Functional interplay of visual, sensitizing and screening pigments in the eyes of Drosophila and other red-eyed dipteran flies.

Several fly species have distinctly red-coloured eyes, meaning that the screening pigments that provide a restricted angular sensitivity of the photoreceptors may perform poorly in the longer wavelength range. The functional reasons for the red transparency and possible negative visual effects of the spectral properties of the eye-colouring screening pigments are discussed within the context of the photochemistry, arrestin binding and turnover of the visual pigments located in the various photoreceptor types. A phylogenetic survey of the spectral properties of the main photoreceptors of the Diptera indicates that the transition of the brown eye colour of the Nematocera and lower Brachycera to a much redder eye colour of the higher Brachycera occurred around the emergence of the Tabanidae family.

Rapid light-induced activation of retinal microglia in mice lacking Arrestin-1.

Microglia dynamically prune synaptic contacts during development, and digest waste that accumulates in degeneration and aging. In many neurodegenerative diseases, microglial activation and phagocytosis gradually increase over months or years, with poorly defined initial triggering events. Here, we describe rapid retinal microglial activation in response to physiological light levels in a mouse model of photoreceptor degeneration that arises from defective rhodopsin deactivation and prolonged signaling. Activation, migration and proliferation of microglia proceeded along a well-defined time course apparent within 12 h of light onset. Retinal imaging in vivo with optical coherence tomography revealed dramatic increases in light-scattering from photoreceptors prior to the outer nuclear layer thinning classically used as a measure of retinal neurodegeneration. This model is valuable for mechanistic studies of microglial activation in a well-defined and optically accessible neural circuit, and for the development of novel methods for detecting early signs of pending neurodegeneration in vivo.

Arrestin-dependent but G-protein coupled receptor kinase-independent uncoupling of D2-dopamine receptors.

We reconstituted D2 like dopamine receptor (D2R) and the delta opioid receptor (DOR) coupling to G-protein gated inwardly rectifying potassium channels (K(ir)3) and directly compared the effects of co-expression of G-protein coupled receptor kinase (GRK) and arrestin on agonist-dependent desensitization of the receptor response. We found, as described previously, that co-expression of a GRK and an arrestin synergistically increased the rate of agonist-dependent desensitization of DOR. In contrast, only arrestin expression was required to produce desensitization of D2R responses. Furthermore, arrestin-dependent GRK-independent desensitization of D2R-K(ir)3 coupling could be transferred to DOR by substituting the third cytoplasmic loop of DOR with that of D2R. The arrestin-dependent GRK-independent desensitization of D2R desensitization was inhibited by staurosporine treatment, and blocked by alanine substitution of putative protein kinase C phosphorylation sites in the third cytoplasmic loop of D2R. Finally, the D2R construct in which putative protein kinase C phosphorylation sites were mutated did not undergo significant agonist-dependent desensitization even after GRK co-expression, suggesting that GRK phosphorylation of D2R does not play an important role in uncoupling of the receptor.

Parathyroid hormone (PTH) and PTH-related peptide domains contributing to activation of different PTH receptor-mediated signaling pathways.

Parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP), acting through the osteoblast PTH1 receptor (PTH1R), play important roles in bone remodeling. Intermittent administration of PTH(1-34) (teriparatide) leads to bone formation, whereas continuous administration paradoxically leads to bone resorption. Activation of PTH1R promotes regulation of multiple signaling pathways, including G(s)/cAMP/protein kinase A, G(q)/calcium/protein kinase C, ß-arrestin recruitment, and extracellular signal-related kinase (ERK)1/2 phosphorylation, as well as receptor internalization, but their role in promoting anabolic and catabolic actions of PTH(1-34) are unclear. In the present investigation, a collection of PTH(1-34) and PTHrP(1-34) peptide analogs were evaluated in orthogonal human PTH1R (hPTH1R) functional assays capturing G(s)- and G(q)-signaling, ß-arrestin recruitment, ERK1/2 phosphorylation, and receptor internalization to further define the patterns of PTH1R signaling that they stimulate and further establish peptide domains contributing to agonist activity. Results indicate that both N- and C-terminal domains of PTH and PTHrP are critical for activation of signaling pathways. However, modifications of both regions lead to more substantial decreases in agonist potency and efficacy to stimulate G(q)-signaling, ß-arrestin recruitment, ERK1/2 phosphorylation, and receptor internalization than to stimulate G(s)-signaling. The substantial contribution of the peptide C-terminal domain in activation of hPTH1R signaling suggests a role in positioning of the peptide N-terminal region into the receptor J-domain. Several PTH and PTHrP peptides evaluated in this study promote different patterns of biased agonist signaling and may serve as useful tools to further elucidate therapeutically relevant PTH1R signaling in osteoblasts. With a better understanding of therapeutically relevant signaling, novel biased peptides with desired signaling could be designed for safer and more effective treatment of osteoporosis.

Functional selectivity of adenosine A1 receptor ligands?

The concept of functional selectivity offers great potential for the development of drugs that selectively activate a specific intracellular signaling pathway. During the last few years, it has become possible to systematically analyse compound libraries on G protein-coupled receptors (GPCRs) for this 'biased' form of signaling. We screened over 800 compounds targeting the class of adenosine A(1) receptors using a ß-arrestin-mediated signaling assay in U2OS cells as a G protein-independent readout for GPCR activation. A selection of compounds was further analysed in a G protein-mediated GTPγS assay. Additionally, receptor affinity of these compounds was determined in a radioligand binding assay with the agonist [(3)H]CCPA. Of all compounds tested, only LUF5589 9 might be considered as functionally selective for the G protein-dependent pathway, particularly in view of a likely overestimation of ß-arrestin signaling in the U2OS cells. Altogether, our study shows that functionally selective ligands for the adenosine A(1) receptor are rare, if existing at all. A thorough analysis of biased signaling on other GPCRs also reveals that only very few compounds can be considered functionally selective. This might indicate that the concept of functional selectivity is less common than speculated.

[Regulation of receptors coupled to G proteins (GPCRs)]. / Regulace receptoru sprazených sG proteiny.

Regulation of G protein coupled receptors (GPCRs) looks to be not only classically lineary regulated but it is now discovered to be a complex network of interconnected proteins. Taking these in mind it is highly probable that there is multilevel system of regulation that is able to maintain control of signaling. We review here main mechanisms of GPCRs regulation.

Metarhodopsin control by arrestin, light-filtering screening pigments, and visual pigment turnover in invertebrate microvillar photoreceptors.

The visual pigments of most invertebrate photoreceptors have two thermostable photo-interconvertible states, the ground state rhodopsin and photo-activated metarhodopsin, which triggers the phototransduction cascade until it binds arrestin. The ratio of the two states in photoequilibrium is determined by their absorbance spectra and the effective spectral distribution of illumination. Calculations indicate that metarhodopsin levels in fly photoreceptors are maintained below ~35% in normal diurnal environments, due to the combination of a blue-green rhodopsin, an orange-absorbing metarhodopsin and red transparent screening pigments. Slow metarhodopsin degradation and rhodopsin regeneration processes further subserve visual pigment maintenance. In most insect eyes, where the majority of photoreceptors have green-absorbing rhodopsins and blue-absorbing metarhodopsins, natural illuminants are predicted to create metarhodopsin levels greater than 60% at high intensities. However, fast metarhodopsin decay and rhodopsin regeneration also play an important role in controlling metarhodopsin in green receptors, resulting in a high rhodopsin content at low light intensities and a reduced overall visual pigment content in bright light. A simple model for the visual pigment-arrestin cycle is used to illustrate the dependence of the visual pigment population states on light intensity, arrestin levels and pigment turnover.

Arrestin and the multi-PDZ domain-containing protein MPZ-1 interact with phosphatase and tensin homolog (PTEN) and regulate Caenorhabditis elegans longevity.

Arrestins are multifunctional adaptor proteins best known for their role in regulating G protein-coupled receptor signaling. Arrestins also regulate other types of receptors, including the insulin-like growth factor receptor (IGF-1R), although the mechanism by which this occurs is not well understood. In Caenorhabditis elegans, the IGF-1R ortholog DAF-2 regulates dauer formation, stress resistance, metabolism, and lifespan through a conserved signaling cascade. To further elucidate the role of arrestin in IGF-1R signaling, we employed an in vivo approach to investigate the role of ARR-1, the sole arrestin ortholog in C. elegans, on longevity. Here, we report that ARR-1 functions to positively regulate DAF-2 signaling in C. elegans. arr-1 mutant animals exhibit increased longevity and enhanced nuclear localization of DAF-16, an indication of decreased DAF-2 signaling, whereas animals overexpressing ARR-1 have decreased longevity. Genetic and biochemical analysis reveal that ARR-1 functions to regulate DAF-2 signaling via direct interaction with MPZ-1, a multi-PDZ domain-containing protein, via a C-terminal PDZ binding domain in ARR-1. Interestingly, ARR-1 and MPZ-1 are found in a complex with the phosphatase and tensin homolog (PTEN) ortholog DAF-18, which normally serves as a suppressor of DAF-2 signaling, suggesting that these three proteins work together to regulate DAF-2 signaling. Our results suggest that the ARR-1-MPZ-1-DAF-18 complex functions to regulate DAF-2 signaling in vivo and provide insight into a novel mechanism by which arrestin is able to regulate IGF-1R signaling and longevity.

Arrestin-mediated endocytosis of yeast plasma membrane transporters.

Many plasma membrane transporters in yeast are endocytosed in response to excess substrate or certain stresses and degraded in the vacuole. Endocytosis invariably requires ubiquitination by the HECT domain ligase Rsp5. In the cases of the manganese transporter Smf1 and the amino acid transporters Can1, Lyp1 and Mup1 it has been shown that ubiquitination is mediated by arrestin-like adaptor proteins that bind to Rsp5 and recognize specific transporters. As yeast contains a large family of arrestins, this has been suggested as a general model for transporter regulation; however, analysis is complicated by redundancy amongst the arrestins. We have tested this model by removing all the arrestins and examining the requirements for endocytosis of four more transporters, Itr1 (inositol), Hxt6 (glucose), Fur4 (uracil) and Tat2 (tryptophan). This reveals functions for the arrestins Art5/Ygr068c and Art4/Rod1, and additional roles for Art1/Ldb19, Art2/Ecm21 and Art8/Csr2. It also reveals functional redundancy between arrestins and the arrestin-like adaptors Bul1 and Bul2. In addition, we show that delivery to the vacuole often requires multiple additional ubiquitin ligases or adaptors, including the RING domain ligase Pib1, and the adaptors Bsd2, Ear1 and Ssh4, some acting redundantly. We discuss the similarities and differences in the requirements for regulation of different transporters.

Diversity in arrestin function.

The termination of heptahelical receptor signaling is a multilevel process coordinated, in large part, by members of the arrestin family of proteins. Arrestin binding to agonist-occupied receptors promotes desensitization by interrupting receptor-G protein coupling, while simultaneously recruiting machinery for receptor endocytosis, vesicular trafficking, and receptor fate determination. By simultaneously binding other proteins, arrestins also act as ligand-regulated scaffolds that recruit protein and lipid kinase, phosphatase, phosphodiesterase, and ubiquitin ligase activity into receptor-based multiprotein 'signalsome' complexes. Arrestin-binding thus 'switches' receptors from a transient G protein-coupled state to a persistent arrestin-coupled state that continues to signal as the receptor transits intracellular compartments. While it is clear that signalsome assembly has profound effects on the duration and spatial characteristics of heptahelical receptor signals, the physiologic functions of this novel signaling mechanism are poorly understood. Growing evidence suggests that signalsomes regulate such diverse processes as endocytosis and exocytosis, cell migration, survival, and contractility.

Structure and function of the visual arrestin oligomer.

A distinguishing feature of rod arrestin is its ability to form oligomers at physiological concentrations. Using visible light scattering, we show that rod arrestin forms tetramers in a cooperative manner in solution. To investigate the structure of the tetramer, a nitroxide side chain (R1) was introduced at 18 different positions. The effects of R1 on oligomer formation, EPR spectra, and inter-spin distance measurements all show that the structures of the solution and crystal tetramers are different. Inter-subunit distance measurements revealed that only arrestin monomer binds to light-activated phosphorhodopsin, whereas both monomer and tetramer bind microtubules, which may serve as a default arrestin partner in dark-adapted photoreceptors. Thus, the tetramer likely serves as a 'storage' form of arrestin, increasing the arrestin-binding capacity of microtubules while readily dissociating to supply active monomer when it is needed to quench rhodopsin signaling.

Regulation of alpha2AR trafficking and signaling by interacting proteins.

The continuing discovery of new G protein-coupled receptor (GPCR) interacting proteins and clarification of the functional consequences of these interactions has revealed multiple roles for these events. Some of these interactions serve to scaffold GPCRs to particular cellular micro-compartments or to tether them to defined signaling molecules, while other GPCR-protein interactions control GPCR trafficking and the kinetics of GPCR-mediated signaling transduction. This review provides a general overview of the variety of GPCR-protein interactions reported to date, and then focuses on one prototypical GPCR, the alpha(2)AR, and the in vitro and in vivo significance of its reciprocal interactions with arrestin and spinophilin. It seems appropriate to recognize the life and career of Arthur Hancock with a summary of studies that both affirm and surprise our preconceived notions of how nature is designed, as his career-long efforts similarly affirmed the complexity of human biology and attempted to surprise pathological changes in that biology with novel, discovery-based therapeutic interventions. Dr. Hancock's love of life, of family, and of commitment to making the world a better place are a model of the life well lived, and truly missed by those who were privileged to know, and thus love, him.

Arrestin can act as a regulator of rhodopsin photochemistry.

We report that visual arrestin can regulate retinal release and late photoproduct formation in rhodopsin. Our experiments, which employ a fluorescently labeled arrestin and rhodopsin solubilized in detergent/phospholipid micelles, indicate that arrestin can trap a population of retinal in the binding pocket with an absorbance characteristic of Meta II with the retinal Schiff-base intact. Furthermore, arrestin can convert Metarhodopsin III (formed either by thermal decay or blue-light irradiation) to a Meta II-like absorbing species. Together, our results suggest arrestin may be able to play a more complex role in the rod cell besides simply quenching transducin activity. This possibility may help explain why arrestin deficiency leads to problems like stationary night blindness (Oguchi disease) and retinal degeneration.

Arrestin is required for agonist-induced trafficking of voltage-dependent calcium channels.

Many metabotropic receptors in the nervous system act through signaling pathways that result in the inhibition of voltage-dependent calcium channels. Our previous findings showed that activation of seven-transmembrane receptors results in the internalization of calcium channels. This internalization takes place within a few seconds, raising the question of whether the endocytic machinery is in close proximity to the calcium channel to cause such rapid internalization. Here we show that voltage-dependent calcium channels are pre-associated with arrestin, a protein known to play a role in receptor trafficking. Upon GABAB receptor activation, receptors are recruited to the arrestin-channel complex and internalized. beta-Arrestin 1 selectively binds to the SNARE-binding region of the calcium channel. Peptides containing the arrestin-binding site of the channel disrupt agonist-induced channel internalization. Taken together these data suggest a novel neuronal role for arrestin.

Eukaryotic expression of human arresten gene and its effect on the proliferation of vascular smooth muscle cells.

The eukaryotic expression of human arresten gene and its effect on the proliferation of in vitro cultured vascular smooth cells (VSMCs) in vitro were investigated. COS-7 cells were transfected with recombinant eukaryotic expression plasmid pSecTag2-AT or control plasmid pSecTag2 mediated by liposome. Forty-eight h after transfection, reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expression of arresten mRNA in the cells, while Western blot assay was applied to detect the expression of arresten protein in concentrated supernatant. Primary VSMCs from thoracic aorta of male Sprague-Dawley rats were cultured using the tissue explant method, and identified by immunohistochemical staining with a smooth muscle-specific anti-alpha-actin monoclonal antibody before serial subcultivation. VSMCs were then co-cultured with the concentrated supernatant and their proliferation was detected using Cell Counting Kit-8 (CCK-8) in vitro. The results showed that RT-PCR revealed that the genome of arresten-transfected cells contained a 449 bp specific fragment of arresten gene, suggesting the successful transfection. Successful protein expression in supernatants was confirmed by Western blot. CCK-8 assay showed that the proliferation of VSMCs were inhibited significantly by arresten protein as compared with control cells (F=40.154, P<0.01). It was concluded that arresten protein expressed in eukaryotic cells can inhibit proliferation of VSMCs effectively in vitro, which would provide possibility to the animal experiments.

Deactivation of phosphorylated and nonphosphorylated rhodopsin by arrestin splice variants.

Arrestins constitute a family of small cytoplasmic proteins that mediate deactivation of G-protein-coupled receptors (GPCRs) and are known to be essential for cascade inactivation and receptor desensitization. Alternative splicing produces an array of arrestin gene products that have widely different specificities for their cognate receptors in vitro, but the differential functions of these splice variants in vivo are essentially unknown. Bovine rod photoreceptors express two splice variants of visual arrestin (p44 and p48) that display different affinities for the GPCR rhodopsin. To determine the functions of these splice variants in intact cells, we expressed a transgene encoding either a truncated form of murine arrestin (mArr(1-369), or m44) or the long (p48) isoform in mouse rods lacking endogenous arrestin (Arr-/-). Morphological analysis showed that expression of either variant attenuated the light-induced degeneration that is thought to result from excessive cascade activity in Arr-/-rods. Suction electrode recordings from individual rods indicated that the expression of either m44 or p48 splice variants could restore normal kinetics to Arr-/- dim flash responses, indicating that both isoforms can bind to and quench phosphorylated rhodopsin rapidly. To our surprise, only the full-length variant was able to alter the kinetics of responses in rods lacking both arrestin and rhodopsin kinase, indicating that p48 can also quench the activity of nonphosphorylated rhodopsin.

A Drosophila nonvisual arrestin is required for the maintenance of olfactory sensitivity.

Nonvisual arrestins are a family of multifunctional adaptor molecules that regulate the activities of diverse families of receptors including G protein-coupled receptors, frizzled, and transforming growth factor-beta receptors. These activities indicate broad roles in both physiology and development for nonvisual arrestins. Drosophila melanogaster has a single nonvisual arrestin, kurtz, which is found at high levels within the adult olfactory receptor neurons (ORNs), suggesting a role for this gene in modulating olfactory sensitivity. Using heat-induced expression of a krz cDNA through development, we rescued krz(1) lethality. The resulting adults lacked detectable levels of krz in the olfactory system. The rescued krz(1) homozygotes have an incompletely penetrant antennal structural defect that was completely rescued by the neural expression of a krz cDNA. The krz(1) loss-of-function adults without visible antennal defects displayed diminished behavioral responsiveness to both aversive and attractive odors and also demonstrated reduced olfactory receptor potentials. Both the behavioral and electrophysiological phenotypes were rescued by the targeted expression of the krz cDNA within postdevelopmental ORNs. Thus, krz is required within the nervous system for antennal development and is required later in the ORNs for the maintenance of olfactory sensitivity in Drosophila. The reduced receptor potentials in krz(1) antenna indicate that nonvisual arrestins are required for the early odor-induced signaling events within the ORNs.

Morphine promotes rapid, arrestin-dependent endocytosis of mu-opioid receptors in striatal neurons.

Morphine activates mu-opioid receptors (MORs) without promoting their rapid endocytosis in a number of cell types. A previous study suggested that morphine can drive rapid redistribution of MORs in the nucleus accumbens, but it was not possible in this in vivo study to identify a specific membrane trafficking pathway affected by morphine, to exclude possible indirect actions of morphine via opiate-regulated neural circuitry, or to define the mechanism of this morphine-dependent regulation. In the present study, we addressed these questions using dissociated primary cultures of rat striatal neurons as a model system. Morphine promoted a rapid redistribution of both endogenous and recombinant MORs within 30 min after drug addition to the culture medium. This effect was mediated by rapid endocytosis and occurred in a cell-autonomous manner, as indicated by its detection in cells plated at low density and in cultures in which depolarization was blocked by tetrodotoxin. Morphine-induced endocytosis of MORs was quantitatively similar to that induced by the enkephalin analog D-Ala2-N-Me-Phe4-Glycol5-enkephalin, and endocytosis induced by both ligands was inhibited by a dominant-negative mutant version of arrestin-3 (beta-arrestin-2). These results extend previous in vivo results and indicate that morphine is indeed capable of driving rapid endocytosis of mu-opioid receptors in an important subset of opiate-responsive CNS neurons. They also suggest a cellular mechanism by which beta-arrestins may modulate the physiological effects of morphine in vivo.

The role of opioid receptor internalization and beta-arrestins in the development of opioid tolerance.

Opioid tolerance, a phenomenon characterized by decreased analgesic effects obtained by the same dose of opioids after repeated use of the opioids, is a significant clinical problem. Traditional theory attributes receptor desensitization and internalization and post-receptor adaptation to the development of opioid tolerance. However, morphine, a commonly used opioid, induces tolerance but is not an effective drug to induce opioid receptor desensitization and internalization. Recent studies found that internalized opioid receptors can become competent receptors and recycle back to the cell surface membrane after dephosphorylation. Thus, receptor internalization may be a way to reduce opioid tolerance. Multiple studies have suggested a key role of beta-arrestins in opioid receptor desensitization and internalization and opioid tolerance. Although beta-arrestin 1 and beta-arrestin 2 are important for these effects induced by opioids with high intrinsic efficacy such as etorphine and fentanyl, morphine tolerance may be mediated mainly via beta-arrestin 2. Modification of opioid receptor internalization by affecting the interaction between opioid receptors and beta-arrestins may be a therapeutic target for reducing opioid tolerance.

Caenorhabditus elegans arrestin regulates neural G protein signaling and olfactory adaptation and recovery.

Although regulation of G protein-coupled receptor signaling by receptor kinases and arrestins is a well established biochemical process, the physiological significance of such regulation remains poorly understood. To better understand the in vivo consequences of arrestin function, we have examined the function of the sole arrestin in Caenorhabditis elegans (ARR-1). ARR-1 is primarily expressed in the nervous system, including the HSN neuron and various chemosensory neurons involved in detecting soluble and volatile odorants. arr-1 null mutants exhibit normal chemotaxis but have significant defects in olfactory adaptation and recovery to volatile odorants. In contrast, adaptation is enhanced in animals overexpressing ARR-1. Both the adaptation and recovery defects of arr-1 mutants are rescued by transgenic expression of wild-type ARR-1, whereas expression of a C-terminally truncated ARR-1 effectively rescues only the adaptation defect. A potential mechanistic basis for these findings is revealed by in vitro studies demonstrating that wild-type ARR-1 binds proteins of the endocytic machinery and promotes receptor endocytosis, whereas C-terminally truncated ARR-1 does not. These results demonstrate that ARR-1 functions to regulate chemosensory signaling, enabling organisms to adapt to a variety of environmental cues, and provide an in vivo link between arrestin, receptor endocytosis, and temporal recovery from adaptation.