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

Baillie, G.S. et al. (2007) 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, 404(1), pp. 71-80. (doi: 10.1042%2FBJ20070005)

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.

• Item Type: Articles
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Vadrevu, Dr Suryakiran and Houslay, Professor Miles and Baillie, Professor George and Milligan, Professor Graeme and Bhari, Mrs Narinder and Dunlop, Dr Allan and Houslay, Mr Thomas
• Authors: Baillie, G.S., Adams, D.R., Bhari, N., Houslay, T.M., Vadrevu, S., Meng, D., Li, X., Dunlop, A., Milligan, G., Bolger, G.B., Klussmann, E., and Houslay, M.D.
• Subjects: Q Science > QH Natural history > QH345 Biochemistry
• College/School: College of Medical Veterinary and Life Sciences; College of Medical Veterinary and Life Sciences > School of Life Sciences
• Journal Name: Biochemical Journal
• Publisher: Portland Press Ltd.
• ISSN: 0264-6021
• ISSN (Online): 1470-8728