title: Mutations of beta-arrestin 2 that limit self-association also interfere with interactions with the beta2-adrenoceptor and the ERK1/2 MAPKs: implications for beta2-adrenoceptor signalling via the ERK1/2 MAPKs.
abstract: FRET (fluorescence resonance energy transfer) and co-immunoprecipitation 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 beta2-adrenoceptor-mediated phosphorylation of the ERK1/2 (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 beta2-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-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 beta-arrestin 2 also play an important role in the interaction with both the beta2-adrenoceptor and the ERK1/2 MAPKs. Regulation of beta-arrestin 2 self-association may therefore control beta-arrestin 2-mediated beta2-adrenoceptor-ERK1/2 MAPK signalling.
authors.affiliation: Sir Henry Welcome Functional Genomics Facility, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, UK.
authors.forename: Tian-Rui
authors.initials: TR
authors.lastname: Xu
authors.forename: George S
authors.initials: GS
authors.lastname: Baillie
authors.forename: Narinder
authors.initials: N
authors.lastname: Bhari
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Houslay
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Pitt
authors.forename: David R
authors.initials: DR
authors.lastname: Adams
authors.forename: Walter
authors.initials: W
authors.lastname: Kolch
authors.forename: Miles D
authors.initials: MD
authors.lastname: Houslay
authors.forename: Graeme
authors.initials: G
authors.lastname: Milligan
mesh_terms: D000595:Amino Acid Sequence; D019390:Arrestins / Q000235:genetics* / Q000378:metabolism*; D002460:Cell Line; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D008958:Models, Molecular; D010766:Phosphorylation; D017354:Point Mutation; D040081:Protein Array Analysis; D011485:Protein Binding; D011487:Protein Conformation; D018343:Receptors, Adrenergic, beta-2 / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins
chemical_list: C000605254:ARRB2 protein, human; D019390:Arrestins; D018343:Receptors, Adrenergic, beta-2; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; D048049:Extracellular Signal-Regulated MAP Kinases