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Graphical abstract, Highlights • A hybrid stochastic/deterministic model of mouse rod phototransduction is presented. • Rod photocurrent to photovoltage conversion in darkness is accurately characterized. • Photoresponses to dim and bright stimuli and in various mutants are well reproduced. • Recently debated molecular mechanisms of the phototransduction cascade are examined., The phototransduction cascade is paradigmatic for signaling pathways initiated by G protein-coupled receptors and is characterized by a fine regulation of photoreceptor sensitivity and electrical response to a broad range of light stimuli. Here, we present a biochemically comprehensive model of phototransduction in mouse rods based on a hybrid stochastic and deterministic mathematical framework, and a quantitatively accurate description of the rod impedance in the dark. The latter, combined with novel patch clamp recordings from rod outer segments, enables the interconversion of dim flash responses between photovoltage and photocurrent and thus direct comparison with the simulations. The model reproduces the salient features of the experimental photoresponses at very dim and bright stimuli, for both normal photoreceptors and those with genetically modified cascade components. Our modelling approach recapitulates a number of recent findings in vertebrate phototransduction. First, our results are in line with the recently established requirement of dimeric activation of PDE6 by transducin and further show that such conditions can be fulfilled at the expense of a significant excess of G protein activated by rhodopsin. Secondly, simulations suggest a crucial role of the recoverin-mediated Ca2+-feedback on rhodopsin kinase in accelerating the shutoff, when light flashes are delivered in the presence of a light background. Finally, stochastic simulations suggest that transient complexes between dark rhodopsin and transducin formed prior to light stimulation increase the reproducibility of single photon responses. Current limitations of the model are likely associated with the yet unknown mechanisms governing the shutoff of the cascade.

G protein-coupled receptor 35 (GPR35) is poorly characterized but nevertheless has been revealed to have diverse roles in areas including lower gut inflammation and pain. The development of novel reagents and tools will greatly enhance analysis of GPR35 functions in health and disease. Here, we used mass spectrometry, mutagenesis, and [32P] orthophosphate labeling to identify that all five hydroxy-amino acids in the C-terminal tail of human GPR35a became phosphorylated in response to agonist occupancy of the receptor and that, apart from Ser294, each of these contributed to interactions with arretin-3, which inhibits further G protein-coupled receptor signaling. We found that Ser303 was key to such interactions; the serine corresponding to human GPR35a residue 303 also played a dominant role in arrestin-3 interactions for both mouse and rat GPR35. We also demonstrated that fully phospho-site-deficient mutants of human GPR35a and mouse GPR35 failed to interact effectively with arrestin-3, and the human phospho-deficient variant was not internalized from the surface of cells in response to agonist treatment. Even in cells stably expressing species orthologues of GPR35, a substantial proportion of the expressed protein(s) was determined to be immature. Finally, phospho-site-specific antisera targeting the region encompassing Ser303 in human (Ser301 in mouse) GPR35a identified only the mature forms of GPR35 and provided effective sensors of the activation status of the receptors both in immunoblotting and immunocytochemical studies. Such antisera may be useful tools to evaluate target engagement in drug discovery and target validation programs.

Morphine, fentanyl, and oxycodone are widely used as analgesics, and recently hydromorphone has been approved in Japan. Although all of these are selective for μ-opioid receptors (MORs) and have similar structures, their analgesic potencies and adverse effects (AEs) are diverse. Recent molecular analyses of MOR signaling revealed that the G protein-mediated signaling pathway causes analgesic effects and the β-arrestin-mediated signaling pathway is responsible for AEs. We used several cell-based analyses that selectively measure cellular responses activated by either G protein- or β-arrestin-mediated pathways. GloSensor™ cAMP, CellKey™, and receptor internalization assays were performed with four different types of cells stably expressing differentially labelled MOR. EC50 values measured by cAMP and CellKey™ assays had potencies in the order fentanyl ≤ hydromorphone

The four vertebrate arrestins play a key role in the desensitization and internalization of G protein-coupled receptors (GPCRs) and also mediate receptor-dependent signaling. Recent work has shown that bias for arrestin vs G protein signaling could offer certain therapeutic advantages (or disadvantages) in different systems, making assays that measure arrestin binding to receptors important for drug discovery efforts. Herein, we briefly review several commonly used techniques for measuring arrestin binding to receptors, as well as provide an in-depth and methodologically focused review of two methods that do not require receptor modification. The first approach measures direct binding between purified arrestin and rhodopsin, and the second measures the recruitment of arrestin to receptors in living cells.

Opioid analgesics are elective for treating moderate to severe pain but their use is restricted by severe side effects. Signaling bias has been proposed as a viable means for improving this situation. To exploit this opportunity, continuous efforts are devoted to understand how ligand-specific modulations of receptor functions could mediate the different in vivo effects of opioids. Advances in the field have led to the development of biased agonists based on hypotheses that allocated desired and undesired effects to specific signaling pathways. However, the prevalent hypothesis associating β-arrestin to opioid side effects was recently challenged and multiple of the newly developed biased drugs may not display the superior side effects profile that was sought. Moreover, biased agonism at opioid receptors is now known to be time- and cell-dependent, which adds a new layer of complexity for bias estimation. Here, we first review the signaling mechanisms underlying desired and undesired effects of opioids. We then describe biased agonism at opioid receptors and discuss the different perspectives that support the desired and undesired effects of opioids in view of exploiting biased signaling for therapeutic purposes. Finally, we explore how signaling kinetics and cellular background can influence the magnitude and directionality of bias at those receptors.

Neurotensin (NT) is a tridecapeptide displaying interesting antinociceptive properties through its action on its receptors, NTS1 and NTS2. Neurotensin-like compounds have been shown to exert better antinociceptive properties than morphine at equimolar doses. In this article, we characterized the molecular effects of a novel neurotensin (8–13) (NT(8–13)) analog containing an unnatural amino acid. This compound, named JMV2009, displays a Silaproline in position 10 in replacement of a proline in the native NT(8–13). We first examined the binding affinities of this novel NT(8–13) derivative at both NTS1 and NTS2 receptor sites by performing competitive displacement of iodinated NT on purified cell membranes. Then, we evaluated the ability of JMV2009 to activate NTS1-related G proteins as well as to promote the recruitment of β-arrestins 1 and 2 by using BRET-based cellular assays in live cells. We next assessed its ability to induce p42/p44 MAPK phosphorylation and NT receptors internalization using western blot and cell-surface ELISA, respectively. Finally, we determined the in vitro plasma stability of this NT derivative. This article is associated with the original article “Pain relief devoid of opioid side effects following central action of a silylated neurotensin analog” published in European Journal of Pharmacology [1] . The reader is directed to the associated article for results interpretation, comments, and discussion.

β-arrestins (βarrs) are multifunctional proteins that interact with activated and phosphorylated G protein-coupled receptors (GPCRs) to regulate their signaling and trafficking. Understanding the intricate details of GPCR-βarr interaction continues to be a key research area in the field of GPCR biology. Bimane fluorescence spectroscopy has been one of the key approaches among a broad range of methods employed to study GPCR-βarr interaction using purified and reconstituted system. Here, we present a step-by-step protocol for labeling βarrs with monobromobimane (mBBr) in a site-directed fashion for measuring their interaction with GPCRs and the resulting conformational changes. This simple protocol can be directly applied to other protein-protein interaction modules as well for measuring interactions and conformational changes in reconstituted systems in vitro.

Most vertebrates express four arrestin subtypes: two visual ones in photoreceptor cells and two non-visuals expressed ubiquitously. The latter two interact with hundreds of G protein-coupled receptors, certain receptors of other types, and numerous non-receptor partners. Arrestins have no enzymatic activity and work by interacting with other proteins, often assembling multi-protein signaling complexes. Arrestin binding to every partner affects cell signaling, including pathways regulating cell survival, proliferation, and death. Thus, targeting individual arrestin interactions has therapeutic potential. This requires precise identification of protein-protein interaction sites of both participants and the choice of the side of each interaction which would be most advantageous to target. The interfaces involved in each interaction can be disrupted by small molecule therapeutics, as well as by carefully selected peptides of the other partner that do not participate in the interactions that should not be targeted.

The arrestins are multifunctional adapter proteins that regulate G protein-coupled receptor (GPCR) signaling. There are four isoforms of arrestin: arrestins 1 and 4 are expressed exclusively in the visual system and are primarily associated with the visual receptors rhodopsin and cone opsins, whereas arrestin-2 and 3, better known as β-arrestin1 and 2, respectively, are expressed ubiquitously and bind to most GPCRs. The name “arrestin” was derived from its ability to inhibit rhodopsin signaling, but over the past decades, roles have been discovered for the arrestins in promoting signaling and regulating receptor trafficking through their action as scaffolding proteins. More recent studies have completely reversed the paradigm of arrestins as desensitizers of G protein signaling, showing that in certain situations they can actually promote G protein signaling. While there is still controversy as to the exact role that the arrestins play in signaling by GPCRs, novel paradigms for their activity continue to be discovered. Here we review the importance of these multifunctional adapters in promoting signaling by GPCRs.

G protein-coupled seven-transmembrane receptors (GPCRs) mediate responses to hormones, metabolites, lipids, and neurotransmitters at the cell membrane, and so they are prominent drug targets. Although many structural, biochemical, cell biological, and biophysical studies made remarkable progress to understand mechanisms of GPCR signaling, there still are many unanswered questions about arrestin-dependent GPCR signaling. In this chapter, we focus on optical assays to see muscarinic receptor-arrestin interactions with ensemble FRET and single-molecule TIRF imaging in live cells and finally to integrate the information to simulate hypothesized steps in Virtual Cell.

βarrestin1 and -2 (also known as arrestin2 and -3, respectively) are G protein-coupled receptor (GPCR) adapter proteins, performing three major functions in the cell: functional desensitization, i.e., G protein uncoupling from the receptor, GPCR internalization via clathrin-coated pits, and formation of signalosomes. The βarrestins elicit a large part of the G protein-independent signaling emanating from GPCRs. Several methodologies have been developed over the past 15 years or so to quantify the GPCR-arrestin interaction/binding, especially since the latter's roles in signal transduction were discovered. One of the simplest and most traditional of these methodologies is the assay of co-immunoprecipitation (co-IP), followed by western blotting. This assay is also one of the most reliable ones, since it does not require any chemical modification of either component in the complex (i.e., neither of the receptor nor of the arrestin). Therefore, it is the only assay that can detect and semi-quantify interactions between native GPCRs and native arrestins. The caveat of this assay is of course that its reliability depends on the quality (specificity and sensitivity) of the utilized antibodies. Here, we describe a simple protocol for performing this co-IP assay to get a measurement of the steady-state levels of agonist-elicited GPCR-arrestin interaction in cells.

Metastatic progression is strongly influenced by the connection between hyperactivated signaling pathways. G-protein coupled receptors (GPCRs) through β-arrestins (β-arrs), which serve as intracellular signaling molecules, integrate different pathways to control multiple aspects of metastatic process. As primary component of a core-scaffold, β-arr-dependent signaling represents a mean to direct spatiotemporal specificity of multi-protein complexes in invasion and extracellular matrix (ECM) degradation. Under this paradigm, β-arrs engage a growing number of signaling molecules and organizing protein networks controlling multiple pathways, and cytoskeleton modifications, permitting adaptation to the tumor microenvironment to sustain metastatic dissemination. These findings implicate GPCR/β-arr function as a regulatory tethering hub to orchestrate diverse cellular mechanisms of cancer cell migration and invasion that are critical for metastatic progression. In this chapter, we outline the most recent findings on GPCR/β-arr-guided molecular interactions in specific intracellular compartments to drive metastasis, while discussing new perspectives for the selection of most effective therapeutic options for a personalized medicine.

Rapid developments in genome editing, based largely on CRISPR/Cas9 technologies, are offering unprecedented opportunities to eliminate the expression of single or multiple gene products in intact organisms and in model cell systems. Elimination of individual G protein-coupled receptors (GPCRs), both single and multiple G protein subunits, and arrestin adaptor proteins is providing new and sometimes unanticipated insights into molecular details of the regulation of cell signalling pathways and the behaviour of receptor ligands. Genome editing is certain to become a central component of therapeutic target validation, and will provide pharmacologists with new understanding of the complexities of action of novel and previously studied ligands, as well as of the transmission of signals from individual cell-surface receptors to intracellular signalling cascades.

Arrestins were discovered as proteins that bind active phosphorylated G protein-coupled receptors (GPCRs) and block their interactions with G proteins, i.e., for their role in homologous desensitization of GPCRs. Mammals express only four arrestin subtypes, two of which are largely restricted to the retina. Two nonvisual arrestins are ubiquitous and interact with hundreds of different GPCRs and dozens of other binding partners. Changes of just a few residues on the receptor-binding surface were shown to dramatically affect GPCR preference of inherently promiscuous nonvisual arrestins. Mutations on the cytosol-facing side of arrestins modulate their interactions with individual downstream signaling molecules. Thus, it appears feasible to construct arrestin mutants specifically linking particular GPCRs with signaling pathways of choice or mutants that sever the links between selected GPCRs and unwanted pathways. Signaling-biased “designer arrestins” have the potential to become valuable molecular tools for research and therapy.

During these last years, the CB cannabinoid receptor has emerged as a potential anti-inflammatory target in diseases such as multiple sclerosis, amyotrophic lateral sclerosis, Huntington's disease, ischemic stroke, autoimmune diseases, osteoporosis, and cancer. However, the development of clinically useful CB agonists reveals to be very challenging. Allosterism and biased-signaling mechanisms at CB receptor may offer new avenues for the development of improved CB receptor-targeted therapies. Although there has been some exploration of CB receptor activation by new CB allosteric or biased-signaling ligands, the CB receptor is still at initial stages in this domain. In an effort to understand the molecular basis behind these pharmacological approaches, we have analyzed and summarized the structural data reported so far at CB receptor., N. J. and P. G. thanks supports from the Spanish Ministry MINECO/ FEDER SAF2015-68580-C2-2-R and Spanish Research Council CSIC IP201880E070.

Eukaryotic α-arrestins connect environmental or stress signaling pathways to the endocytosis of plasma membrane transporters or receptors. The Saccharomyces cerevisiae lactate transporter Jen1p has been used as a model cargo for elucidating the mechanisms underlying endocytic turnover in response to carbon sources. Here, we discover a novel pathway of Jen1p endocytosis mediated by the α-arrestin Bul1p in response to the presence of cycloheximide or rapamycin, or prolonged growth in lactate. While cycloheximide or rapamycin modify cells pleiotropically, the major effect of prolonged growth in lactate was shown to be external pH alkalinization. Importantly, employment of specific inactive Jen1p versions showed that Bul1p-dependent endocytosis requires lactate transport, according to the signal imposed. Our results support a model where conformational changes of Jen1p, associated with substrate/H+ symport, are critical for the efficiency of Bul1p-dependent Jen1p turnover., UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) and the project PTDC/BIAMIC/5184/2014 funded by national funds through the FCT I.P. and by the ERDF through the COMPETE 2020—Programa Operacional Competitividade e Internacionalização. Work in Belgium was supported by a PDR grant (No. 23655065) from the FNRS (Fédération Wallonie-Bruxelles, Belgium). Work in Greece was supported by the “Stavros Niarchos Foundation.” G. Talaia is the recipient of a PhD fellowship from the Portuguese Foundation for Science and Technology (SFRH/BD/86221/2012). C.G. is a postdoctoral researcher of the Fonds de la Recherche Scientifique de Belgique (F.R.S-FNRS), info:eu-repo/semantics/publishedVersion

Within any cellular signaling system membrane trafficking is a critical mechanism for cells to translate complex networks into specific downstream responses, including the signal pathways activated by the superfamily of G protein-coupled receptors (GPCRs). Classically, membrane trafficking is viewed as a mechanism to regulate ligand sensitivity of a target tissue by controlling the level of surface receptors. Recent studies, however, have not only highlighted that GPCR trafficking is a tightly regulated process critical for spatio-temporal control of signaling, but that heterotrimeric G protein signaling can also be reactivated or continue to signal from distinct endocytic compartments, and even endosomal microdomains. The significance of spatio-temporal control will be discussed, not only with respect to how these novel molecular pathways impact our basic understanding of cellular regulation, but also our view of how aberrant signaling can result in disease. Furthermore, these mechanisms offer the potential application for novel therapeutic strategies to identify GPCR compounds with high specificity in their actions.

Rapid inactivation of light-induced signaling in rod photoreceptors is crucial for their single-photon sensitivity and sub-second temporal resolution. Every step in the signaling pathway has a dedicated inactivation mechanism. Light-activated rhodopsin is quenched via phosphorylation by rhodopsin kinase, followed by arrestin binding. Transducin is inactivated by its intrinsic GTPase activity, which is greatly accelerated by its effector PDE and RGS9-1. The decrease in cytoplasmic cGMP closes cGMP-gated Na + /Ca 2+ channels, hyper-polarizing the cell. By doing so, it reduces intracellular Ca 2+ . The replacement of Ca 2+ with Mg 2+ on GCAPs activates RetGC, which promptly replenishes cGMP, opening the channels and restoring membrane potential.

Thioredoxin interacting protein (TXNIP) of the arrestin family is a powerful inhibitor of tumour growth and angiogenesis and suppressor of metastasis. The inhibition of angiogenesis might occur via suppression of Akt-mediated upregulation of vascular endothelial growth factors (VEGFs). TXNIP also inhibits the NF-κB survival pathway and promotes apoptosis, and suppresses EMT. It is downregulated in many tumours. Thioredoxin reductase (TRXR) is an arrestin that activates thioredoxin (TRX) aiding cell proliferation with inhibition of apoptosis. TRX also induces hypoxia-inducible factor (HIF)-1 and VEGF and promotes angiogenesis. TXNIP inhibits the active reduced form of TRX from exerting these biological effects. So, attempts have been made to circumvent the deficiency of TXNIP by using TRX inhibitors such as PX-12 (1-methylpropyl 2-imidazolyl disulphide) or by inhibiting TRXR using pleurotin. Both inhibit HIF-1 and HIF-induced VEGF production. Some potential benefits of PX-12 have been identified.

Production and secretion of catecholamines and aldosterone from the adrenal gland are regulated by various adrenal G protein-coupled receptors (GPCRs), including α2-adrenergic (auto)receptors (ARs) and angiotensin receptors. These hormones, in turn, regulate cardiac function and play important pathophysiological roles in heart failure (HF). Recent studies have identified the GPCR adapter proteins β-arrestins, specifically β-arrestin1, as an important mediator of the GPCR-dependent production of these hormones in the adrenal gland. Upon GPCR agonist activation, β-arrestins translocate from the cytoplasm to the plasma membrane to interact with the phosphorylated and active receptor and mediate its G protein-independent signaling. Here, we describe a simple and fast Western blotting-based technique for measuring adrenal β-arrestin plasma membrane recruitment in vivo, as an (indirect) index of its activity toward various agonist-activated adrenal GPCRs in vivo.

In this chapter, we deal with G-protein-coupled receptors (GPCRs) that operate as sensory receptors for light (vision) and odorants (olfaction). We detail the composition of the retina, the different light receptors (visual pigments), and the role of 11- cis -retinal. We describe the photoisomerization process and how it changes the conformation of the helix bundle of rhodopsin, allowing binding of transducin (Gαt). We describe the activation of phosphodiesterase and how the removal of cGMP leads to a lowering of the membrane potential and a subsequent inhibition of glutamate release. We describe the action of the transducin GAP complex, GRK1 and arrestin and how they terminate the activation cycle. We give a brief overview of signaling in the Drosophila compound eye. We then describe the olfactory epithelium and how odorant receptors activate adenylyl cyclase and how this, through an increase in cAMP, increases permeability of the CNGA2 channel and subsequent Ca 2+ -mediated opening of the ANO2 channel. The chapter ends with an excursion about the GPCR family of receptors.

This chapter deals with G-protein-coupled receptors and their interaction with G-proteins and how these activate adenylyl cyclase and phospholipase C (PLC). The subject is placed in the context of muscle contraction and its regulation by adrenoceptors (adrenergic receptors). It provides a brief, graphical, overview of the (ortho)sympathetic and parasympathetic nervous system, their synapses and neurotransmitters. It then lists the different adrenoceptors and their interaction with heterotrimeric G-proteins and downstream effectors. The chapter describes the adenylyl cyclase and PLC pathway and how GRK and arrestin bring about a switch in signaling events. We discuss, in detail, the structure of the transmembrane helix-bundle, how it interacts with ligand (agonist, inverse agonist, biased agonist) and with the Gα subunit and how both interactions change the energetic landscape. The chapter then describes the anatomy of the contractile machinery of striated and smooth muscle and discusses the signaling pathways that increase the contractile force and rhythmicity of the heart and cause contraction of arterial smooth muscle.

We describe methods based on live cell fluorescent microscopy and mass spectrometry to characterize the mechanism of endosomal cAMP production and its regulation using the parathyroid hormone (PTH) type 1 receptor as a prime example. These methods permit to measure rapid changes of cAMP levels in response to PTH, kinetics of endosomal ligand-receptor interaction, pH changes associated with receptor trafficking, and to identify the endosomal receptor interactome.

Chemokine receptors recruit the multifunctional scaffolding protein beta arrestin in response to binding of their chemokine ligands. Given that arrestin recruitment represents a signaling axis that is in part independent from G-protein signaling, it has become a hallmark of G protein-coupled receptor functional selectivity. Therefore, quantification of arrestin recruitment has become a requirement for the delineation of chemokine and drug candidate activity along different signaling axes. Bioluminescence resonance energy transfer (BRET) techniques provide methodology for such quantification that can reveal differences between nonredundant chemokines binding the same receptor, and that can be upscaled for high-throughput testing. We here provide protocols for the careful setup of BRET-based arrestin recruitment assays, and examples for the application of such systems in dose-response or time-course experiments. Suggestions are given for troubleshooting, optimizing test systems, and the interpretation of results obtained with BRET-based assays, which indeed yield an intricate blend of quantitative and qualitative information.

Arrestins specifically bind active phosphorylated G protein-coupled receptors (GPCRs). Receptor binding induces the release of the arrestin C-tail, which in non-visual arrestins contains high-affinity binding sites for clathrin and its adaptor AP2. Thus, serving as a physical link between the receptor and key components of the internalization machinery of the coated pit is the best-characterized function of non-visual arrestins in GPCR trafficking. However, arrestins also regulate GPCR trafficking less directly by orchestrating their ubiquitination and deubiquitination. Several reports suggest that arrestins play additional roles in receptor trafficking. Non-visual arrestins appear to be required for the recycling of internalized GPCRs, and the mechanisms of their function in this case remain to be elucidated. Moreover, visual and non-visual arrestins were shown to directly bind N-ethylmaleimide-sensitive factor, an important ATPase involved in vesicle trafficking, but neither molecular details nor the biological role of these interactions is clear. Considering how many different proteins arrestins appear to bind, we can confidently expect the elucidation of additional trafficking-related functions of these versatile signaling adaptors.

β-Adrenergic receptors (βARs), prototypical G-protein-coupled receptors, play a pivotal role in regulating neuronal and cardiovascular responses to catecholamines during stress. Agonist-induced receptor endocytosis is traditionally considered as a primary mechanism to turn off the receptor signaling (or receptor desensitization). However, recent progress suggests that intracellular trafficking of βAR presents a mean to translocate receptor signaling machinery to intracellular organelles/compartments while terminating the signaling at the cell surface. Moreover, the apparent multidimensionality of ligand efficacy in space and time in a cell has forecasted exciting pathophysiological implications, which are just beginning to be explored. As we begin to understand how these pathways impact downstream cellular programs, this will have significant implications for a number of pathophysiological conditions in heart and other systems, that in turn open up new therapeutic opportunities.

Antidepressant drugs remain poorly understood, especially with respect to pharmacological mechanisms of action. This lack of knowledge results from the extreme complexity inherent to psychopharmacology, as well as to a corresponding lack of knowledge regarding depressive disorder pathophysiology. While the final analysis is likely to be multifactorial and heterogeneous, compelling evidence exists for upregulation of brain α2 adrenergic receptors (ARs) in depressed patients. This evidence has sparked a line of research into actions of a particular antidepressant drug class, the tricyclic antidepressants (TCAs), as direct ligands at α(2A)ARs. Our findings, as outlined herein, demonstrate that TCAs function as arrestin-biased ligands at α(2A)ARs. Importantly, TCA-induced α(2A)AR/arrestin recruitment leads to receptor endocytosis and downregulation of α(2A)AR expression with prolonged exposure. These findings represent a novel mechanism linking α(2)AR trafficking with antidepressant pharmacology.

The multireceptor multiligand system of neuropeptide Y receptors and their ligands is involved in the regulation of a multitude of physiological and pathophysiological processes. Specific expression patterns, ligand-binding modes, and signaling properties contribute to the complex network regulating distinct cellular responses. Intracellular trafficking processes are important key steps that are regulated in context with accessory proteins. These proteins exert their influence by interacting directly or indirectly with the receptors, causing modification of the receptors, or operating as scaffolds for the assembly of larger signaling complexes. On the intracellular receptor faces, sequence-specific motifs have been identified that play an important role in this process. Interestingly, it is also possible to influence the receptor internalization by modification of the peptide ligand.

Photoreceptor cell death is an irreversible, pathologic event in many blinding retinal diseases including retinitis pigmentosa, age-related macular disease, and retinal detachment. Light exposure can exacerbate a variety of human retinal diseases by increasing the rate of photoreceptor cell death. In the present study, we characterize the kinetics of photoreceptor cell death in Tubby (homozygous tub/tub, which have inherited, progressive retinal degeneration) mice born and raised in a bright cyclic light environment. Our data show that raising tub/tub mice in a bright cyclic light environment induces rapid loss of photoreceptors. This effect can be slowed, but not prevented, by raising animals in constant darkness, which suggests the involvement of phototransduction in the accelerated death of photoreceptors in this animal. We further demonstrated that the activities of cytosolic cytochrome c and caspases-3 and -9 were significantly increased in the retinas of tub/tub mice. Raising animals in darkness significantly reduced the increased activities of caspases-3 and -9, as well as cytosolic cytochrome c. We also observed that rhodopsin, a phototransduction protein, is not restricted to the rod outer segment, but is distributed throughout the rod cell, including the inner segments, cell bodies, and synapses. In addition, the light-dependent translocation and compartmentalization of arrestin and transducin are affected by the tubby mutation. Our results support the interpretation that problems in protein trafficking in the photoreceptors of the tub/tub mouse may contribute to retinal degeneration.

The two homologous mammalian proteins called β-arrestin1 (also known as arrestin2) and β-arrestin2 (also called arrestin3) are now widely accepted as endocytic and signaling adaptors for G protein-coupled receptors (GPCRs), growth factor receptors, and ion channels. The sustained interactions of β-arrestins with activated GPCRs have been shown to correlate with the agonist-induced ubiquitination on distinct domains in the β-arrestin molecule. Additionally, ubiquitination of β-arrestin promotes its interaction with proteins that mediate endocytosis (e.g., clathrin) and signaling (e.g., c-RAF). Recent studies have demonstrated that deubiquitination of β-arrestin by specific deubiquitinating enzymes (DUBs) acts as an important regulatory mechanism, which determines the stability of β-arrestin-GPCR binding and fine-tunes β-arrestin-dependent signaling to downstream kinases. Accordingly, ubiquitination/deubiquitination of β-arrestin can serve as an on/off switch for its signaling and endocytic functions. Moreover, by regulating the stability and localization of signalosomes, deubiquitination of β-arrestins by DUBs imparts spatial and temporal resolution in GPCR signaling.