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

George S. Baillie, David R. Adams, Narinder Bhari, Thomas M. Houslay, Suryakiran Vadrevu, Dong Meng, Xiang Li, Allan Dunlop, Graeme Milligan, Graeme B. Bolger, Enno Klussmann, Miles D. Houslay

Research output: Contribution to journalArticle

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 languageEnglish
Pages (from-to)71-80
Number of pages10
JournalBiochemical Journal
Volume404
Issue number1
DOIs
Publication statusPublished - 15 May 2007

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Arrestin
Phosphoric Diester Hydrolases
Binding Sites
Peptides
Adrenergic Receptors
Cyclic AMP-Dependent Protein Kinases
Isoproterenol
Alanine
Protein Isoforms
Phosphorylation
beta-Arrestin 1
Peptide Library
Cellulose
Transfection
Embryonic Structures
Fibroblasts
Technology

Keywords

  • β-arrestin
  • β 2 -adrenoceptor
  • cAMP
  • Desensitization
  • Peptide array
  • Phosphodiesterase 4 (PDE4)

Cite this

Baillie, George S. ; Adams, David R. ; Bhari, Narinder ; Houslay, Thomas M. ; Vadrevu, Suryakiran ; Meng, Dong ; Li, Xiang ; Dunlop, Allan ; Milligan, Graeme ; Bolger, Graeme B. ; Klussmann, Enno ; Houslay, Miles D. / Mapping binding sites for the PDE4D5 cAMP-specific phosphodiesterase to the N- and C-domains of β-arrestin using spot-immobilized peptide arrays. In: Biochemical Journal. 2007 ; Vol. 404, No. 1. pp. 71-80.
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abstract = "{\ss}2-ARs ({\ss}2-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 {\ss}2-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 {\ss}2-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 {\ss}2-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 {\ss}2-AR regulation. {\circledC} The Authors.",
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Baillie, GS, Adams, DR, Bhari, N, Houslay, TM, Vadrevu, S, Meng, D, Li, X, Dunlop, A, Milligan, G, Bolger, GB, Klussmann, E & Houslay, MD 2007, 'Mapping binding sites for the PDE4D5 cAMP-specific phosphodiesterase to the N- and C-domains of β-arrestin using spot-immobilized peptide arrays', Biochemical Journal, vol. 404, no. 1, pp. 71-80. https://doi.org/10.1042/BJ20070005

Mapping binding sites for the PDE4D5 cAMP-specific phosphodiesterase to the N- and C-domains of β-arrestin using spot-immobilized peptide arrays. / Baillie, George S.; Adams, David R.; Bhari, Narinder; Houslay, Thomas M.; Vadrevu, Suryakiran; Meng, Dong; Li, Xiang; Dunlop, Allan; Milligan, Graeme; Bolger, Graeme B.; Klussmann, Enno; Houslay, Miles D.

In: Biochemical Journal, Vol. 404, No. 1, 15.05.2007, p. 71-80.

Research output: Contribution to journalArticle

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T1 - Mapping binding sites for the PDE4D5 cAMP-specific phosphodiesterase to the N- and C-domains of β-arrestin using spot-immobilized peptide arrays

AU - Baillie, George S.

AU - Adams, David R.

AU - Bhari, Narinder

AU - Houslay, Thomas M.

AU - Vadrevu, Suryakiran

AU - Meng, Dong

AU - Li, Xiang

AU - Dunlop, Allan

AU - Milligan, Graeme

AU - Bolger, Graeme B.

AU - Klussmann, Enno

AU - Houslay, Miles D.

PY - 2007/5/15

Y1 - 2007/5/15

N2 - 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.

AB - 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.

KW - β-arrestin

KW - β 2 -adrenoceptor

KW - cAMP

KW - Desensitization

KW - Peptide array

KW - Phosphodiesterase 4 (PDE4)

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