Scanning peptide array analyses identify overlapping binding sites for the signalling scaffold proteins, β-arrestin and RACK1, in cAMP-specific phosphodiesterase PDE4D5

Abstract

The cAMP-specific phosphodiesterase PDE4D5 can interact with the signalling scaffold proteins RACK (receptors for activated C-kinase) 1 and beta-arrestin. Two-hybrid and co-immunoprecipitation analyses showed that RACK1 and beta-arrestin interact with PDE4D5 in a mutually exclusive manner. Overlay studies with PDE4D5 scanning peptide array libraries showed that RACK1 and beta-arrestin interact at overlapping sites within the unique N-terminal region of PDE4D5 and at distinct sites within the conserved PDE4 catalytic domain. Screening scanning alanine substitution peptide arrays, coupled with mutagenesis and truncation studies, allowed definition of RACK1 and beta-arrestin interaction sites. Modelled on the PDE4D catalytic domain, these form distinct well-defined surface-exposed patches on helices-15-16, for RACK1, and helix-17 for beta-arrestin. siRNA (small interfering RNA)-mediated knockdown of RACK1 in HEK-293 (human embryonic kidney) B2 cells increased beta-arrestin-scaffolded PDE4D5 approx. 5-fold, increased PDE4D5 recruited to the beta2AR (beta2-adrenergic receptor) upon isoproterenol challenge approx. 4-fold and severely attenuated (approx. 4-5 fold) both isoproterenol-stimulated PKA (protein kinase A) phosphorylation of the beta2AR and activation of ERK (extracellular-signal-regulated kinase). The ability of a catalytically inactive form of PDE4D5 to exert a dominant negative effect in amplifying isoproterenol-stimulated ERK activation was ablated by a mutation that blocked the interaction of PDE4D5 with beta-arrestin. In the present study, we show that the signalling scaffold proteins RACK1 and beta-arrestin compete to sequester distinct 'pools' of PDE4D5. In this fashion, alterations in the level of RACK1 expression may act to modulate signal transduction mediated by the beta2AR.

Compartmentalization of cAMP-Dependent Signaling by Phosphodiesterase-4D Is Involved in the Regulation of Vasopressin-Mediated Water Reabsorption in Renal Principal Cells

“…Peptide array analyses indicate that AKAP18␦ interacts with two surface-exposed sites on the conserved PDE4D catalytic unit (16,46), suggesting that all PDE4D isoforms potentially could interact with AKAP18␦. Therefore AKAP18␦ may act to tether both PDE4D3 and PDE4D9 to AQP2-bearing vesicles.…”

“…Peptides were SPOT-synthesized on cellulose membranes and overlain with recombinant AKAP18␦-GST, PDE4D3-GST, or GST (10 g/ml) (16). Interactions were detected with rabbit anti-GST and secondary horseradish peroxidase antibody by a procedure identical to Western blotting.…”

Protein–protein interactions of PDE4 family members — Functions, interactions and therapeutic value

“…The 88 amino acids long unique N terminus of PDE4D5 contains a RACK-binding site (RIAD1) [36,37], and also the binding site for β-arrestin. The binding of PDE4D5 to RACK1 and β-arrestin is mutually exclusive [38]. In HEK293 cells, the interaction of PDE4D5 with these two binding partners seems in equilibrium.…”

“…Disturbing this equilibrium, e.g. by knockdown of RACK1 causes increased association of PDE4D5 with β-arrestin, which upon stimulation of β-adrenoceptors leads to a decrease in PKA phosphorylation of the β-adrenoceptor since β-arrestin causes an enhanced accumulation of PDE4D5 at the receptors [38].…”

“…and β-arrestin-1 [47] have been mapped using this technology; and the technology also allowed for defining overlapping binding sites for β-arrestin and RACK1 in PDE4D5 [38]. Maybe the most promising strategy to reach this goal is displacement of PDE4 enzymes from their cognate cellular location to which they are tethered through a protein-protein interaction.…”