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

Item Type:	Article
Title:	Mapping binding sites for the PDE4D5 cAMP-specific phosphodiesterase to the N- and C-domains of beta-arrestin using spot-immobilized peptide arrays
Creators Name:	Baillie, G.S. and Adams, D.R. and Bhari, N. and Houslay, T.M. and Vadrevu, S. and Meng, D. and Li, X. and Dunlop, A. and Milligan, G. and Bolger, G.B. and Klussmann, E. and Houslay, M.D.
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.
Keywords:	b2-Adrenoceptor, b-Arrestin, cAMP, Desensitization, Peptide Array, Phosphodiesterase 4 (PDE4)
Source:	Biochemical Journal
ISSN:	0264-6021
Publisher:	Portland Press
Volume:	404
Number:	1
Page Range:	71-80
Date:	15 May 2007
Official Publication:	https://doi.org/10.1042/BJ20070005
PubMed:	View item in PubMed

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