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BMC Biochem. 2002 Aug 23;3:24.

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

Author information

1
Veterans Affairs Medical Center, Huntsman Cancer Institute, Division of Oncology, Department of Medicine, University of Utah Health Sciences Center, Salt Lake City, UT 84148, USA. Graeme.Bolger@ccc.uab.edu

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.

PMID:
12193273
PMCID:
PMC126212
DOI:
10.1186/1471-2091-3-24
[Indexed for MEDLINE]
Free PMC Article

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