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 > School of Molecular Biosciences 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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