title: Receptor-specific ubiquitination of beta-arrestin directs assembly and targeting of seven-transmembrane receptor signalosomes.
abstract: Angiotensin II type 1a (AT1a), vasopressin V2, and neurokinin 1 (NK1) receptors are seven-transmembrane receptors (7TMRs) that bind and co-internalize with the multifunctional adaptor protein, beta-arrestin. These receptors also lead to robust and persistent activation of extracellular-signal regulated kinase 1/2 (ERK1/2) localized on endosomes. Recently, the co-trafficking of receptor-beta-arrestin complexes to endosomes was demonstrated to require stable beta-arrestin ubiquitination (Shenoy, S. K., and Lefkowitz, R. J. (2003) J. Biol. Chem. 278, 14498-14506). We now report that lysines at positions 11 and 12 in beta-arrestin2 are specific and required sites for its AngII-mediated sustained ubiquitination. Thus, upon AngII stimulation the mutant beta-arrestin2(K11,12R) is only transiently ubiquitinated, does not form stable endocytic complexes with the AT1aR, and is impaired in scaffolding-activated ERK1/2. Fusion of a ubiquitin moiety in-frame to beta-arrestin2(K11,12R) restores AngII-mediated trafficking and signaling. Wild type beta-arrestin2 and beta-arrestin2(K11R,K12R)-Ub, but not beta-arrestin2(K11R,K12R), prevent nuclear translocation of pERK. These findings imply that sustained beta-arrestin ubiquitination not only directs co-trafficking of receptor-beta-arrestin complexes but also orchestrates the targeting of "7TMR signalosomes" to microcompartments within the cell. Surprisingly, binding of beta-arrestin2(K11R,K12R) to V2R and NK1R is indistinguishable from that of wild type beta-arrestin2. Moreover, ubiquitination patterns and ERK scaffolding of beta-arrestin2(K11,12R) are unimpaired with respect to V2R stimulation. In contrast, a quintuple lysine mutant (beta-arrestin2(K18R,K107R,K108R,K207R,K296R)) is impaired in endosomal trafficking in response to V2R but not AT1aR stimulation. Our findings delineate a novel regulatory mechanism for 7TMR signaling, dictated by the ubiquitination of beta-arrestin on specific lysines that become accessible for modification due to the specific receptor-bound conformational states of beta-arrestin2.
authors.affiliation: Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: Sudha K
authors.initials: SK
authors.lastname: Shenoy
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Lefkowitz
mesh_terms: D021581:Active Transport, Cell Nucleus; D000804:Angiotensin II / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D019390:Arrestins / Q000737:chemistry* / Q000378:metabolism; D019556:COS Cells; D002460:Cell Line; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D008239:Lysine / Q000737:chemistry; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008957:Models, Genetic; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D021381:Protein Transport; D015398:Signal Transduction; D025801:Ubiquitin / Q000737:chemistry* / Q000378:metabolism; D000071557:beta-Arrestins
chemical_list: D019390:Arrestins; D025801:Ubiquitin; D000071557:beta-Arrestins; D000804:Angiotensin II; D049452:Green Fluorescent Proteins; D048049:Extracellular Signal-Regulated MAP Kinases; D008239:Lysine