A role for the light-dependent phosphorylation of visual arrestin

Arrestins are regulatory proteins that participate in the termination of G protein-mediated signal transduction. The major arrestin in the Drosophila visual system, Arrestin 2 (Arr2), is phosphorylated in a light-dependent manner by a Ca2+/calmodulin-dependent protein kinase and has been shown to be essential for the termination of the visual signaling cascade in vivo. Here, we report the isolation of nine alleles of the Drosophila photoreceptor cell-specific arr2 gene. Flies carrying each of these alleles underwent light-dependent retinal degeneration and displayed electrophysiological defects typical of previously identified arrestin mutants, including an allele encoding a protein that lacks the major Ca2+/calmodulin-dependent protein kinase site. The phosphorylation mutant had very low levels of phosphorylation and lacked the light-dependent phosphorylation observed with wild-type Arr2. Interestingly, we found that the Arr2 phosphorylation mutant was still capable of binding to rhodopsin; however, it was unable to release from membranes once rhodopsin had converted back to its inactive form. This finding suggests that phosphorylation of arrestin is necessary for the release of arrestin from rhodopsin. We propose that the sequestering of arrestin to membranes is a possible mechanism for retinal disease associated with previously identified rhodopsin alleles in humans.

The proliferative and antiapoptotic effects of substance P are facilitated by formation of a β-arrestin-dependent scaffolding complex

A requirement for scaffolding complexes containing internalized G protein-coupled receptors and β-arrestins in the activation and subcellular localization of extracellular signal-regulated kinases 1 and 2 (ERK1/2) has recently been proposed. However, the composition of these complexes and the importance of this requirement for function of ERK1/2 appear to differ between receptors. Here we report that substance P (SP) activation of neurokinin-1 receptor (NK1R) stimulates the formation of a scaffolding complex comprising internalized receptor, β-arrestin, src, and ERK1/2 (detected by gel filtration, immunoprecipitation, and immunofluorescence). Inhibition of complex formation, by expression of dominant-negative β-arrestin or a truncated NK1R that fails to interact with β-arrestin, inhibits both SP-stimulated endocytosis of the NK1R and activation of ERK1/2, which is required for the proliferative and antiapoptotic effects of SP. Thus, formation of a β-arrestin-containing complex facilitates the proliferative and antiapoptotic effects of SP, and these effects of SP could be diminished in cells expressing truncated NK1R corresponding to a naturally occurring variant.

β-Arrestin 1 and 2 differentially regulate heptahelical receptor signaling and trafficking

The two widely coexpressed isoforms of β-arrestin (termed βarrestin 1 and 2) are highly similar in amino acid sequence. The β-arrestins bind phosphorylated heptahelical receptors to desensitize and target them to clathrin-coated pits for endocytosis. To better define differences in the roles of β-arrestin 1 and 2, we prepared mouse embryonic fibroblasts from knockout mice that lack one of the β-arrestins (βarr1-KO and βarr2-KO) or both (βarr1/2-KO), as well as their wild-type (WT) littermate controls. These cells were analyzed for their ability to support desensitization and sequestration of the β2-adrenergic receptor (β2-AR) and the angiotensin II type 1A receptor (AT1A-R). Both βarr1-KO and βarr2-KO cells showed similar impairment in agonist-stimulated β2-AR and AT1A-R desensitization, when compared with their WT control cells, and the βarr1/2-KO cells were even further impaired. Sequestration of the β2-AR in the βarr2-KO cells was compromised significantly (87% reduction), whereas in the βarr1-KO cells it was not. Agonist-stimulated internalization of the AT1A-R was only slightly reduced in the βarr1-KO but was unaffected in the βarr2-KO cells. In the βarr1/2-KO cells, the sequestration of both receptors was dramatically reduced. Comparison of the ability of the two β-arrestins to sequester the β2-AR revealed β-arrestin 2 to be 100-fold more potent than β-arrestin 1. Down-regulation of the β2-AR was also prevented in the βarr1/2-KO cells, whereas no change was observed in the single knockout cells. These findings suggest that sequestration of various heptahelical receptors is regulated differently by the two β-arrestins, whereas both isoforms are capable of supporting receptor desensitization and down-regulation.

Activation and targeting of extracellular signal-regulated kinases by β-arrestin scaffolds

Using both confocal immunofluorescence microscopy and biochemical approaches, we have examined the role of β-arrestins in the activation and targeting of extracellular signal-regulated kinase 2 (ERK2) following stimulation of angiotensin II type 1a receptors (AT1aR). In HEK-293 cells expressing hemagglutinin-tagged AT1aR, angiotensin stimulation triggered β-arrestin-2 binding to the receptor and internalization of AT1aR-β-arrestin complexes. Using red fluorescent protein-tagged ERK2 to track the subcellular distribution of ERK2, we found that angiotensin treatment caused the redistribution of activated ERK2 into endosomal vesicles that also contained AT1aR-β-arrestin complexes. This targeting of ERK2 reflects the formation of multiprotein complexes containing AT1aR, β-arrestin-2, and the component kinases of the ERK cascade, cRaf-1, MEK1, and ERK2. Myc-tagged cRaf-1, MEK1, and green fluorescent protein-tagged ERK2 coprecipitated with Flag-tagged β-arrestin-2 from transfected COS-7 cells. Coprecipitation of cRaf-1 with β-arrestin-2 was independent of MEK1 and ERK2, whereas the coprecipitation of MEK1 and ERK2 with β-arrestin-2 was significantly enhanced in the presence of overexpressed cRaf-1, suggesting that binding of cRaf-1 to β-arrestin facilitates the assembly of a cRaf-1, MEK1, ERK2 complex. The phosphorylation of ERK2 in β-arrestin complexes was markedly enhanced by coexpression of cRaf-1, and this effect is blocked by expression of a catalytically inactive dominant inhibitory mutant of MEK1. Stimulation with angiotensin increased the binding of both cRaf-1 and ERK2 to β-arrestin-2, and the association of β-arrestin-2, cRaf-1, and ERK2 with AT1aR. These data suggest that β-arrestins function both as scaffolds to enhance cRaf-1 and MEK-dependent activation of ERK2, and as targeting proteins that direct activated ERK to specific subcellular locations.

beta-Arrestin1 modulates lymphoid enhancer factor transcriptional activity through interaction with phosphorylated dishevelled proteins.

One aspect of the function of the beta-arrestins is to serve as scaffold or adapter molecules coupling G-protein coupled receptors (GPCRs) to signal transduction pathways distinct from traditional second messenger pathways. Here we report the identification of Dishevelled 1 and Dishevelled 2 (Dvl1 and Dvl2) as beta-arrestin1 (betaarr1) interacting proteins. Dvl proteins participate as key intermediates in signal transmission from the seven membrane-spanning Frizzled receptors leading to inhibition of glycogen synthase kinase-3beta (GSK-3beta), stabilization of beta-catenin, and activation of the lymphoid enhancer factor (LEF) transcription factor. We find that phosphorylation of Dvl strongly enhances its interaction with betaarr1, suggesting that regulation of Dvl phosphorylation and subsequent interaction with betaarr1 may play a key role in the activation of the LEF transcription pathway. Because coexpression of the Dvl kinases, CK1epsilon and PAR-1, with Dvl synergistically activates LEF reporter gene activity, we reasoned that coexpression of betaarr1 with Dvl might also affect LEF-dependent gene activation. Interestingly, whereas betaarr1 or Dvl alone leads to low-level stimulation of LEF (2- to 5-fold), coexpression of betaarr1 with either Dvl1 or Dvl2 leads to a synergistic activation of LEF (up to 16-fold). Additional experiments with LiCl as an inhibitor of GSK-3beta kinase activity indicate that the step affected by betaarr1 is upstream of GSK-3beta and most likely at the level of Dvl. These results identify betaarr1 as a regulator of Dvl-dependent LEF transcription and suggest that betaarr1 might serve as an adapter molecule that can couple Frizzled receptors and perhaps other GPCRs to these important transcription pathways.

β-Arrestin-mediated Regulation of the Human Ether-a-go-go-Related Gene (hERG) Potassium Channel

The human ether-a-go-go-related gene (hERG) encodes the voltage-gated K+ channel, hERG (Kv11.1). This channel passes the rapidly-activating delayed rectifier K+ current (IKr), which is important for cardiac repolarization. A reduction in IKr due to loss-of-function mutations or drug interactions causes long QT syndrome (LQTS), which can lead to cardiac arrhythmias and sudden cardiac death. The density of hERG channels in the plasma membrane is a key determinant of normal physiological function, and is balanced by trafficking to and from the cell surface. Many LQTS-associated hERG mutations result in a trafficking deficiency of otherwise functional channels. Thus, elucidating mechanisms of hERG regulation at the plasma membrane is useful for the prevention and treatment of LQTS. We previously demonstrated that M3 muscarinic receptor activation increases mature hERG expression through a Gq protein-dependent protein kinase C (PKC) pathway. In addition to conventional Gq protein-coupling, M3 receptors recruit β-arrestins upon agonist binding. Traditionally known for their role in receptor desensitization and internalization, β-arrestins also act as adaptor proteins to facilitate G protein-independent signaling. In the present work, I investigated the exclusive effect of β-arrestin signaling on hERG expression by utilizing an arrestin-biased M3 designer receptor (M3D-arr) exclusively activated by clozapine-N-oxide (CNO). By expressing M3D-arr in hERG-HEK cells and treating with CNO under various conditions, I found that M3D-arr activation increased mature hERG expression and current. Within this paradigm, M3D-arr recruited β-arrestin to the plasma membrane, and promoted the PI3K-dependent activation of Akt. I further found that the activated Akt acted through phosphatidylinositol 3-phosphate 5-kinase (PIKfyve) and Rab11 to facilitate endosomal recycling of hERG channels to the plasma membrane.

Gut hormone GPCRs: structure, function, drug discovery

Crystallization and determination of the high resolution three-dimensional structure of the β2-adrenergic receptor in 2007 was followed by structure elucidation of a number of other receptors, including those for neurotensin and glucagon. These major advances foster the understanding of structure-activity relationship of these receptors and structure-based rational design of new ligands having more predictable activity. At present, structure determination of gut hormone receptors in complex with their ligands (natural, synthetic) and interacting signalling proteins, for example, G-proteins, arrestins, represents a challenge which promises to revolutionize gut hormone endocrinonology.