Cnidocyte discharge is regulated by light and opsin-mediated phototransduction

Cnidocyte discharge is regulated by light and opsin-mediated phototransduction Plachetzki et al. Plachetzki et al. BMC Biology 2012, 10:1

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

Internalization Dissociates β2-Adrenergic Receptors

G protein-coupled receptors (GPCRs) self-associate as dimers or higher-order oligomers in living cells. The stability of associated GPCRs has not been extensively studied, but it is generally thought that these receptors move between the plasma membrane and intracellular compartments as intact dimers or oligomers. Here we show that β2-adrenergic receptors (β2ARs) that self-associate at the plasma membrane can dissociate during agonist-induced internalization. We use bioluminescence-resonance energy transfer (BRET) to monitor movement of β2ARs between subcellular compartments. BRET between β2ARs and plasma membrane markers decreases in response to agonist activation, while at the same time BRET between β2ARs and endosome markers increases. Energy transfer between β2ARs is decreased in a similar manner if either the donor- or acceptor-labeled receptor is mutated to impair agonist binding and internalization. These changes take place over the course of 30 minutes, persist after agonist is removed, and are sensitive to several inhibitors of arrestin- and clathrin-mediated endocytosis. The magnitude of the decrease in BRET between donor- and acceptor-labeled β2ARs suggests that at least half of the receptors that contribute to the BRET signal are physically segregated by internalization. These results are consistent with the possibility that β2ARs associate transiently with each other in the plasma membrane, or that β2AR dimers or oligomers are actively disrupted during internalization

Filamin A Binds to CCR2B and Regulates Its Internalization

The chemokine (C-C motif) receptor 2B (CCR2B) is one of the two isoforms of the receptor for monocyte chemoattractant protein-1 (CCL2), the major chemoattractant for monocytes, involved in an array of chronic inflammatory diseases. Employing the yeast two-hybrid system, we identified the actin-binding protein filamin A (FLNa) as a protein that associates with the carboxyl-terminal tail of CCR2B. Co-immunoprecipitation experiments and in vitro pull down assays demonstrated that FLNa binds constitutively to CCR2B. The colocalization of endogenous CCR2B and filamin A was detected at the surface and in internalized vesicles of THP-1 cells. In addition, CCR2B and FLNa were colocalized in lamellipodia structures of CCR2B-expressing A7 cells. Expression of the receptor in filamin-deficient M2 cells together with siRNA experiments knocking down FLNa in HEK293 cells, demonstrated that lack of FLNa delays the internalization of the receptor. Furthermore, depletion of FLNa in THP-1 monocytes by RNA interference reduced the migration of cells in response to MCP-1. Therefore, FLNa emerges as an important protein for controlling the internalization and spatial localization of the CCR2B receptor in different dynamic membrane structures

Lack of effects of typical and atypical antipsychotics in DARPP-32 and NCS-1 levels in PC12 cells overexpressing NCS-1

<p>Abstract</p> <p>Background</p> <p>Schizophrenia is the major psychiatry disorder, which the exact cause remains unknown. However, it is well known that dopamine-mediated neurotransmission imbalance is associated with this pathology and the main target of antipsychotics is the dopamine receptor D₂. Recently, it was described alteration in levels of two dopamine signaling related proteins in schizophrenic prefrontal cortex (PFC): Neuronal Calcium Sensor-1 (NCS-1) and DARPP-32. NCS-1, which is upregulated in PFC of schizophrenics, inhibits D₂internalization. DARPP-32, which is decreased in PFC of schizophrenics, is a key downstream effector in transducing dopamine signaling. We previously demonstrated that antipsychotics do not change levels of both proteins in rat's brain. However, since NCS-1 and DARPP-32 levels are not altered in wild type rats, we treated wild type PC12 cells (PC12 WT) and PC12 cells stably overexpressing NCS-1 (PC12 Clone) with antipsychotics to investigate if NCS-1 upregulation modulates DARPP-32 expression in response to antipsychotics treatment.</p> <p>Results</p> <p>We chronically treated both PC12 WT and PC12 Clone cells with typical (Haloperidol) or atypical (Clozapine and Risperidone) antipsychotics for 14 days. Using western blot technique we observed that there is no change in NCS-1 and DARPP-32 protein levels in both PC12 WT and PC12 Clone cells after typical and atypical antipsychotic treatments.</p> <p>Conclusions</p> <p>Because we observed no alteration in NCS-1 and DARPP-32 levels in both PC12 WT and Clone cells treated with typical or atypical antipsychotics, we suggest that the alteration in levels of both proteins in schizophrenic's PFC is related to psychopathology but not with antipsychotic treatment.</p

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

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

Quantitative Modeling of GRK-Mediated β2AR Regulation

We developed a unified model of the GRK-mediated β2 adrenergic receptor (β2AR) regulation that simultaneously accounts for six different biochemical measurements of the system obtained over a wide range of agonist concentrations. Using a single deterministic model we accounted for (1) GRK phosphorylation in response to various full and partial agonists; (2) dephosphorylation of the GRK site on the β2AR; (3) β2AR internalization; (4) recycling of the β2AR post isoproterenol treatment; (5) β2AR desensitization; and (6) β2AR resensitization. Simulations of our model show that plasma membrane dephosphorylation and recycling of the phosphorylated receptor are necessary to adequately account for the measured dephosphorylation kinetics. We further used the model to predict the consequences of (1) modifying rates such as GRK phosphorylation of the receptor, arrestin binding and dissociation from the receptor, and receptor dephosphorylation that should reflect effects of knockdowns and overexpressions of these components; and (2) varying concentration and frequency of agonist stimulation “seen” by the β2AR to better mimic hormonal, neurophysiological and pharmacological stimulations of the β2AR. Exploring the consequences of rapid pulsatile agonist stimulation, we found that although resensitization was rapid, the β2AR system retained the memory of the previous stimuli and desensitized faster and much more strongly in response to subsequent stimuli. The latent memory that we predict is due to slower membrane dephosphorylation, which allows for progressive accumulation of phosphorylated receptor on the surface. This primes the receptor for faster arrestin binding on subsequent agonist activation leading to a greater extent of desensitization. In summary, the model is unique in accounting for the behavior of the β2AR system across multiple types of biochemical measurements using a single set of experimentally constrained parameters. It also provides insight into how the signaling machinery can retain memory of prior stimulation long after near complete resensitization has been achieved

Neutrophil Paralysis in Plasmodium vivax Malaria

Plasmodium vivax is responsible for approximately 60–80% of the malaria cases in the world, and contributes to significant social and economic instability in the developing countries of Latin America and Asia. The pathogenesis of P. vivax malaria is a consequence of host derived inflammatory mediators. Hence, a better understanding of the mechanisms involved in induction of systemic inflammation during P. vivax malaria is critical for the clinical management and prevention of severe disease. The innate immune receptors recognize Plasmodium sp. and initiate a broad spectrum of host defense mechanisms that mediate resistance to infection. However, the innate immune response is the classic “two-edged sword”, and clinical malaria is associated with high levels of circulating pro-inflammatory cytokines. Our findings show that both monocytes and neutrophils are highly activated during malaria. Monocytes produced high levels of IL-1β, IL-6 and TNF-α during acute malaria. On the other hand, neutrophils were a poor source of cytokines, but displayed an enhanced phagocytic activity and superoxide production. Unexpectedly, we noticed an impaired chemotaxis of neutrophils towards an IL-8 (CXCL8) gradient. We proposed that neutrophil paralysis is in part responsible for the enhanced susceptibility to bacterial infection observed in malaria patients

IL-24 Inhibits lung cancer cell migration and invasion by disrupting the SDF-1/CXCR4 signaling axis

© 2015 Panneerselvam et al. Background The stromal cell derived factor (SDF)-1/chemokine receptor (CXCR)-4 signaling pathway plays a key role in lung cancer metastasis and is molecular target for therapy. In the present study we investigated whether interleukin (IL)-24 can inhibit the SDF-1/CXCR4 axis and suppress lung cancer cell migration and invasion in vitro. Further, the efficacy of IL-24 in combination with CXCR4 antagonists was investigated. Methods Human H1299, A549, H460 and HCC827 lung cancer cell lines were used in the present study. The H1299 lung cancer cell line was stably transfected with doxycycline-inducible plasmid expression vector carrying the human IL-24 cDNA and used in the present study to determine the inhibitory effects of IL-24 on SDF-1/CXCR4 axis. H1299 and A549 cell lines w ere used in transient transfection studies. The inhibitory effects of IL-24 on SDF1/CXCR4 and its downstream targets were analyzed by quantitative RT-PCR, western blot, luciferase reporter assay, flow cytometry and immunocytochemistry. Functional studies included cell migration and invasion assays. Principal Findings Endogenous CXCR4 protein expression levels varied among the four human lung cancer cell lines. Doxycycline-induced IL-24 expression in the H1299-IL24 cell line resulted in reduced CXCR4 mRNA and protein expression. IL-24 post-transcriptionally regulated CXCR4 mRNA expression by decreasing the half-life of CXCR4 mRNA ( > 40%). Functional studies showed IL-24 inhibited tumor cell migration and invasion concomitant with reduction in CXCR4 and its downstream targets (pAKTS 473 , pmTORS 2448 , pPRAS40 T246 and HIF-1α). Additionally, IL-24 inhibited tumor cell migration both in the presence and absence of the CXCR4 agonist, SDF-1. Finally, IL-24 when combined with CXCR4 inhibitors (AMD3100, SJA5) or with CXCR4 siRNA demonstrated enhanced inhibitory activity on tumor cell migration. Conclusions IL-24 disrupts the SDF-1/CXCR4 signaling pathway and inhibits lung tumor cell migration and invasion. Additionally, IL-24, when combined with CXCR4 inhibitors exhibited enhanced anti-metastatic activity and is an attractive therapeutic strategy for lung metastasi