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  • Title: 1H NMR structural and functional characterisation of a cAMP-specific phosphodiesterase-4D5 (PDE4D5) N-terminal region peptide that disrupts PDE4D5 interaction with the signalling scaffold proteins, beta-arrestin and RACK1.
    Author: Smith KJ, Baillie GS, Hyde EI, Li X, Houslay TM, McCahill A, Dunlop AJ, Bolger GB, Klussmann E, Adams DR, Houslay MD.
    Journal: Cell Signal; 2007 Dec; 19(12):2612-24. PubMed ID: 17900862.
    Abstract:
    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.
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