title: Mapping binding sites for the PDE4D5 cAMP-specific phosphodiesterase to the N- and C-domains of beta-arrestin using spot-immobilized peptide arrays.
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.
authors.affiliation: Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, UK.
authors.forename: George S
authors.initials: GS
authors.lastname: Baillie
authors.forename: David R
authors.initials: DR
authors.lastname: Adams
authors.forename: Narinder
authors.initials: N
authors.lastname: Bhari
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Houslay
authors.forename: Suryakiran
authors.initials: S
authors.lastname: Vadrevu
authors.forename: Dong
authors.initials: D
authors.lastname: Meng
authors.forename: Xiang
authors.initials: X
authors.lastname: Li
authors.forename: Allan
authors.initials: A
authors.lastname: Dunlop
authors.forename: Graeme
authors.initials: G
authors.lastname: Milligan
authors.forename: Graeme B
authors.initials: GB
authors.lastname: Bolger
authors.forename: Enno
authors.initials: E
authors.lastname: Klussmann
authors.forename: Miles D
authors.initials: MD
authors.lastname: Houslay
mesh_terms: D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000378:metabolism*; D019390:Arrestins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002460:Cell Line; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D006801:Humans; D007668:Kidney; D008958:Models, Molecular; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry* / Q000235:genetics; D040081:Protein Array Analysis; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D014162:Transfection; D000071559:beta-Arrestin 1; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins
chemical_list: C000605253:ARRB1 protein, human; C000605254:ARRB2 protein, human; C000605255:Arrb1 protein, mouse; C000605258:Arrb2 protein, mouse; D019390:Arrestins; D011994:Recombinant Proteins; D000071559:beta-Arrestin 1; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; D010727:Phosphoric Diester Hydrolases; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; C517376:PDE4D protein, human