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

George S Baillie , David R Adams , Narinder Bahri , Thomas M Houslay , Suryakiran Vadrevu , Dong Meng , Xiang Li , Allan Dunlop , Graeme Milligan , Graeme B Bolger , Enno Klussmann , Miles D Houslay (1)

1 Biochemistry and Molecular Biology

β 2}-adrenoceptors (β 2}-AR) become rapidly desensitised upon recruitment of cytosolic βarrestin. The cAMP phosphodiesterase-4D isoform, PDE4D5 can be recruited in complex with βarrestin, whereupon it regulates PKA phosphorylation of the β 2}-AR. Here we use novel technology, employing a library of overlapping peptides (25 mers) immobilized on cellulose membranes that scan the entire sequence of βarrestin2, to define the interaction sites on βarrestin2 for binding of PDE4D5 and the cognate long isoform, PDE4D3. We identify a binding site in the βarrestin2 N-domain for the common PDE4D catalytic unit and two regions in the βarrestin2 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 Arg26Ala substitution and reduced interaction upon either Lys18Ala or Thr20ala substitution. Similar analysis of the βarrestin2 C-domain identified Arg286 and Asp291, together with the Leu215-His220 region, as being of importance in binding PDE4D5 but not PDE4D3. Transfection with wild-type βarrestin2 profoundly decreased isoprenaline-stimulated PKA phosphorylation of the β 2}-AR in mouse embryo fibroblasts (MEFs) lacking both βarrestin1 and βarrestin2. This effect was negated using either the Arg26Ala or Arg286Ala mutant forms of βarrestin2 or one with substitution of Leu215-His220 for an alanine cassette, which showed little/no PDE4D5 binding but were still recruited to the β 2}-AR upon isoprenaline challenge. These data show that the interaction of PDE4D5 with both the N- and C- domains of βarrestin2 are essential for β 2}-AR regulation.

Mots clés: Life Sciences

George S Baillie, David R Adams, Narinder Bahri, Thomas M Houslay, Suryakiran Vadrevu, et al.. Mapping binding sites for the PDE4D5 cAMP-specific phosphodiesterase to the N- and C-domains of {beta}-arrestin using spot-immobilized peptide arrays. Biochemical Journal, 2007, 404 (1), pp.71-80. ⟨10.1042/BJ20070005⟩. ⟨hal-00478731⟩