Mutations of β-arrestin 2 that limit self-association also interfere with interactions with the β2-adrenoceptor and the ERK1/2 MAPKs: Implications for β2-adrenoceptor signalling via the ERK1/2 MAPKs

Tian R. 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

27 Citations (Scopus)

Abstract

FRET (fluorescence resonance energy transfer) and co-immunoprecipitation studies confirmed the capacity of ß-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, Lys285, Arg286 and Lys295 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 ß-arrestin 2-eCFP (where eCFP is enhanced cyan fluorescent protein) and ß-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 ß2-adrenoceptor-mediated phosphorylation of the ERK1/2 (extracellular-signal-regulated kinase 1/2) MAPKs (mitogen-activated protein kinases). Both ß-arrestin 2 mutants displayed limited capacity to co-immunoprecipitate ERK1/2 and further spot-immobilized peptide arrays indicated each of Lys285, Arg286 and particularly Lys 295 to be important for this interaction. Direct interactions between ß-arrestin 2 and the ß2-adrenoceptor were also compromised by both K285A/R286A and K295A mutations of ß-arrestin 2. These were not non-specific effects linked to improper folding of ß-arrestin 2 as limited proteolysis was unable to distinguish the K285A/R286A or K295A mutants from wild-type ß-arrestin 2, and the interaction of ß-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 ß-arrestin 2 also play an important role in the interaction with both the ß2-adrenoceptor and the ERK1/2 MAPKs. Regulation of ß-arrestin 2 self-association may therefore control ß-arrestin 2-mediated ß2-adrenoceptor-ERK1/2 MAPK signalling. © The Authors.

Original languageEnglish
Pages (from-to)51-60
Number of pages10
JournalBiochemical Journal
Volume413
Issue number1
DOIs
Publication statusPublished - 1 Jul 2008

Keywords

  • β-arrestin 2
  • Scaffolding
  • Self association

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