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

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.

Application of genomics, proteomics and metabolomics in drug discovery, development and clinic

Genomics, proteomics and metabolomics are three areas that are routinely applied throughout the drug-development process as well as after a product enters the market. This review discusses all three 'omics, reporting on the key applications, techniques, recent advances and expectations of each. Genomics, mainly through the use of novel and next-generation sequencing techniques, has advanced areas of drug discovery and development through the comparative assessment of normal and diseased-state tissues, transcription and/or expression profiling, side-effect profiling, pharmacogenomics and the identification of biomarkers. Proteomics, through techniques including isotope coded affinity tags, stable isotopic labeling by amino acids in cell culture, isobaric tags for relative and absolute quantification, multidirectional protein identification technology, activity-based probes, protein/peptide arrays, phage displays and two-hybrid systems is utilized in multiple areas through the drug development pipeline including target and lead identification, compound optimization, throughout the clinical trials process and after market analysis. Metabolomics, although the most recent and least developed of the three 'omics considered in this review, provides a significant contribution to drug development through systems biology approaches. Already implemented to some degree in the drug-discovery industry and used in applications spanning target identification through to toxicological analysis, metabolic network understanding is essential in generating future discoveries.

Application of genomics, proteomics and metabolomics in drug discovery, development and clinic

Genomics, proteomics and metabolomics are three areas that are routinely applied throughout the drug-development process as well as after a product enters the market. This review discusses all three 'omics, reporting on the key applications, techniques, recent advances and expectations of each. Genomics, mainly through the use of novel and next-generation sequencing techniques, has advanced areas of drug discovery and development through the comparative assessment of normal and diseased-state tissues, transcription and/or expression profiling, side-effect profiling, pharmacogenomics and the identification of biomarkers. Proteomics, through techniques including isotope coded affinity tags, stable isotopic labeling by amino acids in cell culture, isobaric tags for relative and absolute quantification, multidirectional protein identification technology, activity-based probes, protein/peptide arrays, phage displays and two-hybrid systems is utilized in multiple areas through the drug development pipeline including target and lead identification, compound optimization, throughout the clinical trials process and after market analysis. Metabolomics, although the most recent and least developed of the three 'omics considered in this review, provides a significant contribution to drug development through systems biology approaches. Already implemented to some degree in the drug-discovery industry and used in applications spanning target identification through to toxicological analysis, metabolic network understanding is essential in generating future discoveries.