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

被引:32
|
作者
Xu, Tian-Rui [1 ,2 ]
Baillie, George S. [1 ]
Bhari, Narinder [1 ,2 ]
Holislay, Thomas M. [1 ]
Pitt, Andrew M. [1 ,2 ]
Adams, David R. [3 ]
Kolch, Walter [2 ,4 ]
Houslay, Miles D. [1 ]
Milligan, Graeme [1 ]
机构
[1] Univ Glasgow, Inst Biomed & Life Sci, Div Biochem & Mol Biol, Mol Pharmacol Grp, Glasgow G12 8QQ, Lanark, Scotland
[2] Univ Glasgow, Div Biochem & Mol Biol, Inst Biomed & Life Sci, Sir Henry Welcome Funct Genom Facil, Glasgow G12 8QQ, Lanark, Scotland
[3] Heriot Watt Univ, Dept Chem, Edinburgh EH14 4AS, Midlothian, Scotland
[4] Beatson Inst Canc Res, Glasgow G61 1BD, Lanark, Scotland
基金
英国医学研究理事会;
关键词
beta-arrestin; 2; scaffolding; self association;
D O I
10.1042/BJ20080685
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
FRET (fluorescence resonance energy transfer) and co-immuno-precipitation 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 beta(2)-adrenoceptor-mediated phosphorylation of the ERK 1/2 (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 beta(2)-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-affestin 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 beta(2)-adrenoceptor and the ERK1/2 MAPKs. Regulation of beta-arrestin 2 self-association may therefore control beta-arrestin 2-mediated beta(2)-adrenoceptor-ERK1/2 MAPK signalling.
引用
收藏
页码:51 / 60
页数:10
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