过程中发挥着核心作用，从而调节脱敏、再敏化和隔离中的受体功能。尽管 gravin 对于 G 蛋白连接受体生物学至关重要，但与该支架相互作用所需的受体的分子特征尚不清楚。在此，我们绘制了与 gravin 结合所需的 β (2)-肾上腺素能受体区域，细胞内环 1、2 和 3 似乎不参与受体与支架的结合。相比之下，无论使用体外还是体内测定，受体的 C 端胞质区域（Arg-329 至 Leu-413）很容易与 gravin 竞争 β (2)-肾上腺素能受体的结合。 C 端截短肽的序列范围为 Arg-329 至 Leu-342（13 个氨酰基残基）、Asn-352（23 个残基）、Tyr-366（37 个残基）、Asp-380（51 个残基）或 His-390（61 个残基），以及 N 端截短肽从 Gln-391 至Leu-413（23个残基）或Leu-381至Leu-413（33个残基）没有表现出阻断受体与gravin结合的能力，然而，Arg-329与His-390肽以及Gln-391与Leu-413肽的组合重建了片段化但全长的C端区域，并且还有效地阻断了gravin结合β的能力(2)-肾上腺素能受体。通过共聚焦显微镜检查了 gravin-受体对激动剂的相互作用。值得注意的是，在激动剂诱导的隔离过程中，受体与 gravin 的结合并未受到破坏。受体-支架复合物在激动剂诱导的隔离过程中得以维持。这些数据与生化数据一致，表明 gravin 通过 β2-肾上腺素能受体 C 端胞质结构域与受体结合，并且随着受体内化，这种相互作用得以维持。这是 AKAP 支架蛋白与其受体易位的首次报道，在本例中是 G 蛋白偶联受体。
The cyclic AMP-dependent kinase-anchoring proteins (AKAPs) function as scaffolds for a wide-range of protein-protein interactions. The 250-kDa AKAP known as gravin plays a central role in organizing G-protein-coupled receptors to the protein kinases and phosphatases that regulate receptor function in desensitization, resensitization, and sequestration. Although gravin is critical for G-protein-linked receptor biology, the molecular features of the receptor necessary for interaction with this scaffold are not known. Herein, we map the regions of the beta (2)-adrenergic receptor that are required for binding to gravin, Intracellular loops 1, 2, and 3 appear not to participate in the binding of the receptor to the scaffold. In contrast, the C-terminal cytoplasmic region of the receptor (Arg-329 to Leu-413) competes readily for the binding of the beta (2)-adrenergic receptor by gravin, both using in vitro and in vivo assays. C-terminally truncated peptides with sequences ranging from Arg-329 to Leu-342 (13 aminoacyl residues), to Asn-352 (23 residues), to Tyr-366 (37 residues), to Asp-380 (51 residues), or to His-390 (61 residues), as well as N-terminally truncated peptides from Gln-391 to Leu-413 (23 residues) or Leu-381 to Leu-413 (33 residues) displayed no ability to block binding of receptor to gravin, The combination of Arg-329 to His-390 peptide and Gln-391 to Leu-413 peptide, however, reconstitutes a fragmented but full-length C-terminal region and also potently blocks the ability of gravin to bind the beta (2)-adrenergic receptor. The gravin-receptor interaction was examined in response to agonist by confocal microscopy, Remarkably, the association of the receptor with gravin was not disrupted during agonist-induced sequestration. The receptor-scaffold complex was maintained during agonist-induced sequestration. These data, in agreement with the biochemical data, reveal that gravin binds the receptor through the beta2-adrenergic receptor C-terminal cytoplasmic domain and that this interaction is maintained as the receptor is internalized. This is the first report of an AKAP scaffold protein translocating with its receptor, in this case a G-protein-coupled receptor.