Independent beta-arrestin 2 and G protein-mediated pathways for angiotensin II activation of extracellular signal-regulated kinases 1 and 2.

Stimulation of a mutant angiotensin type 1A receptor (DRY/AAY) with angiotensin II (Ang II) or of a wild-type receptor with an Ang II analog ([sarcosine1,Ile4,Ile8]Ang II) fails to activate classical heterotrimeric G protein signaling but does lead to recruitment of beta-arrestin 2-GFP and activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) (maximum stimulation approximately 50% of wild type). This G protein-independent activation of mitogen-activated protein kinase is abolished by depletion of cellular beta-arrestin 2 but is unaffected by the PKC inhibitor Ro-31-8425. In parallel, stimulation of the wild-type angiotensin type 1A receptor with Ang II robustly stimulates ERK1/2 activation with approximately 60% of the response blocked by the PKC inhibitor (G protein dependent) and the rest of the response blocked by depletion of cellular beta-arrestin 2 by small interfering RNA (beta-arrestin dependent). These findings imply the existence of independent G protein- and beta-arrestin 2-mediated pathways leading to ERK1/2 activation and the existence of distinct "active" conformations of a seven-membrane-spanning receptor coupled to each

Defining an olfactory receptor function in airway smooth muscle cells

Pathways that control, or can be exploited to alter, the increase in airway smooth muscle (ASM) mass and cellular remodeling that occur in asthma are not well defined. Here we report the expression of odorant receptors (ORs) belonging to the superfamily of G-protein coupled receptors (GPCRs), as well as the canonical olfaction machinery (G olf and AC3) in the smooth muscle of human bronchi. In primary cultures of isolated human ASM, we identified mRNA expression for multiple ORs. Strikingly, OR51E2 was the most highly enriched OR transcript mapped to the human olfactome in lung-resident cells. In a heterologous expression system, OR51E2 trafficked readily to the cell surface and showed ligand selectivity and sensitivity to the short chain fatty acids (SCFAs) acetate and propionate. These endogenous metabolic byproducts of the gut microbiota slowed the rate of cytoskeletal remodeling, as well as the proliferation of human ASM cells. These cellular responses in vitro were found in ASM from non-asthmatics and asthmatics, and were absent in OR51E2-deleted primary human ASM. These results demonstrate a novel chemo-mechanical signaling network in the ASM and serve as a proof-of-concept that a specific receptor of the gut-lung axis can be targeted to treat airflow obstruction in asthma.open0

Beta-Arrestin Functionally Regulates the Non-Bleaching Pigment Parapinopsin in Lamprey Pineal

The light response of vertebrate visual cells is achieved by light-sensing proteins such as opsin-based pigments as well as signal transduction proteins, including visual arrestin. Previous studies have indicated that the pineal pigment parapinopsin has evolutionally and physiologically important characteristics. Parapinopsin is phylogenetically related to vertebrate visual pigments. However, unlike the photoproduct of the visual pigment rhodopsin, which is unstable, dissociating from its chromophore and bleaching, the parapinopsin photoproduct is stable and does not release its chromophore. Here, we investigated arrestin, which regulates parapinopsin signaling, in the lamprey pineal organ, where parapinopsin and rhodopsin are localized to distinct photoreceptor cells. We found that beta-arrestin, which binds to stimulated G protein-coupled receptors (GPCRs) other than opsin-based pigments, was localized to parapinopsin-containing cells. This result stands in contrast to the localization of visual arrestin in rhodopsin-containing cells. Beta-arrestin bound to cultured cell membranes containing parapinopsin light-dependently and translocated to the outer segments of pineal parapinopsin-containing cells, suggesting that beta-arrestin binds to parapinopsin to arrest parapinopsin signaling. Interestingly, beta-arrestin colocalized with parapinopsin in the granules of the parapinopsin-expressing cell bodies under light illumination. Because beta-arrestin, which is a mediator of clathrin-mediated GPCR internalization, also served as a mediator of parapinopsin internalization in cultured cells, these results suggest that the granules were generated light-dependently by beta-arrestin-mediated internalization of parapinopsins from the outer segments. Therefore, our findings imply that beta-arrestin-mediated internalization is responsible for eliminating the stable photoproduct and restoring cell conditions to the original dark state. Taken together with a previous finding that the bleaching pigment evolved from a non-bleaching pigment, vertebrate visual arrestin may have evolved from a “beta-like” arrestin by losing its clathrin-binding domain and its function as an internalization mediator. Such changes would have followed the evolution of vertebrate visual pigments, which generate unstable photoproducts that independently decay by chromophore dissociation

An Essential Role of the Cytoplasmic Tail of CXCR4 in G-Protein Signaling and Organogenesis

CXCR4 regulates cell proliferation, enhances cell survival and induces chemotaxis, yet molecular mechanisms underlying its signaling remain elusive. Like all other G-protein coupled receptors (GPCRs), CXCR4 delivers signals through G-protein-dependent and -independent pathways, the latter involving its serine-rich cytoplasmic tail. To evaluate the signaling and biological contribution of this G-protein-independent pathway, we generated mutant mice that express cytoplasmic tail-truncated CXCR4 (ΔT) by a gene knock-in approach. We found that ΔT mice exhibited multiple developmental defects, with not only G-protein-independent but also G-protein-dependent signaling events completely abolished, despite ΔT's ability to still associate with G-proteins. These results reveal an essential positive regulatory role of the cytoplasmic tail in CXCR4 signaling and suggest the tail is crucial for mediating G-protein activation and initiating crosstalk between G-protein-dependent and G-protein-independent pathways for correct GPCR signaling

Recombination in West Nile Virus: minimal contribution to genomic diversity

Recombination is known to play a role in the ability of various viruses to acquire sequence diversity. We consequently examined all available West Nile virus (WNV) whole genome sequences both phylogenetically and with a variety of computational recombination detection algorithms. We found that the number of distinct lineages present on a phylogenetic tree reconstruction to be identical to the 6 previously reported. Statistically-significant evidence for recombination was only observed in one whole genome sequence. This recombination event was within the NS5 polymerase coding region. All three viruses contributing to the recombination event were originally isolated in Africa at various times, with the major parent (SPU116_89_B), minor parent (KN3829), and recombinant sequence (AnMg798) belonging to WNV taxonomic lineages 2, 1a, and 2 respectively. This one isolated recombinant genome was out of a total of 154 sequences analyzed. It therefore does not seem likely that recombination contributes in any significant manner to the overall sequence variation within the WNV genome

Pharmacological Evaluation of the Long-Term Effects of Xanomeline on the M1 Muscarinic Acetylcholine Receptor

Xanomeline is a unique agonist of muscarinic receptors that possesses functional selectivity at the M1 and M4 receptor subtypes. It also exhibits wash-resistant binding to and activation of the receptor. In the present work we investigated the consequences of this type of binding of xanomeline on the binding characteristics and function of the M1 muscarinic receptor. Pretreatment of CHO cells that stably express the M1 receptor for 1 hr with increasing concentrations of xanomeline followed by washing and waiting for an additional 23 hr in control culture media transformed xanomeline-induced inhibition of [3H]NMS binding from monophasic to biphasic. The high-affinity xanomeline binding site exhibited three orders of magnitude higher affinity than in the case of xanomeline added directly to the binding assay medium containing control cells. These effects were associated with a marked decrease in maximal radioligand binding and attenuation of agonist-induced increase in PI hydrolysis and were qualitatively similar to those caused by continuous incubation of cells with xanomeline for 24 hr. Attenuation of agonist-induced PI hydrolysis by persistently-bound xanomeline developed with a time course that parallels the return of receptor activation by prebound xanomeline towards basal levels. Additional data indicated that blockade of the receptor orthosteric site or the use of a non-functional receptor mutant reversed the long-term effects of xanomeline, but not its persistent binding at an allosteric site. Furthermore, the long-term effects of xanomeline on the receptor are mainly due to receptor down-regulation rather than internalization

The role of GRK6 in animal models of Parkinson's Disease and L-DOPA treatment

G protein-coupled Receptor Kinase 6 (GRK6) belongs to a family of kinases that phosphorylate GPCRs. GRK6 levels were found to be altered in Parkinson's Disease (PD) and D2 dopamine receptors are supersensitive in mice lacking GRK6 (GRK6-KO mice). To understand how GRK6 modulates the behavioral manifestations of dopamine deficiency and responses to L-DOPA, we used three approaches to model PD in GRK6-KO mice: 1) the cataleptic response to haloperidol; 2) introducing GRK6 mutation to an acute model of absolute dopamine deficiency, DDD mice; 3) hemiparkinsonian 6-OHDA model. Furthermore, dopamine-related striatal signaling was analyzed by assessing the phosphorylation of AKT/GSK3β and ERK1/2. GRK6 deficiency reduced cataleptic behavior, potentiated the acute effect of L-DOPA in DDD mice, reduced rotational behavior in hemi-parkinsonian mice, and reduced abnormal involuntary movements induced by chronic L-DOPA. These data indicate that approaches to regulate GRK6 activity could be useful in modulating both therapeutic and side-effects of L-DOPA

Role of the Drosophila Non-Visual ß-Arrestin Kurtz in Hedgehog Signalling

The non-visual ß-arrestins are cytosolic proteins highly conserved across species that participate in a variety of signalling events, including plasma membrane receptor degradation, recycling, and signalling, and that can also act as scaffolding for kinases such as MAPK and Akt/PI3K. In Drosophila melanogaster, there is only a single non-visual ß-arrestin, encoded by kurtz, whose function is essential for neuronal activity. We have addressed the participation of Kurtz in signalling during the development of the imaginal discs, epithelial tissues requiring the activity of the Hedgehog, Wingless, EGFR, Notch, Insulin, and TGFβ pathways. Surprisingly, we found that the complete elimination of kurtz by genetic techniques has no major consequences in imaginal cells. In contrast, the over-expression of Kurtz in the wing disc causes a phenotype identical to the loss of Hedgehog signalling and prevents the expression of Hedgehog targets in the corresponding wing discs. The mechanism by which Kurtz antagonises Hedgehog signalling is to promote Smoothened internalization and degradation in a clathrin- and proteosomal-dependent manner. Intriguingly, the effects of Kurtz on Smoothened are independent of Gprk2 activity and of the activation state of the receptor. Our results suggest fundamental differences in the molecular mechanisms regulating receptor turnover and signalling in vertebrates and invertebrates, and they could provide important insights into divergent evolution of Hedgehog signalling in these organisms

PKA regulatory subunits mediate synergy among conserved G-protein-coupled receptor cascades

G-protein-coupled receptors sense extracellular chemical or physical stimuli and transmit these signals to distinct trimeric G-proteins. Activated Gα-proteins route signals to interconnected effector cascades, thus regulating thresholds, amplitudes and durations of signalling. Gαs- or Gαi-coupled receptor cascades are mechanistically conserved and mediate many sensory processes, including synaptic transmission, cell proliferation and chemotaxis. Here we show that a central, conserved component of Gαs-coupled receptor cascades, the regulatory subunit type-II (RII) of protein kinase A undergoes adenosine 3′-5′-cyclic monophosphate (cAMP)-dependent binding to Gαi. Stimulation of a mammalian Gαi-coupled receptor and concomitant cAMP-RII binding to Gαi, augments the sensitivity, amplitude and duration of Gαi:βγ activity and downstream mitogen-activated protein kinase signalling, independent of protein kinase A kinase activity. The mechanism is conserved in budding yeast, causing nutrient-dependent modulation of a pheromone response. These findings suggest a direct mechanism by which coincident activation of Gαs-coupled receptors controls the precision of adaptive responses of activated Gαi-coupled receptor cascades

Protein Phosphatase 2A Interacts with the Na+,K+-ATPase and Modulates Its Trafficking by Inhibition of Its Association with Arrestin

Background: The P-type ATPase family constitutes a collection of ion pumps that form phosphorylated intermediates during ion transport. One of the best known members of this family is the Na +,K +-ATPase. The catalytic subunit of the Na +,K +-ATPase includes several functional domains that determine its enzymatic and trafficking properties. Methodology/Principal Findings: Using the yeast two-hybrid system we found that protein phosphatase 2A (PP2A) catalytic C-subunit is a specific Na +,K +-ATPase interacting protein. PP-2A C-subunit interacted with the Na +,K +-ATPase, but not with the homologous sequences of the H +,K +-ATPase. We confirmed that the Na +,K +-ATPase interacts with a complex of A- and C-subunits in native rat kidney. Arrestins and G-protein coupled receptor kinases (GRKs) are important regulators of G-protein coupled receptor (GPCR) signaling, and they also regulate Na +,K +-ATPase trafficking through direct association. PP2A inhibits association between the Na +,K +-ATPase and arrestin, and diminishes the effect of arrestin on Na +,K +-ATPase trafficking. GRK phosphorylates the Na +,K +-ATPase and PP2A can at least partially reverse this phosphorylation. Conclusions/Significance: Taken together, these data demonstrate that the sodium pump belongs to a growing list of io