Scanning peptide array analysis identify overlapping binding sites for the signaling scaffold proteins, beta-arestin and RACK1 in the cAMP-specific phosphodiesterase, PDE4D5

Bolger, G.B. et al. (2006) Scanning peptide array analysis identify overlapping binding sites for the signaling scaffold proteins, beta-arestin and RACK1 in the cAMP-specific phosphodiesterase, PDE4D5. Biochemical Journal, 398(1), pp. 23-36. (doi:10.1042/BJ20060423)

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Abstract

The cAMP-specific phosphodiesterase PDE4D5 can interact with the signalling scaffold proteins RACK (receptors for activated C-kinase) 1 and β-arrestin. Two-hybrid and co-immunoprecipitation analyses showed that RACK1 and β-arrestin interact with PDE4D5 in a mutually exclusive manner. Overlay studies with PDE4D5 scanning peptide array libraries showed that RACK1 and β-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 β-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 β-arrestin. siRNA (small interfering RNA)-mediated knockdown of RACK1 in HEK-293 (human embryonic kidney) B2 cells increased β-arrestin-scaffolded PDE4D5 approx. 5-fold, increased PDE4D5 recruited to the β2AR (β2-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 β2AR 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 β-arrestin. In the present study, we show that the signalling scaffold proteins RACK1 and β-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 β2AR.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Houslay, Professor Miles and Baillie, Professor George and Herzyk, Dr Pawel and McCahill, Dr Angela and Lynch, Dr Martin
Authors: Bolger, G.B., Baillie, G.S., Li, X., Lynch, M.J., Herzyk, P., Mohamed, A., Mitchell, L., McCahill, A., Hundsrucker, C., Klussmann, E., Adams, D.R., and Houslay, M.D.
Subjects:Q Science > QR Microbiology > QR355 Virology
College/School:College of Medical Veterinary and Life Sciences
College of Medical Veterinary and Life Sciences > Institute of Molecular Cell and Systems Biology
Journal Name:Biochemical Journal
Publisher:Portland Press Ltd.
ISSN:0264-6021
ISSN (Online):1470-8728

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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
208361PDE4 cAMP phosphodiesterses: intracellular targeting, SH3 domain interaction and phosphorylation by stress kinasesMiles HouslayMedical Research Council (MRC)G8604010Institute of Neuroscience and Psychology
294081PDE4 Cyclic AMP phosphodiesterasesMiles HouslayMedical Research Council (MRC)G8604010Institute of Neuroscience and Psychology
30692Molecular interaction between receptor signal transduction systems involving guanine nucleotide regulatory proteinsMiles HouslayMedical Research Council (MRC)G8604010Institute of Neuroscience and Psychology
343641PDE4 cAMP phosphodiesterases - intracellular targeting, SH3 Domain Interaction & phosphorylation by stress kinasesMiles HouslayMedical Research Council (MRC)G8604010Institute of Neuroscience and Psychology
97371Programme Grant (PG 8604010) Supplementation (Equipment). Molecular interaction between receptor transduction systems involving guanine nuclMiles HouslayMedical Research Council (MRC)G8604010Institute of Neuroscience and Psychology