Mutations of beta-arrestin 2 that limit self-association also interfere with interactions with the beta2-adrenoceptor and the ERK1/2 MAPKs: implications for beta2-adrenoceptor signalling via the ERK1/2 MAPKs

Tian-Rui Xu, George S. Baillie, Narinder Bhari, Thomas M. Houslay, Andrew M Pitt, David R. Adams, Walter Kolch, Miles D. Houslay, Graeme Milligan

Research output: Contribution to journalArticle

Abstract

FRET (fluorescence resonance energy transfer) and co-immunoprecipitation studies confirmed the capacity of beta-arrestin 2 to self-associate. Amino acids potentially involved in direct protein-protein interaction were identified via combinations of spot-immobilized peptide arrays and mapping of surface exposure. Among potential key amino acids, Lys(285), Arg(286) and Lys(295) are part of a continuous surface epitope located in the polar core between the N- and C-terminal domains. Introduction of K285A/R286A mutations into beta-arrestin 2-eCFP (where eCFP is enhanced cyan fluorescent protein) and beta-arrestin 2-eYFP (where eYFP is enhanced yellow fluorescent protein) constructs substantially reduced FRET, whereas introduction of a K295A mutation had a more limited effect. Neither of these mutants was able to promote beta2-adrenoceptor-mediated phosphorylation of the ERK1/2 (extracellular-signal-regulated kinase 1/2) MAPKs (mitogen-activated protein kinases). Both beta-arrestin 2 mutants displayed limited capacity to co-immunoprecipitate ERK1/2 and further spot-immobilized peptide arrays indicated each of Lys(285), Arg(286) and particularly Lys(295) to be important for this interaction. Direct interactions between beta-arrestin 2 and the beta2-adrenoceptor were also compromised by both K285A/R286A and K295A mutations of beta-arrestin 2. These were not non-specific effects linked to improper folding of beta-arrestin 2 as limited proteolysis was unable to distinguish the K285A/R286A or K295A mutants from wild-type beta-arrestin 2, and the interaction of beta-arrestin 2 with JNK3 (c-Jun N-terminal kinase 3) was unaffected by the K285A/R286A or L295A mutations. These results suggest that amino acids important for self-association of beta-arrestin 2 also play an important role in the interaction with both the beta2-adrenoceptor and the ERK1/2 MAPKs. Regulation of beta-arrestin 2 self-association may therefore control beta-arrestin 2-mediated beta2-adrenoceptor-ERK1/2 MAPK signalling.
Original languageEnglish
Pages (from-to)51-60
Number of pages10
JournalBiochemical Journal
Volume413
Issue number1
DOIs
Publication statusPublished - 1 Jul 2008

Fingerprint

Arrestin
Mitogen-Activated Protein Kinase 3
Mitogen-Activated Protein Kinase 1
Mitogen-Activated Protein Kinases
Adrenergic Receptors
Association reactions
Mutation
Fluorescence Resonance Energy Transfer
Amino Acids
beta-Arrestin 2
Mitogen-Activated Protein Kinase 10
Proteolysis
Peptides
Phosphorylation
Proteins
Peptide Mapping
Immunoprecipitation
Epitopes

Keywords

  • amino acid sequence
  • arrestins
  • cell line
  • extracellular signal-regulated MAP kinases
  • fluorescence resonance energy transfer
  • humans
  • molecular models
  • phosphorylation
  • point mutation
  • protein array analysis
  • protein binding
  • protein conformation
  • adrenergic receptors
  • signal transduction

Cite this

Xu, Tian-Rui ; Baillie, George S. ; Bhari, Narinder ; Houslay, Thomas M. ; Pitt, Andrew M ; Adams, David R. ; Kolch, Walter ; Houslay, Miles D. ; Milligan, Graeme. / Mutations of beta-arrestin 2 that limit self-association also interfere with interactions with the beta2-adrenoceptor and the ERK1/2 MAPKs : implications for beta2-adrenoceptor signalling via the ERK1/2 MAPKs. In: Biochemical Journal. 2008 ; Vol. 413, No. 1. pp. 51-60.
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Mutations of beta-arrestin 2 that limit self-association also interfere with interactions with the beta2-adrenoceptor and the ERK1/2 MAPKs : implications for beta2-adrenoceptor signalling via the ERK1/2 MAPKs. / Xu, Tian-Rui; Baillie, George S.; Bhari, Narinder; Houslay, Thomas M.; Pitt, Andrew M; Adams, David R.; Kolch, Walter; Houslay, Miles D.; Milligan, Graeme.

In: Biochemical Journal, Vol. 413, No. 1, 01.07.2008, p. 51-60.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Mutations of beta-arrestin 2 that limit self-association also interfere with interactions with the beta2-adrenoceptor and the ERK1/2 MAPKs

T2 - implications for beta2-adrenoceptor signalling via the ERK1/2 MAPKs

AU - Xu, Tian-Rui

AU - Baillie, George S.

AU - Bhari, Narinder

AU - Houslay, Thomas M.

AU - Pitt, Andrew M

AU - Adams, David R.

AU - Kolch, Walter

AU - Houslay, Miles D.

AU - Milligan, Graeme

PY - 2008/7/1

Y1 - 2008/7/1

N2 - FRET (fluorescence resonance energy transfer) and co-immunoprecipitation studies confirmed the capacity of beta-arrestin 2 to self-associate. Amino acids potentially involved in direct protein-protein interaction were identified via combinations of spot-immobilized peptide arrays and mapping of surface exposure. Among potential key amino acids, Lys(285), Arg(286) and Lys(295) are part of a continuous surface epitope located in the polar core between the N- and C-terminal domains. Introduction of K285A/R286A mutations into beta-arrestin 2-eCFP (where eCFP is enhanced cyan fluorescent protein) and beta-arrestin 2-eYFP (where eYFP is enhanced yellow fluorescent protein) constructs substantially reduced FRET, whereas introduction of a K295A mutation had a more limited effect. Neither of these mutants was able to promote beta2-adrenoceptor-mediated phosphorylation of the ERK1/2 (extracellular-signal-regulated kinase 1/2) MAPKs (mitogen-activated protein kinases). Both beta-arrestin 2 mutants displayed limited capacity to co-immunoprecipitate ERK1/2 and further spot-immobilized peptide arrays indicated each of Lys(285), Arg(286) and particularly Lys(295) to be important for this interaction. Direct interactions between beta-arrestin 2 and the beta2-adrenoceptor were also compromised by both K285A/R286A and K295A mutations of beta-arrestin 2. These were not non-specific effects linked to improper folding of beta-arrestin 2 as limited proteolysis was unable to distinguish the K285A/R286A or K295A mutants from wild-type beta-arrestin 2, and the interaction of beta-arrestin 2 with JNK3 (c-Jun N-terminal kinase 3) was unaffected by the K285A/R286A or L295A mutations. These results suggest that amino acids important for self-association of beta-arrestin 2 also play an important role in the interaction with both the beta2-adrenoceptor and the ERK1/2 MAPKs. Regulation of beta-arrestin 2 self-association may therefore control beta-arrestin 2-mediated beta2-adrenoceptor-ERK1/2 MAPK signalling.

AB - FRET (fluorescence resonance energy transfer) and co-immunoprecipitation studies confirmed the capacity of beta-arrestin 2 to self-associate. Amino acids potentially involved in direct protein-protein interaction were identified via combinations of spot-immobilized peptide arrays and mapping of surface exposure. Among potential key amino acids, Lys(285), Arg(286) and Lys(295) are part of a continuous surface epitope located in the polar core between the N- and C-terminal domains. Introduction of K285A/R286A mutations into beta-arrestin 2-eCFP (where eCFP is enhanced cyan fluorescent protein) and beta-arrestin 2-eYFP (where eYFP is enhanced yellow fluorescent protein) constructs substantially reduced FRET, whereas introduction of a K295A mutation had a more limited effect. Neither of these mutants was able to promote beta2-adrenoceptor-mediated phosphorylation of the ERK1/2 (extracellular-signal-regulated kinase 1/2) MAPKs (mitogen-activated protein kinases). Both beta-arrestin 2 mutants displayed limited capacity to co-immunoprecipitate ERK1/2 and further spot-immobilized peptide arrays indicated each of Lys(285), Arg(286) and particularly Lys(295) to be important for this interaction. Direct interactions between beta-arrestin 2 and the beta2-adrenoceptor were also compromised by both K285A/R286A and K295A mutations of beta-arrestin 2. These were not non-specific effects linked to improper folding of beta-arrestin 2 as limited proteolysis was unable to distinguish the K285A/R286A or K295A mutants from wild-type beta-arrestin 2, and the interaction of beta-arrestin 2 with JNK3 (c-Jun N-terminal kinase 3) was unaffected by the K285A/R286A or L295A mutations. These results suggest that amino acids important for self-association of beta-arrestin 2 also play an important role in the interaction with both the beta2-adrenoceptor and the ERK1/2 MAPKs. Regulation of beta-arrestin 2 self-association may therefore control beta-arrestin 2-mediated beta2-adrenoceptor-ERK1/2 MAPK signalling.

KW - amino acid sequence

KW - arrestins

KW - cell line

KW - extracellular signal-regulated MAP kinases

KW - fluorescence resonance energy transfer

KW - humans

KW - molecular models

KW - phosphorylation

KW - point mutation

KW - protein array analysis

KW - protein binding

KW - protein conformation

KW - adrenergic receptors

KW - signal transduction

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