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, βarrestin and RACK1

Kevin John Smith, G Baillie, Eva Hyde, X Li, T Houslay, A McCahill, A Dunlop, G Bolger, E Klussmann, D Adams, MD Houslay

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46 Citations (Scopus)

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, beta arrestin 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. H-1 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 a-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 (Thr1 1-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 RAID] 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 2AR) and its activation of ERK, whilst the Glu27Ala peptide was ineffective in both these regards. (C) 2007 Elsevier Inc. All rights reserved.
Original languageEnglish
Pages (from-to)2612-2624
Number of pages13
JournalCellular Signalling
Volume19
Issue number12
Early online date1 Sept 2007
DOIs
Publication statusPublished - 1 Dec 2007

Keywords

  • Rolipram
  • RACK1
  • beta arrestin
  • spot immobilised peptide arrays
  • protein-protein interaction
  • cyclic AMP
  • PKA
  • ERK
  • beta(2)-adrenergic receptors (beta(2)AR)
  • signalling scaffold
  • peptide displacement
  • NMR structure

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