Probing Arrestin Function Using Intramolecular FlAsH-BRET Biosensors.

Information contained in the structure of extracellular ligands is transmitted across the cell membrane through allosterically induced changes in G protein-coupled receptor (GPCR) conformation that occur upon ligand binding. These changes, in turn, are imprinted upon intracellular effectors like arrestins and help determine which of its many functions are performed. Intramolecular fluorescein arsenical hairpin (FlAsH) bioluminescence resonance energy transfer (BRET), in which both the fluorescence donor and acceptor are contained within the same protein, can be used to report on activation-induced changes in protein conformation. Here, we describe a method using a series of Rluc-arrestin3-FlAsH-BRET biosensors to measure stimulus-induced changes in arrestin conformation in live cells. Each Rluc-arrestin3-FlAsH-BRET construct contains an N-terminal Renilla luciferase fluorescence donor that excites a fluorescent arsenical targeted to a different position within the protein by mutational insertion of a tetracysteine tag motif. Changes in net BRET upon GPCR stimulation can thus be viewed from multiple vantage points within the protein and used to develop an arrestin3 "conformational signature" that is receptor- and ligand-specific. This method can be used to determine how differences in GPCR and ligand structure influence information transfer across the plasma membrane and to classify GPCRs and/or ligands based on their capacity to induce different arrestin3 activation modes.

Angiotensin II receptors and peritoneal dialysis-induced peritoneal fibrosis.

The vasoactive hormone angiotensin II initiates its major hemodynamic effects through interaction with AT1 receptors, a member of the class of G protein-coupled receptors. Acting through its AT1R, angiotensin II regulates blood pressure and renal salt and water balance. Recent evidence points to additional pathological influences of activation of AT1R, in particular inflammation, fibrosis and atherosclerosis. The transcription factor nuclear factor κB, a key mediator in inflammation and atherosclerosis, can be activated by angiotensin II through a mechanism that may involve arrestin-dependent AT1 receptor internalization. Peritoneal dialysis is a therapeutic modality for treating patients with end-stage kidney disease. The effectiveness of peritoneal dialysis at removing waste from the circulation is compromised over time as a consequence of peritoneal dialysis-induced peritoneal fibrosis. The non-physiological dialysis solution used in peritoneal dialysis, i.e. highly concentrated, hyperosmotic glucose, acidic pH as well as large volumes infused into the peritoneal cavity, contributes to the development of fibrosis. Numerous trials have been conducted altering certain components of the peritoneal dialysis fluid in hopes of preventing or delaying the fibrotic response with limited success. We hypothesize that structural activation of AT1R by hyperosmotic peritoneal dialysis fluid activates the internalization process and subsequent signaling through the transcription factor nuclear factor κB, resulting in the generation of pro-fibrotic/pro-inflammatory mediators producing peritoneal fibrosis.

The conformational signature of ß-arrestin2 predicts its trafficking and signalling functions.

Arrestins are cytosolic proteins that regulate G-protein-coupled receptor (GPCR) desensitization, internalization, trafficking and signalling. Arrestin recruitment uncouples GPCRs from heterotrimeric G proteins, and targets the proteins for internalization via clathrin-coated pits. Arrestins also function as ligand-regulated scaffolds that recruit multiple non-G-protein effectors into GPCR-based 'signalsomes'. Although the dominant function(s) of arrestins vary between receptors, the mechanism whereby different GPCRs specify these divergent functions is unclear. Using a panel of intramolecular fluorescein arsenical hairpin (FlAsH) bioluminescence resonance energy transfer (BRET) reporters to monitor conformational changes in ß-arrestin2, here we show that GPCRs impose distinctive arrestin 'conformational signatures' that reflect the stability of the receptor-arrestin complex and role of ß-arrestin2 in activating or dampening downstream signalling events. The predictive value of these signatures extends to structurally distinct ligands activating the same GPCR, such that the innate properties of the ligand are reflected as changes in ß-arrestin2 conformation. Our findings demonstrate that information about ligand-receptor conformation is encoded within the population average ß-arrestin2 conformation, and provide insight into how different GPCRs can use a common effector for different purposes. This approach may have application in the characterization and development of functionally selective GPCR ligands and in identifying factors that dictate arrestin conformation and function.

Arrestin-dependent activation of ERK and Src family kinases.

The four members of the mammalian arrestin family, two visual and two nonvisual, share the property of stimulus-dependent docking to G protein-coupled receptors. This conformational selectivity permits them to function in receptor desensitization, as arrestin binding sterically inhibits G protein coupling. The two nonvisual arrestins further act as adapter proteins, linking receptors to the clathrin-dependent endocytic machinery and regulating receptor sequestration, intracellular trafficking, recycling, and degradation. Arrestins also function as ligand-regulated scaffolds, recruiting catalytically active proteins into receptor-based multiprotein "signalsome" complexes. Arrestin binding thus marks the transition from a transient G protein-coupled state on the plasma membrane to a persistent arrestin-coupled state that continues to signal as the receptor internalizes. Two of the earliest discovered and most studied arrestin-dependent signaling pathways involve regulation of Src family nonreceptor tyrosine kinases and the ERK1/2 mitogen-activated kinase cascade. In each case, arrestin scaffolding imposes constraints on kinase activity that dictate signal duration and substrate specificity. Evidence suggests that arrestin-bound ERK1/2 and Src not only play regulatory roles in receptor desensitization and trafficking but also mediate longer term effects on cell growth, migration, proliferation, and survival.

Cryoinjury models of the adult and neonatal mouse heart for studies of scarring and regeneration.

A major limitation in studies of the injured heart is animal-to-animal variability in wound size resulting from commonly used techniques such as left anterior descending coronary artery ligation. This variability can make standard errors sufficiently large that mean separation between treatment and control groups can be difficult without replicating numbers (n) of animals in groups by excessive amounts. Here, we describe the materials and protocol necessary for delivering a standardized non-transmural cryoinjury to the left ventricle of an adult mouse heart that may in part obviate the issue of injury variance between animals. As reported previously, this cryoinjury model generates a necrotic wound to the ventricle of consistent size and shape that resolves into a scar of uniform size, shape, and organization. The cryo-model also provides an extended injury border zone that exhibits classic markers of remodeling found in surviving cardiac tissue at the edge of a myocardial infarction, including connexin43 (Cx43) lateralization. In a further extension of the method, we describe how we have adapted the model to deliver a cryoinjury to the apex of the heart of neonatal mice-a modification that may be useful for studies of myocardial regeneration in mammals.

The beta-arrestin pathway-selective type 1A angiotensin receptor (AT1A) agonist [Sar1,Ile4,Ile8]angiotensin II regulates a robust G protein-independent signaling network.

The angiotensin II peptide analog [Sar(1),Ile(4),Ile(8)]AngII (SII) is a biased AT(1A) receptor agonist that stimulates receptor phosphorylation, ß-arrestin recruitment, receptor internalization, and ß-arrestin-dependent ERK1/2 activation without activating heterotrimeric G-proteins. To determine the scope of G-protein-independent AT(1A) receptor signaling, we performed a gel-based phosphoproteomic analysis of AngII and SII-induced signaling in HEK cells stably expressing AT(1A) receptors. A total of 34 differentially phosphorylated proteins were detected, of which 16 were unique to SII and eight to AngII stimulation. MALDI-TOF/TOF mass fingerprinting was employed to identify 24 SII-sensitive phosphoprotein spots, of which three (two peptide inhibitors of protein phosphatase 2A (I1PP2A and I2PP2A) and prostaglandin E synthase 3 (PGES3)) were selected for validation and further study. We found that phosphorylation of I2PP2A was associated with rapid and transient inhibition of a ß-arrestin 2-associated pool of protein phosphatase 2A, leading to activation of Akt and increased phosphorylation of glycogen synthase kinase 3ß in an arrestin signalsome complex. SII-stimulated PGES3 phosphorylation coincided with an increase in ß-arrestin 1-associated PGES3 and an arrestin-dependent increase in cyclooxygenase 1-dependent prostaglandin E(2) synthesis. These findings suggest that AT(1A) receptors regulate a robust G protein-independent signaling network that affects protein phosphorylation and autocrine/paracrine prostaglandin production and that these pathways can be selectively modulated by biased ligands that antagonize G protein activation.