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

Bolger, G.B., McCahill, A., Yarwood, S., Steele, M.R., Warwicker, J. and Houslay, M. (2002) Delineation of RAID1, the RACK1 interaction domain located within the unique N-terminal region of the cAMP-specific phosphodiesterase, PDE4D5. BMC Biochemistry, 3(24), (doi: 10.1186/1471-2091-3-24)

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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-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 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.

Item Type:Articles
Keywords:Rolipram; Protein kinase C binding protein; signalling scaffold; cyclic AMP
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:McCahill, Dr Angela and Yarwood, Dr Stephen and Houslay, Professor Miles
Authors: Bolger, G.B., McCahill, A., Yarwood, S., Steele, M.R., Warwicker, J., and Houslay, M.
Subjects:Q Science > QH Natural history > QH301 Biology
College/School:College of Medical Veterinary and Life Sciences
Research Group:Molecular Pharmacology Group
Journal Name:BMC Biochemistry
Publisher:Biomed Central
ISSN:14722091

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