title: beta-arrestin-dependent, G protein-independent ERK1/2 activation by the beta2 adrenergic receptor.
abstract: Physiological effects of beta adrenergic receptor (beta2AR) stimulation have been classically shown to result from G(s)-dependent adenylyl cyclase activation. Here we demonstrate a novel signaling mechanism wherein beta-arrestins mediate beta2AR signaling to extracellular-signal regulated kinases 1/2 (ERK 1/2) independent of G protein activation. Activation of ERK1/2 by the beta2AR expressed in HEK-293 cells was resolved into two components dependent, respectively, on G(s)-G(i)/protein kinase A (PKA) or beta-arrestins. G protein-dependent activity was rapid, peaking within 2-5 min, was quite transient, was blocked by pertussis toxin (G(i) inhibitor) and H-89 (PKA inhibitor), and was insensitive to depletion of endogenous beta-arrestins by siRNA. beta-Arrestin-dependent activation was slower in onset (peak 5-10 min), less robust, but more sustained and showed little decrement over 30 min. It was insensitive to pertussis toxin and H-89 and sensitive to depletion of either beta-arrestin1 or -2 by small interfering RNA. In G(s) knock-out mouse embryonic fibroblasts, wild-type beta2AR recruited beta-arrestin2-green fluorescent protein and activated pertussis toxin-insensitive ERK1/2. Furthermore, a novel beta2AR mutant (beta2AR(T68F,Y132G,Y219A) or beta2AR(TYY)), rationally designed based on Evolutionary Trace analysis, was incapable of G protein activation but could recruit beta-arrestins, undergo beta-arrestin-dependent internalization, and activate beta-arrestin-dependent ERK. Interestingly, overexpression of GRK5 or -6 increased mutant receptor phosphorylation and beta-arrestin recruitment, led to the formation of stable receptor-beta-arrestin complexes on endosomes, and increased agonist-stimulated phospho-ERK1/2. In contrast, GRK2, membrane translocation of which requires Gbetagamma release upon G protein activation, was ineffective unless it was constitutively targeted to the plasma membrane by a prenylation signal (CAAX). These findings demonstrate that the beta2AR can signal to ERK via a GRK5/6-beta-arrestin-dependent pathway, which is independent of G protein coupling.
authors.affiliation: Howard Hughes Medical Institute at Duke University Medical Center, Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA.
authors.forename: Sudha K
authors.initials: SK
authors.lastname: Shenoy
authors.forename: Matthew T
authors.initials: MT
authors.lastname: Drake
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Nelson
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Houtz
authors.forename: Kunhong
authors.initials: K
authors.lastname: Xiao
authors.forename: Srinivasan
authors.initials: S
authors.lastname: Madabushi
authors.forename: Eric
authors.initials: E
authors.lastname: Reiter
authors.forename: Richard T
authors.initials: RT
authors.lastname: Premont
authors.forename: Olivier
authors.initials: O
authors.lastname: Lichtarge
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Lefkowitz
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019390:Arrestins / Q000378:metabolism*; D019556:COS Cells; D002417:Cattle; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D000242:Cyclic AMP / Q000378:metabolism; D019143:Evolution, Molecular; D054774:G-Protein-Coupled Receptor Kinase 5; D054768:G-Protein-Coupled Receptor Kinases; D019204:GTP-Binding Proteins / Q000737:chemistry*; D006801:Humans; D020120:Iodocyanopindolol / Q000737:chemistry; D007546:Isoquinolines / Q000494:pharmacology; D007700:Kinetics; D051379:Mice; D018345:Mice, Knockout; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D037342:Pertussis Toxin / Q000494:pharmacology; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D021381:Protein Transport; D034741:RNA, Small Interfering / Q000378:metabolism; D018343:Receptors, Adrenergic, beta-2 / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D013449:Sulfonamides / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection; D000071557:beta-Arrestins
chemical_list: D019390:Arrestins; D007546:Isoquinolines; D034741:RNA, Small Interfering; D018343:Receptors, Adrenergic, beta-2; D013449:Sulfonamides; D000071557:beta-Arrestins; D020120:Iodocyanopindolol; D000242:Cyclic AMP; D037342:Pertussis Toxin; D017346:Protein Serine-Threonine Kinases; D054774:G-Protein-Coupled Receptor Kinase 5; D054768:G-Protein-Coupled Receptor Kinases; C089176:G-protein-coupled receptor kinase 6; C517898:GRK5 protein, human; C517895:Grk5 protein, mouse; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D019204:GTP-Binding Proteins; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide