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

摘要

pFRET (fluorescence resonance energy transfer) 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, Lyssup285/sup, Argsup286/sup and Lyssup295/sup 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 β-arrestin 2–eCFP (where eCFP is enhanced cyan fluorescent protein) and β-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 βsub2/sub-adrenoceptor-mediated phosphorylation of the ERK1/2 (extracellular-signal-regulated kinase 1/2) MAPKs (mitogen-activated protein kinases). Both β-arrestin 2 mutants displayed limited capacity to co-immunoprecipitate ERK1/2 and further spot-immobilized peptide arrays indicated each of Lyssup285/sup, Argsup286/sup and particularly Lyssup295/sup to be important for this interaction. Direct interactions between β-arrestin 2 and the βsub2/sub-adrenoceptor were also compromised by both K285A/R286A and K295A 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 K285A/R286A or K295A mutants from wild-type β-arrestin 2, and the interaction of β-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 β-arrestin 2 also play an important role in the interaction with both the βsub2/sub-adrenoceptor and the ERK1/2 MAPKs. Regulation of β-arrestin 2 self-association may therefore control β-arrestin 2-mediated βsub2/sub-adrenoceptor-ERK1/2 MAPK signalling./p