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

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

Isoform-selective susceptibility of DISC1/phosphodiesterase-4 complexes to dissociation by elevated intracellular cAMP levels

Disrupted-in-schizophrenia 1 (DISC1) is a genetic susceptibility factor for schizophrenia and related severe psychiatric conditions. DISC1 is a multifunctional scaffold protein that is able to interact with several proteins, including the independently identified schizophrenia risk factor phosphodiesterase-4B (PDE4B). Here we report that the 100 kDa full-length DISC1 isoform (fl-DISC1) can bind members of each of the four gene, cAMP-specific PDE4 family. Elevation of intracellular cAMP levels, so as to activate protein kinase A, caused the release of PDE4D3 and PDE4C2 isoforms from fl-DISC1 while not affecting binding of PDE4B1 and PDE4A5 isoforms. Using a peptide array strategy, we show that PDE4D3 binds fl-DISC1 through two regions found in common with PDE4B isoforms, the interaction of which is supplemented because of the presence of additional PDE4B-specific binding sites. We propose that the additional binding sites found in PDE4B1 underpin its resistance to release during cAMP elevation. We identify, for the first time, a functional distinction between the 100 kDa long DISC1 isoform and the short 71 kDa isoform. Thus, changes in the expression pattern of DISC1 and PDE4 isoforms offers a means to reprogram their interaction and to determine whether the PDE4 sequestered by DISC1 is released after cAMP elevation. The PDE4B-specific binding sites encompass point mutations in mouse Disc1 that confer phenotypes related to schizophrenia and depression and that affect binding to PDE4B. Thus, genetic variation in DISC1 and PDE4 that influence either isoform expression or docking site functioning may directly affect psychopathology

Dimerization of cAMP phosphodiesterase-4 (PDE4) in living cells requires interfaces located in both the UCR1 and catalytic unit domains

PDE4 family cAMP phosphodiesterases play a pivotal role in determining compartmentalised cAMP signalling through targeted cAMP breakdown. Expressing the widely found PDE4D5 isoform, as both bait and prey in a yeast 2-hybrid system, we demonstrated interaction consistent with the notion that long PDE4 isoforms form dimers. Four potential dimerization sites were uncovered using a scanning peptide array approach, where recombinant purified PDE4D5 fusion protein was used to probe a 25-mer library of overlapping peptides covering the entire PDE4D5 sequence. Key residues involved in PDE4D5 dimerization were defined using a site-directed mutagenesis programme directed by an alanine scanning peptide array approach. Critical residues stabilising PDE4D5 dimerization were defined within the regulatory UCR1 region found in long, but not short, PDE4 isoforms, namely the Arg(173), Asn(174) and Asn(175) (DD1) cluster. Disruption of the DD1 cluster was not sufficient, in itself, to destabilise PDE4D5 homodimers. Instead, disruption of an additional interface, located on the PDE4 catalytic unit, was additionally required to convert PDE4D5 into a monomeric form. This second dimerization site on the conserved PDE4 catalytic unit is dependent upon a critical ion pair interaction. This involves Asp(463) and Arg(499) in PDE4D5, which interact in a trans fashion involving the two PDE4D5 molecules participating in the homodimer. PDE4 long isoforms adopt a dimeric state in living cells that is underpinned by two key contributory interactions, one involving the UCR modules and one involving an interface on the core catalytic domain. We propose that short forms do not adopt a dimeric configuration because, in the absence of the UCR1 module, because residual engagement of the remaining core catalytic domain interface provides insufficient free energy to drive dimerization. The functioning of PDE4 long and short forms is thus poised to be inherently distinct due to this difference in quaternary structure

Mapping binding sites for the PDE4D5 cAMP-specific phosphodiesterase to the N- and C-domains of beta-arrestin using spot-immobilized peptide arrays

Beta2-ARs (beta2-adrenoceptors) become desensitized rapidly upon recruitment of cytosolic beta-arrestin. PDE4D5 (family 4 cAMP-specific phosphodiesterase, subfamily D, isoform 5) can be recruited in complex with beta-arrestin, whereupon it regulates PKA (cAMP-dependent protein kinase) phosphorylation of the beta2-AR. In the present study, we have used novel technology, employing a library of overlapping peptides (25-mers) immobilized on cellulose membranes that scan the entire sequence of beta-arrestin 2, to define the interaction sites on beta-arrestin 2 for binding of PDE4D5 and the cognate long isoform, PDE4D3. We have identified a binding site in the beta-arrestin 2 N-domain for the common PDE4D catalytic unit and two regions in the beta-arrestin 2 C-domain that confer specificity for PDE4D5 binding. Alanine-scanning peptide array analysis of the N-domain binding region identified severely reduced interaction with PDE4D5 upon R26A substitution, and reduced interaction upon either K18A or T20A substitution. Similar analysis of the beta-arrestin 2 C-domain identified Arg286 and Asp291, together with the Leu215-His220 region, as being important for binding PDE4D5, but not PDE4D3. Transfection with wild-type beta-arrestin 2 profoundly decreased isoprenaline-stimulated PKA phosphorylation of the beta2-AR in MEFs (mouse embryo fibroblasts) lacking both beta-arrestin 1 and beta-arrestin 2. This effect was negated using either the R26A or the R286A mutant form of beta-arrestin 2 or a mutant with substitution of an alanine cassette for Leu215-His220, which showed little or no PDE4D5 binding, but was still recruited to the beta2-AR upon isoprenaline challenge. These data show that the interaction of PDE4D5 with both the N- and C-domains of beta-arrestin 2 are essential for beta2-AR regulation