Abstract
ß2-ARs (ß2-adrenoceptors) become desensitized rapidly upon recruitment of cytosolic ß-arrestin. PDE4D5 (family 4 cAMP-specific phosphodiesterase, subfamily D, isoform 5) can be recruited in complex with ß-arrestin, whereupon it regulates PKA (cAMP-dependent protein kinase) phosphorylation of the ß2-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 ß-arrestin 2, to define the interaction sites on ß-arrestin 2 for binding of PDE4D5 and the cognate long isoform, PDE4D3. We have identified a binding site in the ß-arrestin 2 N-domain for the common PDE4D catalytic unit and two regions in the ß-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 ß-arrestin 2 C-domain identified Arg286 and Asp291, together with the Leu 215-His220 region, as being important for binding PDE4D5, but not PDE4D3. Transfection with wild-type ß-arrestin 2 profoundly decreased isoprenaline-stimulated PKA phosphorylation of the ß2-AR in MEFs (mouse embryo fibroblasts) lacking both ß-arrestin 1 and ß-arrestin 2. This effect was negated using either the R26A or the R286A mutant form of ß-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 ß2-AR upon isoprenaline challenge. These data show that the interaction of PDE4D5 with both the N- and C-domains of ß-arrestin 2 are essential for ß2-AR regulation. © The Authors.
Original language | English |
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Pages (from-to) | 71-80 |
Number of pages | 10 |
Journal | Biochemical Journal |
Volume | 404 |
Issue number | 1 |
DOIs | |
Publication status | Published - 15 May 2007 |
Keywords
- β-arrestin
- β 2 -adrenoceptor
- cAMP
- Desensitization
- Peptide array
- Phosphodiesterase 4 (PDE4)