Delineation of RAID1, the RACK1 interaction domain located within the unique N-terminal region of the cAMP-specific phosphodiesterase, PDE4D5

Graeme B. Bolger, Angela McCahill, Stephen J Yarwood, Michael R. Steele, Jim Warwicker, Miles Douglas Houslay

    Research output: Contribution to journalArticlepeer-review

    39 Citations (Scopus)

    Abstract

    BACKGROUND: The cyclic AMP specific phosphodiesterase, PDE4D5 interacts with the beta-propeller protein RACK1 to form a signaling scaffold complex in cells. Two-hybrid analysis of truncation and mutant constructs of the unique N-terminal region of the cAMP-specific phosphodiesterase, PDE4D5 were used to define a domain conferring interaction with the signaling scaffold protein, RACK1.

    RESULTS: Truncation and mutagenesis approaches showed that the RACK1-interacting domain on PDE4D5 comprised a cluster of residues provided by Asn-22/Pro-23/Trp-24/Asn-26 together with a series of hydrophobic amino acids, namely Leu-29, Val-30, Leu-33, Leu-37 and Leu-38 in a 'Leu-Xaa-Xaa-Xaa-Leu' repeat. This was done by 2-hybrid analyses and then confirmed in biochemical pull down analyses using GST-RACK1 and mutant PDE4D5 forms expressed in COS cells. Mutation of Arg-34, to alanine, in PDE4D5 attenuated its interaction with RACK1 both in 2-hybrid screens and in pull down analyses. A 38-mer peptide, whose sequence reflected residues 12 through 49 of PDE4D5, bound to RACK1 with similar affinity to native PDE4D5 itself (Ka circa 6 nM).

    CONCLUSIONS: The RACK1 Interaction Domain on PDE4D5, that we here call RAID1, is proposed to form an amphipathic helical structure that we suggest may interact with the C-terminal beta-propeller blades of RACK1 in a manner akin to the interaction of the helical G-gamma signal transducing protein with the beta-propeller protein, G-beta.

    Original languageEnglish
    Article number24
    JournalBMC Biochemistry
    Volume3
    DOIs
    Publication statusPublished - 2002

    Keywords

    • Adaptor Proteins, Signal Transducing
    • Amino Acid Sequence
    • Animals
    • Binding Sites
    • COS Cells
    • CRADD Signaling Adaptor Protein
    • Cercopithecus aethiops
    • Cyclic AMP
    • Cyclic Nucleotide Phosphodiesterases, Type 3
    • Cyclic Nucleotide Phosphodiesterases, Type 4
    • GTP-Binding Proteins
    • Molecular Sequence Data
    • Neoplasm Proteins
    • Phosphoric Diester Hydrolases
    • Protein Structure, Secondary
    • Protein Structure, Tertiary
    • Receptors, Cell Surface

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