Scanning peptide array analyses identify overlapping binding sites for the signaling scaffold proteins, {beta}-arrestin and RACK1, in cAMP-specific phosphodiesterase PDE4D5

Graeme B Bolger , George S Baillie , Xiang Li , Martin J Lynch , Pawel Herzyk , Ahmed Mohamed , Lisa High Mitchell , Angela Mccahill , Christian Hundsrucker , Enno Klussmann , David R Adams , Miles D Houslay (1)

1 Biochemistry & Molecular Biology

The cAMP-specific phosphodiesterase PDE4D5 can interact with the signaling scaffold proteins RACK1 and {beta}arrestin. 2-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-mediated knockdown of RACK1 in HEK293B2 cells increased {beta}arrestin-scaffolded PDE4D5 approximately 5-fold; increased PDE4D5 recruited to the {beta} 2}AR upon isoproterenol challenge approximately 4-fold and severely attenuated (approximately 4-5 fold) both isoproterenol stimulated PKA phosphorylation of the {beta} 2}AR and activation of ERK. 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. Here we show that the signaling 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 {beta} 2}-adrenergic receptor.

Mots clés: Life Sciences

Graeme B Bolger, George S Baillie, Xiang Li, Martin J Lynch, Pawel Herzyk, et al.. Scanning peptide array analyses identify overlapping binding sites for the signaling scaffold proteins, {beta}-arrestin and RACK1, in cAMP-specific phosphodiesterase PDE4D5. Biochemical Journal, 2006, 398 (1), pp.23-36. ⟨10.1042/BJ20060423⟩. ⟨hal-00478558⟩