Mapping binding sites for the PDE4D5 cAMP-specific phosphodiesterase to the N- and C-domains of β-arrestin using spot-immobilized peptide arrays
Mapping binding sites for the PDE4D5 cAMP-specific phosphodiesterase to the N- and C-domains of β-arrestin using spot-immobilized peptide arrays
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DOI：
10.1042/bj20070005
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发表时间：
2007-05-15
影响因子：
4.1
通讯作者：
Houslay, Miles D.
中科院分区：
文献类型：
--
作者：
Baillie, George S.;Adams, David R.;Houslay, Miles D.
β (2)- ar （β(2)-肾上腺素受体）在吸收细胞质β -阻滞素后迅速脱敏。PDE4D5（家族4 camp特异性磷酸二酯酶，亚家族D，异构体5）可以与β -抑制蛋白复合物募集，从而调节β (2)-AR的PKA （camp依赖性蛋白激酶）磷酸化。在本研究中，我们使用了一种新技术，利用固定在纤维素膜上的重叠肽库（25-mers）扫描β -阻滞蛋白2的整个序列，来确定β -阻滞蛋白2上与PDE4D5和同源长异构体PDE4D3结合的相互作用位点。我们已经在常见的PDE4D催化单元的β -阻滞蛋白2 n结构域中确定了一个结合位点，在-阻滞蛋白2 c结构域中确定了两个与PDE4D5结合特异性的区域。对n结构域结合区的丙氨酸扫描肽阵列分析发现，在R26A取代后，与PDE4D5的相互作用严重减少，在K18A或T20A取代后，相互作用也减少。对β -阻滞蛋白2 c结构域的类似分析发现，Arg（286）和Asp（291）以及Leu(215) -His（220）区域对结合PDE4135很重要，但对PDE4D3不起作用。转染野生型β -抑制素2可显著降低异丙肾上腺素刺激的mef（小鼠胚胎成纤维细胞）中β (2)-AR的PKA磷酸化，mef缺乏β -抑制素1和β -抑制素2。使用β -抑制蛋白2的R26A或R286A突变体形式，或用丙氨酸盒替代Leu(215) -His（220）的突变体，这种作用被否定，这种突变体很少或没有PDE4D5结合，但在异丙肾上腺素刺激下仍被招募到β (2)-AR。这些数据表明PDE4D5与β -抑制蛋白2的N-和c -结构域的相互作用对β (2)- ar调控至关重要。
beta(2)-ARs (beta(2)-adrenoceptors) become desensitized rapidly upon recruitment of cytosolic beta-arrestin. PDE4D5 (family 4 cAMP-specific phosphodiesterase, subfamily D, isoform 5) can be recruited in complex with beta-arrestin, whereupon it regulates PKA (cAMP-dependent protein kinase) phosphorylation of the beta(2)-AR. In the present study, we have used novel technology, employing a library of overlapping peptides (25-mers) immobilized on cellulose membranes that scan the entire sequence of beta-arrestin 2, to define the interaction sites on beta-arrestin 2 for binding of PDE4D5 and the cognate long isoform, PDE4D3. We have identified a binding site in the beta-arrestin 2 N-domain for the common PDE4D catalytic unit and two regions in the -arrestin 2 C-domain that confer specificity for PDE4D5 binding. Alanine-scanning peptide array analysis of the N-domain binding region identified severely reduced interaction with PDE4D5 upon R26A substitution, and reduced interaction upon either K18A or T20A substitution. Similar analysis of the beta-arrestin 2 C-domain identified Arg(286) and Asp(291), together with the Leu(215) -His(220) region, as being important for binding PDE4135, but not PDE4D3. Transfection with wild-type beta-arrestin 2 profoundly decreased isoprenaline-stimulated PKA phosphorylation of the beta(2)-AR in MEFs (mouse embryo fibroblasts) lacking both beta-arrestin 1 and beta-arrestin 2. This effect was negated using either the R26A or the R286A mutant form of beta-arrestin 2 or a mutant with substitution of an alanine cassette for Leu(215) -His(220), which showed little or no PDE4D5 binding, but was still recruited to the beta(2)-AR upon isoprenaline challenge. These data show that the interaction of PDE4D5 with both the N- and C-domains of beta-arrestin 2 are essential for beta(2)-AR regulation.