<sup>1sup>H NMR structural and functional characterisation of a cAMP-specific phosphodiesterase-4D5 (PDE4D5) N-terminal region peptide that disrupts PDE4D5 interaction with the signalling scaffold p
- 作者：K. John Smith ; George S. Baillie ; Eva I. Hyde ; Xiang Li ; Thomas M. Houslay ; Angela McCahill ; Allan J. Dunlop ; Graeme B. Bolger ; Enno Klussmann ; David R. Adams ; Miles D. Houslay
- 关键词：Rolipram ; RACK1 ; β ; arrestin ; NMR structure ; Signalling scaffold ; Cyclic AMP ; Protein– ; protein interaction ; Peptide displacement ; Spot immobilised peptide arrays ; β ; <sub>2sub>-adrenergic receptors (β ; <sub>2sub>AR) ; ERK ; PKA
- 刊名：Cellular Signalling
- 出版年：2007
- 期刊代码：42_08986568
- 类别：bio
- 出版时间：December 2007
- 卷：19
- 期：12
- 页码：2612-2624
- 文件大小：1181 K
摘要
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, βarrestin 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 βarrestin binding site. <sup>1sup>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 α-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<sub>γsub> binding to the WD-repeat protein, G<sub>βsub>. A more extensive section of the PDE4D5 N-terminal sequence (Thr11-Ala85) is involved in βarrestin 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 βarrestin 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 βarrestin binding. A membrane-permeable, stearoylated Val12-Ser49 38-mer peptide disrupted the interaction of both βarrestin 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 β<sub>2sub>-adrenergic receptors (β<sub>2sub>AR) and its activation of ERK, whilst the Glu27Ala peptide was ineffective in both these regards.