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Distinct conformational changes in beta-arrestin report biased agonism at seven-transmembrane receptors.

ABSTRACT: Beta-arrestins critically regulate G protein-coupled receptors (GPCRs), also known as seven-transmembrane receptors (7TMRs), both by inhibiting classical G protein signaling and by initiating distinct beta-arrestin-mediated signaling. The recent discovery of beta-arrestin-biased ligands and receptor mutants has allowed characterization of these independent "G protein-mediated" and "beta-arrestin-mediated" signaling mechanisms of 7TMRs. However, the molecular mechanisms underlying the dual functions of beta-arrestins remain unclear. Here, using an intramolecular BRET (bioluminescence resonance energy transfer)-based biosensor of beta-arrestin 2 and a combination of biased ligands and/or biased mutants of three different 7TMRs, we provide evidence that beta-arrestin can adopt multiple "active" conformations. Surprisingly, phosphorylation-deficient mutants of the receptors are also capable of directing similar conformational changes in beta-arrestin as is the wild-type receptor. This indicates that distinct receptor conformations induced and/or stabilized by different ligands can promote distinct and functionally specific conformations in beta-arrestin even in the absence of receptor phosphorylation. Our data thus highlight another interesting aspect of 7TMR signaling--i.e., functionally specific receptor conformations can be translated to downstream effectors such as beta-arrestins, thereby governing their functional specificity.

SUBMITTER: Shukla AK

PROVIDER: S-EPMC2481318 | biostudies-literature | 2008 Jul

REPOSITORIES: biostudies-literature

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Distinct conformational changes in beta-arrestin report biased agonism at seven-transmembrane receptors.

Shukla Arun K AK Violin Jonathan D JD Whalen Erin J EJ Gesty-Palmer Diane D Shenoy Sudha K SK Lefkowitz Robert J RJ

Proceedings of the National Academy of Sciences of the United States of America 20080711 29

Beta-arrestins critically regulate G protein-coupled receptors (GPCRs), also known as seven-transmembrane receptors (7TMRs), both by inhibiting classical G protein signaling and by initiating distinct beta-arrestin-mediated signaling. The recent discovery of beta-arrestin-biased ligands and receptor mutants has allowed characterization of these independent "G protein-mediated" and "beta-arrestin-mediated" signaling mechanisms of 7TMRs. However, the molecular mechanisms underlying the dual functi ...[more]

PMID: 18621717

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Project description:Beta-arrestin2 and its ubiquitination play crucial roles in both internalization and signaling of seven-transmembrane receptors (7TMRs). To understand the connection between ubiquitination and the endocytic and signaling functions of beta-arrestin, we generated a beta-arrestin2 mutant that is defective in ubiquitination (beta-arrestin2(0K)), by mutating all of the ubiquitin acceptor lysines to arginines and compared its properties with the wild type and a stably ubiquitinated beta-arrestin2-ubiquitin (Ub) chimera. In vitro translated beta-arrestin2 and beta-arrestin2(0K) displayed equivalent binding to recombinant beta(2)-adrenergic receptor (beta(2)AR) reconstituted in vesicles, whereas beta-arrestin2-Ub bound approximately 4-fold more. In cellular coimmunoprecipitation assays, beta-arrestin2(0K) bound nonreceptor partners, such as AP-2 and c-Raf and scaffolded phosphorylated ERK robustly but displayed weak binding to clathrin. Moreover, beta-arrestin2(0K) was recruited only transiently to activated receptors at the membrane, did not enhance receptor internalization, and decreased the amount of phosphorylated ERK assimilated into isolated beta(2)AR complexes. Although the wild type beta-arrestin2 formed ERK signaling complexes with the beta(2)AR at the membrane, a stably ubiquitinated beta-arrestin2-Ub chimera not only stabilized the ERK signalosomes but also led to their endosomal targeting. Interestingly, in cellular fractionation assays, the ubiquitination state of beta-arrestin2 favors its distribution in membrane fractions, suggesting that ubiquitination increases the propensity of beta-arrestin for membrane association. Our findings suggest that although beta-arrestin ubiquitination is dispensable for beta-arrestin cytosol to membrane translocation and its "constitutive" interactions with some cytosolic proteins, it nevertheless is a prerequisite both for the formation of tight complexes with 7TMRs in vivo and for membrane compartment interactions that are crucial for downstream endocytic and signaling processes.

Project description:beta-Arrestin-mediated signaling downstream of seven transmembrane receptors (7TMRs) is a relatively new paradigm for signaling by these receptors. We examined changes in protein phosphorylation occurring when HEK293 cells expressing the angiotensin II type 1A receptor (AT1aR) were stimulated with the beta-arrestin-biased ligand Sar(1), Ile(4), Ile(8)-angiotensin (SII), a ligand previously found to signal through beta-arrestin-dependent, G protein-independent mechanisms. Using a phospho-antibody array containing 46 antibodies against signaling molecules, we found that phosphorylation of 35 proteins increased upon SII stimulation. These SII-mediated phosphorylation events were abrogated after depletion of beta-arrestin 2 through siRNA-mediated knockdown. We also performed an MS-based quantitative phosphoproteome analysis after SII stimulation using a strategy of stable isotope labeling of amino acids in cell culture (SILAC). We identified 1,555 phosphoproteins (4,552 unique phosphopeptides), of which 171 proteins (222 phosphopeptides) showed increased phosphorylation, and 53 (66 phosphopeptides) showed decreased phosphorylation upon SII stimulation of the AT1aR. This study identified 38 protein kinases and three phosphatases whose phosphorylation status changed upon SII treatment. Using computational approaches, we performed system-based analyses examining the beta-arrestin-mediated phosphoproteome including construction of a kinase-substrate network for beta-arrestin-mediated AT1aR signaling. Our analysis demonstrates that beta-arrestin-dependent signaling processes are more diverse than previously appreciated. Notably, our analysis identifies an AT1aR-mediated cytoskeletal reorganization network whereby beta-arrestin regulates phosphorylation of several key proteins, including cofilin and slingshot. This study provides a system-based view of beta-arrestin-mediated phosphorylation events downstream of a 7TMR and opens avenues for research in a rapidly evolving area of 7TMR signaling.

Project description:RATIONALE:MicroRNAs (miRs) are small, non-coding RNAs that function to post-transcriptionally regulate target genes. First transcribed as primary miR transcripts (pri-miRs), they are enzymatically processed by Drosha into premature miRs (pre-miRs) and further cleaved by Dicer into mature miRs. Initially discovered to desensitize ?-adrenergic receptor (?AR) signaling, ?-arrestins are now well-appreciated to modulate multiple pathways independent of G protein signaling, a concept known as biased signaling. Using the ?-arrestin-biased ?AR ligand carvedilol, we previously showed that ?-arrestin1 (not ?-arrestin2)-biased ?1AR (not ?2AR) cardioprotective signaling stimulates Drosha-mediated processing of six miRs by forming a multi-protein nuclear complex, which includes ?-arrestin1, the Drosha microprocessor complex and a single-stranded RNA binding protein hnRNPA1. OBJECTIVE:Here, we investigate whether ?-arrestin-mediated ?AR signaling induced by carvedilol could regulate Dicer-mediated miR maturation in the cytoplasm and whether this novel mechanism promotes cardioprotective signaling. METHODS AND RESULTS:In mouse hearts, carvedilol indeed upregulates three mature miRs, but not their pre-miRs and pri-miRs, in a ?-arrestin 1- or 2-dependent manner. Interestingly, carvedilol-mediated activation of miR-466g or miR-532-5p, and miR-674 is dependent on ?2ARs and ?1ARs, respectively. Mechanistically, ?-arrestin 1 or 2 regulates maturation of three newly identified ?AR/?-arrestin-responsive miRs (?-miRs) by associating with the Dicer maturation RNase III enzyme on three pre-miRs of ?-miRs. Myocardial cell approaches uncover that despite their distinct roles in different cell types, ?-miRs act as gatekeepers of cardiac cell functions by repressing deleterious targets. CONCLUSIONS:Our findings indicate a novel role for ?AR-mediated ?-arrestin signaling activated by carvedilol in Dicer-mediated miR maturation, which may be linked to its protective mechanisms.

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Distinct conformational changes in beta-arrestin report biased agonism at seven-transmembrane receptors.

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Distinct conformational changes in beta-arrestin report biased agonism at seven-transmembrane receptors.

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