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Biased signaling pathways in ?2-adrenergic receptor characterized by 19F-NMR.

ABSTRACT: Extracellular ligand binding to G protein-coupled receptors (GPCRs) modulates G protein and ?-arrestin signaling by changing the conformational states of the cytoplasmic region of the receptor. Using site-specific (19)F-NMR (fluorine-19 nuclear magnetic resonance) labels in the ?(2)-adrenergic receptor (?(2)AR) in complexes with various ligands, we observed that the cytoplasmic ends of helices VI and VII adopt two major conformational states. Changes in the NMR signals reveal that agonist binding primarily shifts the equilibrium toward the G protein-specific active state of helix VI. In contrast, ?-arrestin-biased ligands predominantly impact the conformational states of helix VII. The selective effects of different ligands on the conformational equilibria involving helices VI and VII provide insights into the long-range structural plasticity of ?(2)AR in partial and biased agonist signaling.

SUBMITTER: Liu JJ

PROVIDER: S-EPMC3292700 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature

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Biased signaling pathways in β2-adrenergic receptor characterized by 19F-NMR.

Liu Jeffrey J JJ Horst Reto R Katritch Vsevolod V Stevens Raymond C RC Wüthrich Kurt K

Science (New York, N.Y.) 20120119 6072

Extracellular ligand binding to G protein-coupled receptors (GPCRs) modulates G protein and β-arrestin signaling by changing the conformational states of the cytoplasmic region of the receptor. Using site-specific (19)F-NMR (fluorine-19 nuclear magnetic resonance) labels in the β(2)-adrenergic receptor (β(2)AR) in complexes with various ligands, we observed that the cytoplasmic ends of helices VI and VII adopt two major conformational states. Changes in the NMR signals reveal that agonist bindin ...[more]

PMID: 22267580

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Project description:Reperfusion as a therapeutic intervention for acute myocardial infarction-induced cardiac injury itself induces further cardiomyocyte death. ?-arrestin (?arr)-biased ?-adrenergic receptor (?AR) activation promotes survival signaling responses in vitro; thus, we hypothesize that this pathway can mitigate cardiomyocyte death at the time of reperfusion to better preserve function. However, a lack of efficacious ?arr-biased orthosteric small molecules has prevented investigation into whether this pathway relays protection against ischemic injury in vivo. We recently demonstrated that the pepducin ICL1-9, a small lipidated peptide fragment designed from the first intracellular loop of ?2AR, allosterically engaged pro-survival signaling cascades in a ?arr-dependent manner in vitro. Thus, in this study we tested whether ICL1-9 relays cardioprotection against ischemia/reperfusion (I/R)-induced injury in vivo. Methods: Wild-type (WT) C57BL/6, ?2AR knockout (KO), ?arr1KO and ?arr2KO mice received intracardiac injections of either ICL1-9 or a scrambled control pepducin (Scr) at the time of ischemia (30 min) followed by reperfusion for either 24 h, to assess infarct size and cardiomyocyte death, or 4 weeks, to monitor the impact of ICL1-9 on long-term cardiac structure and function. Neonatal rat ventricular myocytes (NRVM) were used to assess the impact of ICL1-9 versus Scr pepducin on cardiomyocyte survival and mitochondrial superoxide formation in response to either serum deprivation or hypoxia/reoxygenation (H/R) in vitro and to investigate the associated mechanism(s). Results: Intramyocardial injection of ICL1-9 at the time of I/R reduced infarct size, cardiomyocyte death and improved cardiac function in a ?2AR- and ?arr-dependent manner, which led to improved contractile function early and less fibrotic remodeling over time. Mechanistically, ICL1-9 attenuated mitochondrial superoxide production and promoted cardiomyocyte survival in a RhoA/ROCK-dependent manner. RhoA activation could be detected in cardiomyocytes and whole heart up to 24 h post-treatment, demonstrating the stability of ICL1-9 effects on ?arr-dependent ?2AR signaling. Conclusion: Pepducin-based allosteric modulation of ?arr-dependent ?2AR signaling represents a novel therapeutic approach to reduce reperfusion-induced cardiac injury and relay long-term cardiac remodeling benefits.

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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Biased signaling pathways in ?2-adrenergic receptor characterized by 19F-NMR.

Publications

Biased signaling pathways in β2-adrenergic receptor characterized by 19F-NMR.

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