Mammalian Alpha Arrestins Link Activated Seven Transmembrane Receptors to Nedd4 Family E3 Ubiquitin Ligases and Interact with Beta Arrestins

<div><p>The complement of fungal cell surface proteins is widely regulated by ubiquitination of membrane proteins, which results in their endocytosis and vacuolar degradation. For diverse fungal transporters, the specificity of ubiquitination is conferred by alpha arrestin adaptors, which recruit the Nedd4 family E3 ubiquitin ligase Rsp5. A recent study showed that one mammalian alpha arrestin also mediates ubiquitination and lysosomal trafficking of an activated plasma membrane receptor. Here we first screen all five widely-expressed human alpha arrestins for subcellular localization in ligand-stimulated and -unstimulated cells overexpressing the seven transmembrane receptor vasopressin 2. We then characterize the effects of alpha arrestins ARRDC3 and ARRDC4 upon activation of the seven transmembrane receptors vasopressin 2 and beta adrenergic 2. Using biochemical and imaging approaches, we show that ligand-activated receptors interact with alpha arrestins, and this results in recruitment of Nedd4 family E3 ubiquitin ligases and receptor ubiquitination – which are known to result in lysosomal trafficking. Our time course studies show these effects occur in the first 1–5 minutes after ligand activation, the same time that beta arrestins are known to have roles in receptor endocytic trafficking and kinase signaling. We tested the possibility that alpha and beta arrestins function coordinately and found co-immunoprecipitation and colocalization evidence to support this. Others recently reported that Arrdc3 knockout mice are lean and resistant to obesity. In the course of breeding our own Arrdc3-deficient mice, we observed two novel phenotypes in homozygotes: skin abnormalities, and embryonic lethality on normal chow diet, but not on high fat diet. Our findings suggest that alpha and beta arrestins function coordinately to maintain the optimal complement and function of cell surface proteins according to cellular physiological context and external signals. We discuss the implications of the alpha arrestin functions in fungi having evolved into coordinated alpha/beta arrestin functions in animals.</p> </div

Biology of alpha arrestins.

<p>(<i>A</i>) Ubiquitination effect of alpha arrestins on GPCR. Empty vector, pBSR-ARRDC3-Flag, or pBSR-ARRDC3-Flag PY motif mutant construct was cotransfected with pcDNA3-HA-b2AR-V5. After 24 h incubation, cells were serum-starved for 2 h and treated or not with 1 uM Iso for 30 m. The transfected cells were lysed, and lysates were immunoprecipitated (IP) and analyzed by western blot (WB). The same experiment was performed with ARRDC4 and V2R (1 uM AVP treatment for 30 m). (<i>B</i>) The mean values (+/− S.D.) of three independent experiments are included in the histogram (p value levels and lines used as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050557#pone-0050557-g002" target="_blank">Fig. 2</a>). (<i>C.</i>) Effects of siRNA-mediated ADDRC3 knockdown on b2AR ubiquitination and ITCH expression. Scrambled siRNA, and ARRDC3 siRNAs #1 and #2 were cotransfected with HA-b2AR-V5 construct. After 48 h incubation, cells were serum-starved for 2 h and treated with 1 uM Isoproterenol Iso for 30 m. The transfected cells were lysed, and the lysates were immunoprecipitated (IP) and analyzed by western blot (WB). (<i>D</i>) Histograms of mean (+/− S.D.) and p-values calculated from three different experiments by paired, two-tailed t-tests comparing ligand-stimulated cells transfected with ARRDC3 siRNA versus with control siRNA (p value levels used as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050557#pone-0050557-g002" target="_blank">Fig. 2</a>).</p

Subcellular colocalization and coIP of alpha arrestins with activated GPCR, clathrin, and endosome markers.

<p>(<i>A</i>) Subcellular colocalization of alpha-arrestin-mCherry in receptor HA-b2AR permanent cell lines. HEK-293T cells stably expressing HA-b2AR were transiently cotransfected with ARRDC3-mCherry construct. 24 h after transfection, cells were serum-starved for 2 h, treated or not with 1 uM isoproterenol (Iso) ligand for 30 m, and then washed, and fixed and permeabilized. HA-b2AR was stained with rabbit anti-HA antibodies followed by incubation with Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody. Epifluorescence images were captured using AxioVision or Zeiss 510 META confocal microscope (receptor, <i>green</i>; aArr, <i>red</i>). (<i>B</i>) Co-immunoprecipitation of aArr ARRDC3 with b2AR, clathrin and Hrs. HEK-293T cells were transiently cotransfected with HA-b2AR-V5 plus either empty vector, pBSR-ARRDC3-GFP, or pBSR-ARRDC3 PY motif mutant construct respectively. After 24 h incubation, cells were serum-starved for 2 h and treated or not with 1 uM Iso for 30 m. The cells were lysed, and lysates were immunoprecipitated (IP) and analyzed by western blot (WB). (<i>C</i>) Co-immunoprecipitation of aArr ARRDC4 with V2R, clathrin, and Hrs. HEK-293T cells were transiently cotransfected with HA-V2R-V5 plus either empty vector, pcDNA3-ARRDC4-Flag, or pcDNA3-ARRDC4 PY motif mutant construct respectively. After 24 h incubation, cells were serum-starved for 2 h and treated or not with 1 uM AVP for 30 m. The cells were lysed, the lysates were immunoprecipitated (IP), and analyzed as above. (<i>D, E</i>) Histograms of B and C from three independent experiments including the mean (+/− S.D.) and p-values calculated by paired, two-tailed t-tests on signal from untreated and ligand-treated samples versus their respective vector control are denoted by thin lines and treated vs. untreated samples are denoted by bold lines (***, p<0.001; **, p<0.01; *p<0.05). (<i>F</i>) Subcellular colocalization of alpha-arrestin-mCherry with early (Rab5) and late (Rab7) endosomal markers in HA-V2R-V5 permanent cells. HA-V2R-V5 permanent cells were transiently co-transfected with ARRDC3- or ARRDC4-mCherry construct with Rab5- or Rab7-GFP construct. After 24 h, transfected cells were serum-starved, treated or not with 1 uM AVP for 30 m, and fixed with 4% paraformaldehyde. Epifluorescence images were captured using AxioVision confocal microscope (aArr, <i>red</i>; Rab, <i>green</i>).</p

Model of integrated alpha and beta arrestin roles in membrane protein transport in mammals.

<p>In the case of 7TMRs, receptor activation results in heterotrimeric G protein signaling through second messenger systems. This is instantly followed by receptor phosphorylation by GRK, which results in beta arrestin binding (arresting further G protein binding/signaling). Our findings suggest that alpha and beta arrestins heteroassociate and have coordinated functions. Among the biological functions that could be regulated by integrated alpha/beta arrestin functions are endocytic trafficking of cell surface cargoes (recycling or lysosomal degradation), kinase signaling (e.g., metabolism, cell growth, survival/apoptosis) and cytoskeletal dynamics. Alpha and beta arrestins could have a yin yang relationship with both antagonistic (e.g., inactivating and activating, respectively) and cooperative properties depending on context. We envision that arrestins are dynamic adaptors that can integrate internal and external information to mediate programmed ends (i.e., programs created through evolutionary processes). This is largely achieved through reversible posttranslational modifications, such as phosphorylation (not shown) and ubiqutination, and their effects on protein-protein interactions.</p

Time course analysis of ligand-stimulated aArrs.

<p>(<i>A</i>) HEK-293T cells were transiently cotransfected with HA-b2AR-V5 plus pBSR-ARRDC3-Flag, or HA-V2R-V5 plus pcDNA3-ARRDC4-Flag respectively. After 24 h incubation, cells were serum-starved and treated with 1 uM Iso or AVP for 1, 5, 15, 30, and 60 m. The cells were lysed, and lysates were immunoprecipitated (IP) and analyzed by western blot (WB). Note ARRDC4-Flag protein appears as two bands; we believe this is also the case for ARRDC4-mCherry (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050557#pone-0050557-g002" target="_blank">Fig. 2C</a>), but that is less evident due to the large size of the fusion protein. The basis of this is not yet determined. (<i>B, C</i>) The histogram of A with mean (+/− S.D.) and p-values calculated by comparing levels of ligand-treated samples with respective vector controls (paired, two-tailed t-tests from three independent experiments; ***, p<0.001; **, p<0.01; *p<0.05).</p

Subcellular localization of alpha arrestins.

<p>Vasopressin receptor 2 HA-V2R-V5-stably transfected HEK293T cells were transiently transfected with expression constructs of alpha arrestins with C-terminal fusions of fluorescent protein mCherry. Unstimulated or 1 uM arginine vasopressin (AVP)-stimulated (15 m) cells were fixed, and epifluorescence images were captured using an AxioVision confocal microscope.</p

Recruitment and activation of Nedd4 E3 ligases by alpha arrestins.

<p>(<i>A</i>) Interactions of PY motif-containing alpha arrestin tails and WW region fragments demonstrated by yeast two-hybrid analysis. Bait plasmid (pDEST32-ARRDC1 or -ARRDC3) which contain two sets of PY motifs and prey plasmid (pDEST22-ITCH, -WWP2, -SAV1, -PLEKHA5, -Nedd4L, or -MAGI-2) which contain 1–4 sets of WW domains were transformed into competent yeast strain MaV203. pEXP32/Krev1 plus pEXP22/RalGDS-wt was included as strong positive control, pEXP32/Krev1 plus pEXP22/RALGDS-m1 weak positive control, pEXP32/Krev1 plus pEXP22/RALGDS-m2 and empty bait vector pDEST32 plus empty prey vector pDEST22 negative controls. Positive clones growing on SD/-Trp/-Leu/-His/-Ura plates are selected and the activity of lacZ reporter gene is examined with colony-lift filter assay by X-gal. (<i>B</i>) Subcellular colocalization of alpha-arrestin-mCherry and Nedd4 in HA-b2AR permanent cell lines. HA-b2AR permanent cells were transiently co-transfected with ARRDC3 -mCherry and pBJ-Nedd4-myc constructs. After 24 h, transfected cells were serum-starved, treated with 1 uM Iso for 30 m, and fixed. Nedd4 was stained with mouse monoclonal anti-myc antibody followed by incubation with Alexa Fluor 488-conjugated goat anti-mouse second antibody. Confocal epifluorescence images were obtained (aArr, <i>red</i>; Nedd4, <i>green</i>). (<i>C</i>) Co-immunoprecipitation of alpha arestin ARRDC3 and Nedd4 family E3 ubiquitin ligases with overexpressed b2AR. Empty vector, pBSR-ARRDC3-Flag, or pBSR-ARRDC3-Flag PY motif mutant construct was cotransfected respectively with pcDNA3-HA-b2AR-V5. After 24 h incubation, cells were serum-starved and treated or not with 1 uM Iso for 5 m. The transfected cells were lysed, lysates were immunoprecipitated (IP), and analyzed by western blot (WB). (<i>D</i>) Co-immunoprecipitation of aArr ARRDC4 and Nedd4 family E3 ubiquitin ligases with overexpressed V2R, unstimulated or 5 m post 1 uM AVP stimulation. (<i>E, F</i>) Histograms of C and D from three different experiments; p-values were calculated by comparing levels of ligand-treated or non-treated samples with their respective vector control (p value levels and thin/bold lines used as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050557#pone-0050557-g002" target="_blank">Fig. 2</a>).</p

Human Genetic Relevance and Potent Antitumor Activity of Heat Shock Protein 90 Inhibition in Canine Lung Adenocarcinoma Cell Lines

<div><p>Background</p><p>It has been an open question how similar human and canine lung cancers are. This has major implications in availability of human treatments for dogs and in establishing translational models to test new therapies in pet dogs. The prognosis for canine advanced lung cancer is poor and new treatments are needed. Heat shock protein 90 (HSP90) is an ATPase-dependent molecular chaperone ubiquitously expressed in eukaryotic cells. HSP90 is essential for posttranslational conformational maturation and stability of client proteins including protein kinases and transcription factors, many of which are important for the proliferation and survival of cancer cells. We investigated the activity of STA-1474, a HSP90 inhibitor, in two canine lung cancer cell lines, BACA and CLAC.</p><p>Results</p><p>Comparative genomic hybridization analysis of both cell lines revealed genetic relevance to human non-small cell lung cancer. STA-1474 inhibited growth and induced apoptosis of both cell lines in a dose- and time-dependent manner. The ICs₅₀ after 72 h treatment with STA-1474 were 0.08 and 0.11 μM for BACA and CLAC, respectively. When grown as spheroids, the IC₅₀ of STA-1474 for BACA cells was approximately two-fold higher than when grown as a monolayer (0.348 μM <i>vs</i>. 0.168 μM), whereas CLAC spheroids were relatively drug resistant. Treatment of tumor-stromal fibroblasts with STA-1474 resulted in a dose-dependent decrease in their relative cell viability with a low IC₅₀ of 0.28 μM.</p><p>Conclusions</p><p>Here we first established that lung adenocarcinoma in people and dogs are genetically and biochemically similar. STA1474 demonstrated biological activity in both canine lung cancer cell lines and tumor-stromal fibroblasts. As significant decreases in relative cell viability can be achieved with nanomolar concentrations of STA-1474, investigation into the clinical efficacy of this drug in canine lung cancer patients is warranted.</p></div

Protein expression of HSP90-regulated proteins, HSP70 and HSP90 in canine lung cancer cell lines after treatment with small molecule inhibitors.

<p>A set of three plates for each cell line was used for evaluation of total and phosphoproteins of downstream signaling pathways (A) Representative immunoblots of HSP90 client protein expression from BACA whole cell protein lysates after treatment with STA-1474 or toceranib phosphate. (B) Representative immunoblots of HSP90 client protein expression from CLAC whole cell protein lysates after treatment with STA-1474 or toceranib phosphate. Controls were cell lines treated with the drug solvent, DMSO, as represented by the 0 concentration. Evaluation of phosphoprotein forms of the proteins are indicated by “p”. Drug concentrations are μmol/L. The β-actin Western blots serve as loading controls. (C) Immunobloting from whole cell protein lysates of HSP70 and HSP90 of BACA and CLAC lines treated with DMSO (control), STA-1474 and toceranib phosphate.</p

Characterization of BACA and CLAC canine lung cancer lines by RT-PCR.

<p>(A) Reverse transcriptase cDNA transcripts of NKX2-1 (lung cancer cell marker) and HSP90 client proteins in BACA and CLAC cell lines. NDUFA1 serves as a loading control. (B) Reverse transcriptase cDNA transcripts of HSP70 and three HSP90 isoforms in BACA and CLAC cell lines. NDUFA1 serves as a loading control.</p