TY - JOUR
T1 - 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
AU - Smith, Kevin John
AU - Baillie, G
AU - Hyde, Eva
AU - Li, X
AU - Houslay, T
AU - McCahill, A
AU - Dunlop, A
AU - Bolger, G
AU - Klussmann, E
AU - Adams, D
AU - Houslay, MD
PY - 2007/12/1
Y1 - 2007/12/1
N2 - 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.
AB - 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.
KW - Rolipram
KW - RACK1
KW - beta arrestin
KW - spot immobilised peptide arrays
KW - protein-protein interaction
KW - cyclic AMP
KW - PKA
KW - ERK
KW - beta(2)-adrenergic receptors (beta(2)AR)
KW - signalling scaffold
KW - peptide displacement
KW - NMR structure
U2 - 10.1016/j.cellsig.2007.08.015
DO - 10.1016/j.cellsig.2007.08.015
M3 - Article
C2 - 17900862
VL - 19
SP - 2612
EP - 2624
JO - Cellular Signalling
JF - Cellular Signalling
IS - 12
ER -