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

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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., 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.
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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