Smith K John, Baillie George S, Hyde Eva I, Li Xiang, Houslay Thomas M, McCahill Angela, Dunlop Allan J, Bolger Graeme B, Klussmann Enno, Adams David R, Houslay Miles D
School of Biosciences, University of Birmingham, Edgbaston, Birmingham, PO Box 363, B15 2TT, UK.
Cell Signal. 2007 Dec;19(12):2612-24. doi: 10.1016/j.cellsig.2007.08.015. Epub 2007 Sep 1.
The unique 88 amino acid N-terminal region of cAMP-specific phosphodiesterase-4D5 (PDE4D5) contains overlapping binding sites conferring interaction with the signaling scaffold proteins, betaarrestin and RACK1. A 38-mer peptide, whose sequence reflected residues 12 through 49 of PDE4D5, encompasses the entire N-terminal RACK1 Interaction Domain (RAID1) together with a portion of the beta-arrestin binding site. (1)H NMR and CD analyses indicate that this region has propensity to form a helical structure. The leucine-rich hydrophobic grouping essential for RACK1 interaction forms a discrete hydrophobic ridge located along a single face of an amphipathic alpha-helix with Arg34 and Asn36, which also play important roles in RACK1 binding. The Asn22/Pro23/Trp24/Asn26 grouping, essential for RACK1 interaction, was located at the N-terminal head of the amphipathic helix that contained the hydrophobic ridge. RAID1 is thus provided by a distinct amphipathic helical structure. We suggest that the binding of PDE4D5 to the WD-repeat protein, RACK1, may occur in a manner akin to the helix-helix interaction shown for G(gamma) binding to the WD-repeat protein, G(beta). A more extensive section of the PDE4D5 N-terminal sequence (Thr11-Ala85) is involved in beta-arrestin binding. Several residues within the RAID1 helix contribute to this interaction however. We show here that these residues form a focused band around the centre of the RAID1 helix, generating a hydrophobic patch (from Leu29, Val30 and Leu33) flanked by polar/charged residues (Asn26, Glu27, Asp28, Arg34). The interaction with beta-arrestin exploits a greater circumference on the RAID1 helix, and involves two residues (Glu27, Asp28) that do not contribute to RACK1 binding. In contrast, the interaction of RACK1 with RAID1 is extended over a greater length of the helix and includes Leu37/Leu38, which do not contribute to beta-arrestin binding. A membrane-permeable, stearoylated Val12-Ser49 38-mer peptide disrupted the interaction of both beta-arrestin and RACK1 with endogenous PDE4D5 in HEKB2 cells, whilst a cognate peptide with a Glu27Ala substitution selectively failed to disrupt PDE4D5/RACK1 interaction. The stearoylated Val12-Ser49 38-mer peptide enhanced the isoprenaline-stimulated PKA phosphorylation of the beta(2)-adrenergic receptors (beta(2)AR) and its activation of ERK, whilst the Glu27Ala peptide was ineffective in both these regards.
环磷酸腺苷特异性磷酸二酯酶4D5（PDE4D5）独特的88个氨基酸的N端区域包含重叠的结合位点，可与信号支架蛋白β-抑制蛋白和富含脯氨酸的丝氨酸/苏氨酸激酶1（RACK1）相互作用。一个38肽，其序列反映了PDE4D5的第12至49位残基，包含整个N端RACK1相互作用结构域（RAID1）以及一部分β-抑制蛋白结合位点。氢核磁共振（1H NMR）和圆二色性（CD）分析表明，该区域倾向于形成螺旋结构。对于RACK1相互作用至关重要的富含亮氨酸的疏水基团形成了一个离散的疏水脊，沿着两亲性α-螺旋的一个面定位，其中精氨酸34和天冬酰胺36也在RACK1结合中起重要作用。对于RACK1相互作用至关重要的天冬酰胺22/脯氨酸23/色氨酸24/天冬酰胺26基团位于包含疏水脊的两亲性螺旋的N端头部。因此，RAID1由一个独特的两亲性螺旋结构提供。我们认为，PDE4D5与WD重复蛋白RACK1的结合可能以类似于G（γ）与WD重复蛋白G（β）所示的螺旋-螺旋相互作用的方式发生。PDE4D5 N端序列的更广泛部分（苏氨酸11-丙氨酸85）参与β-抑制蛋白结合。然而，RAID1螺旋内的几个残基有助于这种相互作用。我们在此表明，这些残基在RAID1螺旋中心周围形成一个集中带，产生一个疏水补丁（来自亮氨酸29、缬氨酸30和亮氨酸33），两侧是极性/带电荷的残基（天冬酰胺26、谷氨酸27、天冬氨酸28、精氨酸34）。与β-抑制蛋白的相互作用利用了RAID1螺旋上更大的圆周，并且涉及两个对RACK-1结合无贡献的残基（谷氨酸27、天冬氨酸28）。相比之下，RACK1与RAID1的相互作用在螺旋的更长长度上延伸，并且包括对β-抑制蛋白结合无贡献的亮氨酸37/亮氨酸38。一种可透过膜的硬脂酰化缬氨酸12-丝氨酸49 38肽破坏了HEKB2细胞中β-抑制蛋白和RACK1与内源性PDE4D5的相互作用，而具有谷氨酸27丙氨酸取代的同源肽选择性地未能破坏PDE4D5/RACK1相互作用。硬脂酰化缬氨酸12-丝氨酸49 38肽增强了异丙肾上腺素刺激的β2-肾上腺素能受体（β2AR）的蛋白激酶A（PKA）磷酸化及其对细胞外信号调节激酶（ERK）的激活，而谷氨酸27丙氨酸肽在这两方面均无效。
