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

Xu, T.-R., Baillie, G. S. , Bhari, N., Houslay, T. M., Pitt, A. M., Adams, D. R., Kolch, W., Houslay, M. D. and Milligan, G. (2008) 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. Biochemical Journal, 413(1), pp. 51-60. (doi: 10.1042/BJ20080685)

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Publisher's URL: http://dx.doi.org/10.1042/BJ20080685

Abstract

FRET (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, 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 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 β2-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 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 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 β2-adrenoceptor and the ERK1/2 MAPKs. Regulation of β-arrestin 2 self-association may therefore control β-arrestin 2-mediated β2-adrenoceptor-ERK1/2 MAPK signalling.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Xu, Professor Tianrui and Houslay, Professor Miles and Baillie, Professor George and Kolch, Prof Walter and Milligan, Professor Graeme and Pitt, Dr Andrew and Bhari, Mrs Narinder and Houslay, Mr Thomas
Authors: Xu, T.-R., Baillie, G. S., Bhari, N., Houslay, T. M., Pitt, A. M., Adams, D. R., Kolch, W., Houslay, M. D., and Milligan, G.
Subjects:Q Science > QH Natural history > QH345 Biochemistry
College/School:College of Medical Veterinary and Life Sciences > Institute of Molecular Cell and Systems Biology
College of Medical Veterinary and Life Sciences > School of Psychology & Neuroscience
Journal Name:Biochemical Journal
Publisher:Portland Press Ltd.
ISSN:0264-6021
ISSN (Online):1470-8728
Published Online:24 April 2008
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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
438301Phosphodiesterase-4 isoforms - intracellular targeting, regulation and potential therapeutic targetsMiles HouslayMedical Research Council (MRC)G0600765Institute of Neuroscience and Psychology
242491Quantitative Analysis of the Effects of Receptor and G-Protein Mutations and Polymorphisms on Signal InitiationGraeme MilliganMedical Research Council (MRC)G9811527Institute of Neuroscience and Psychology
242492Quantitative Analysis of the Effects of Receptor and G-Protein Mutations and Polymorphisms on Signal InitiationGraeme MilliganMedical Research Council (MRC)G9811527Institute of Neuroscience and Psychology
389781The quaternary structure of G-protein coupled receptors - implications for function drug design. Programme grant support renewalGraeme MilliganMedical Research Council (MRC)G9811527Institute of Neuroscience and Psychology
374281Protein interactions and compartmentalisation in cell signallingWalter KolchMedical Research Council (MRC)G0400053MCSB - BIOCHEMISTRY & CELL BIOLOGY