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

Tian-Rui Xu , George S. Baillie , Narinder Bhari , Thomas M. Houslay , Andrew M Pitt , David R Adams , Walter Kolch , Miles D Houslay , Graeme Milligan (1)

1 Biochemistry and Molecular Biology

Fluorescence resonance energy transfer (FRET) and co-immunoprecipitation studies confirmed the capacity of β-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 Lys285Ala:Arg286Ala mutations into β-arrestin 2-eCFP and β-arrestin 2-eYFP constructs substantially reduced FRET, whilst introduction of a Lys295Ala mutation had a more limited effect. Neither of these mutants was able to promote β2-adrenoceptor-mediated phosphorylation of the ERK1/2 MAP kinases. Both β-arrestin 2 mutants displayed limited capacity to co-immunoprecipitate ERK1/2 and further spot-immobilized peptide arrays indicated each of Lys285, Arg286 and particularly Lys295 to be important for this interaction. Direct interactions between β-arrestin 2 and the β2-adrenoceptor were also compromised by both Lys285Ala:Arg286Ala and Lys295Ala mutations of β-arrestin 2. These were not non-specific effects linked to improper folding of β-arrestin 2 as limited proteolysis was unable to distinguish the Lys285Ala:Arg286Ala or Lys295Ala mutants from wild type β-arrestin 2, whilst interaction of β-arrestin 2 with JNK3 was unaffected by the Lys285Ala:Arg286Ala or Lys295Ala mutations. These data suggest that amino acids important for self-association of β-arrestin 2 also play an important role in interaction with both the β2-adrenoceptor and the ERK1/2 MAP kinases. Regulation of β-arrestin 2 self-association may therefore control β-arrestin 2-mediated β2-adrenoceptor-ERK1/2 MAP kinase signalling.

Mots clés: Life Sciences

Tian-Rui Xu, George S. Baillie, Narinder Bhari, Thomas M. Houslay, Andrew M Pitt, et al.. Mutations of β-arrestin 2 that limit self-association also interfere with interactions with the β2-adrenoceptor and the ERK1/2 MAP kinases: Implications for β2-adrenoceptor-signalling via the ERK1/2 MAP kinases.. Biochemical Journal, 2008, 413 (1), pp.51-60. ⟨10.1042/BJ20080685⟩. ⟨hal-00479005⟩