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

Abstract

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.

Cyclic AMP Phosphodiesterase 4D (PDE4D) Tethers EPAC1 in a Vascular Endothelial Cadherin (VE-Cad)-based Signaling Complex and Controls cAMP-mediated Vascular Permeability

“…In addition, we also demonstrated for the first time that targeted cAMP hydrolysis by sequestered PDE4D plays a pivotal regula- 362 VLVLERASQGAGPSRPPTPGRNRYT 386 in which individual amino acids sequentially substituted with alanine were probed with GST-PDE4D3 fusion proteins (10 g/ml) to allow identification of the residues important for the interaction between PDE4D and EPAC1 was carried out as described in Ref. 23 and under "Experimental Procedures." B and C, HAEC incubated for 2 h with a steroylated EPAC1-based peptide encoding amino acids Val 362 to Thr 386 , designed to disrupt the interaction between EPAC1 and PDE4D (defined herein as a disrupting peptide (DP)), or with a scrambled version of this peptide (defined herein as Control peptide) were either lysed and subjected to ␤-catenin pulldown analysis as described under "Experimental Procedures" and subsequently investigated by immunoblot analysis (B) or fixed and analyzed by immunofluorescence (C).…”

“…Peptide Array Analysis-An EPAC1 peptide library of 25 individual amino acid peptides, each displaced by five amino acids, was immobilized on cellulose membranes using automated SPOT synthesis as described previously (23). The interaction between immobilized peptides and GST (10 g/ml) or a GST-PDE4D3 fusion protein (10 g/ml) was determined by overlaying membranes with recombinant proteins.…”

Isoform-Selective Susceptibility of DISC1/Phosphodiesterase-4 Complexes to Dissociation by Elevated Intracellular cAMP Levels

“…We show that increased cAMP levels cause release of PDE4D isoforms from fl-DISC1, whereas PDE4B isoforms remain stably associated. Insight into the basis of these selective actions comes from our use of a novel peptide array approach (Bolger et al, 2006;Baillie et al, 2007) to show that fl-DISC1 has two binding sites that PDE4 isoforms bind in common, together with additional N-terminal sites specific for PDE4B.…”

Location, location, location: PDE4D5 function is directed by its unique N-terminal region

“…Mutant forms of the scaffold could be designed that do not associate with PDE4s. These mutations did not hinder arrestin translocation to receptors, but did promote PKA phosphorylation of the receptor by virtue of the lack of PDE4 activity within the proximity of the activated receptor [32]. Cellular regulation of the abundance of the PDE4D5-arrestin complex can be controlled by ubiquitination [33].…”

“…As the many functions of beta-arrestin are conferred by its ability to associate with hundreds of binding partners [26], the PDE4 binding sites on arrestin were discovered using similar approaches as those described above [32]. In common with other client proteins of arrestin, PDE4 bound to sites within both the N-and Cdomains of the scaffold [30].…”