Receptor-specific ubiquitination of beta-arrestin directs assembly and targeting of seven-transmembrane receptor signalosomes.
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.
Mesh Terms:
Active Transport, Cell Nucleus, Angiotensin II, Animals, Arrestins, COS Cells, Cell Line, Endocytosis, Endosomes, Extracellular Signal-Regulated MAP Kinases, Green Fluorescent Proteins, Humans, Immunoprecipitation, Lysine, Microscopy, Confocal, Microscopy, Fluorescence, Models, Genetic, Mutation, Protein Binding, Protein Conformation, Protein Transport, Signal Transduction, Ubiquitin
Active Transport, Cell Nucleus, Angiotensin II, Animals, Arrestins, COS Cells, Cell Line, Endocytosis, Endosomes, Extracellular Signal-Regulated MAP Kinases, Green Fluorescent Proteins, Humans, Immunoprecipitation, Lysine, Microscopy, Confocal, Microscopy, Fluorescence, Models, Genetic, Mutation, Protein Binding, Protein Conformation, Protein Transport, Signal Transduction, Ubiquitin
J. Biol. Chem.
Date: Apr. 15, 2005
PubMed ID: 15699045
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