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.
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.
authors: School of Biosciences, University of Birmingham, Edgbaston, Birmingham, PO Box 363, B15 2TT, UK.
authors: K John
authors: KJ
authors: Smith
authors: George S
authors: GS
authors: Baillie
authors: Eva I
authors: EI
authors: Hyde
authors: Xiang
authors: X
authors: Li
authors: Thomas M
authors: TM
authors: Houslay
authors: Angela
authors: A
authors: McCahill
authors: Allan J
authors: AJ
authors: Dunlop
authors: Graeme B
authors: GB
authors: Bolger
authors: Enno
authors: E
authors: Klussmann
authors: David R
authors: DR
authors: Adams
authors: Miles D
authors: MD
authors: Houslay
mesh_terms: D058666:Adrenergic beta-2 Receptor Agonists; D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000595:Amino Acid Sequence; D019390:Arrestins / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D002460:Cell Line; D002942:Circular Dichroism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D004847:Epithelial Cells / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007545:Isoproterenol / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000737:chemistry / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D010455:Peptides / Q000494:pharmacology; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D000075370:Receptors for Activated C Kinase; D018343:Receptors, Adrenergic, beta-2 / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction; D000071557:beta-Arrestins
chemical_list: D058666:Adrenergic beta-2 Receptor Agonists; D000318:Adrenergic beta-Agonists; D019390:Arrestins; D009363:Neoplasm Proteins; D010455:Peptides; C419368:RACK1 protein, human; D000075370:Receptors for Activated C Kinase; D018343:Receptors, Adrenergic, beta-2; D011956:Receptors, Cell Surface; D000071557:beta-Arrestins; D017868:Cyclic AMP-Dependent Protein Kinases; D010727:Phosphoric Diester Hydrolases; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; C517376:PDE4D protein, human; D019204:GTP-Binding Proteins; D007545:Isoproterenol