Scanning peptide array analyses identify overlapping binding sites for the signalling scaffold proteins, β-arrestin and RACK1, in cAMP-specific phosphodiesterase PDE4D5
Scanning peptide array analyses identify overlapping binding sites for the signalling scaffold proteins, β-arrestin and RACK1, in cAMP-specific phosphodiesterase PDE4D5
复制标题
DOI：
10.1042/bj20060423
复制
发表时间：
2006-08-15
影响因子：
4.1
通讯作者：
Houslay, Miles D.
中科院分区：
文献类型：
--
作者：
Bolger, Graeme B.;Baillie, George S.;Houslay, Miles D.
cAMP 特异性磷酸二酯酶 PDE4D5 可以与信号支架蛋白 RACK（活化 C 激酶受体）1 和 β-arrestin 相互作用。两种杂交和免疫共沉淀分析表明，RACK1 和 β-arrestin 与 PDE4D5 以相互排斥的方式相互作用。 PDE4D5 扫描肽阵列文库的重叠研究表明，RACK I 和 P-arrestin 在 PDE4D5 独特 N 末端区域内的重叠位点以及保守 PDE4 催化结构域内的不同位点相互作用。筛选扫描丙氨酸取代肽阵列，结合诱变和截短研究。允许定义 RACK1 和 β-arrestin 相互作用位点。以 PDE4D 催化结构域为模型，它们在 RACK1 的 helices-15-16 和 β-arrestin 的 helix-17 上形成明显明确的表面暴露斑块。 HEK-293（人胚胎肾）B2 细胞中 siRNA（小干扰 RNA）介导的 RACK1 敲低使 β-抑制蛋白支架的 PDE4D5 增加约 10%。异丙肾上腺素激发后，β(2)AR（β(2)-肾上腺素能受体）募集的 PDE4D5 增加约 5 倍。异丙肾上腺素刺激的 PKA（蛋白激酶 A）β(2)AR 磷酸化和 ERK（细胞外信号调节激酶）激活均 4 倍且严重减弱（约 4-5 倍）。 PDE4D5 的催化失活形式在放大异丙肾上腺素刺激的 ERK 激活方面发挥显性负效应的能力，被阻止 PDE4D5 与 β-arrestin 相互作用的突变所消除。在本研究中，我们表明信号支架蛋白 RACK1 和 β-arrestin 竞争隔离不同的 PDE4D5“池”。以这种方式，RACK1表达水平的改变可能起到调节β(2)AR介导的信号转导的作用。
The cAMP-specific phosphodiesterase PDE4D5 can interact with the signalling scaffold proteins RACK (receptors for activated C-kinase) 1 and beta-arrestin. Two-hybrid and co-immunoprecipitation analyses showed that RACK1 and beta-arrestin interact with PDE4D5 in a mutually exclusive manner. Overlay studies with PDE4D5 scanning peptide array libraries showed that RACK I and P-arrestin interact at overlapping sites within the unique N-terminal region of PDE4D5 and at distinct sites within the conserved PDE4 catalytic domain. Screening scanning alanine substitution peptide arrays, coupled with mutagenesis and truncation studies. allowed definition of RACK1 and beta-arrestin interaction sites. Modelled on the PDE4D catalytic domain, these form distinct well-defined surface-exposed patches on helices-15-16, for RACK1, and helix-17 for beta-arrestin. siRNA (small interfering RNA)-mediated knockdown of RACK1 in HEK-293 (human embryonic kidney) B2 cells increased beta-arrestin-scaffolded PDE4D5 approx. 5-fold, increased PDE4D5 recruited to the beta(2)AR (beta(2)-adrenergic receptor) upon isoproterenol challenge approx. 4-fold and severely attenuated (approx. 4-5 fold) both isoproterenol-stimulated PKA (protein kinase A) phosphorylation of the beta(2)AR and activation of ERK (extracellular-signal-regulated kinase). The ability of a catalytically inactive form of PDE4D5 to exert a dominant negative effect in amplifying isoproterenol-stimulated ERK activation was ablated by a mutation that blocked the interaction of PDE4D5 with beta-arrestin. In the present study, we show that the signalling scaffold proteins RACK1 and beta-arrestin compete to sequester distinct 'pools' of PDE4D5. In this fashion, alterations in the level of RACK1 expression may act to modulate signal transduction mediated by the beta(2)AR.