Research article

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

Graeme B Bolger^3,4 , Angela McCahill¹ , Stephen J Yarwood¹ , Michael R Steele³ , Jim Warwicker² and Miles D Houslay^1,2

¹Molecular Pharmacology Group, Division of Biochemistry & Molecular Biology, Davidson Building, Institute of Biomedical & Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, UK

²Dept of Biomolecular Sciences, UMIST, Sackville Street, Manchester M60 1QD, UK

³Veterans Affairs Medical Center, Huntsman Cancer Institute, Departments of Medicine (Division of Oncology) and Oncological Science, University of Utah Health Sciences Center, Salt Lake City, UT 84148 USA

⁴University of Alabama at Birmingham, Comprehensive Cancer Center, WTI 520, 1530 3rd Ave. S., Birmingham AL 35294-3300, USA

author email corresponding author email

BMC Biochemistry 2002, 3:24doi:10.1186/1471-2091-3-24

Published:	23 August 2002

Abstract

Background

The cyclic AMP specific phosphodiesterase, PDE4D5 interacts with the β-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-X_aa-X_aa-X_aa-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 (K_a 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 β-propeller blades of RACK1 in a manner akin to the interaction of the helical G-γ signal transducing protein with the β-propeller protein, G-β.