A new way to rapidly create functional, fluorescent fusion proteins: random insertion of GFP with an in vitro transposition reaction

BACKGROUND: The jellyfish green fluorescent protein (GFP) can be inserted into the middle of another protein to produce a functional, fluorescent fusion protein. Finding permissive sites for insertion, however, can be difficult. Here we describe a transposon-based approach for rapidly creating libraries of GFP fusion proteins. RESULTS: We tested our approach on the glutamate receptor subunit, GluR1, and the G protein subunit, α(s). All of the in-frame GFP insertions produced a fluorescent protein, consistent with the idea that GFP will fold and form a fluorophore when inserted into virtually any domain of another protein. Some of the proteins retained their signaling function, and the random nature of the transposition process revealed permissive sites for insertion that would not have been predicted on the basis of structural or functional models of how that protein works. CONCLUSION: This technique should greatly speed the discovery of functional fusion proteins, genetically encodable sensors, and optimized fluorescence resonance energy transfer pairs

Regulation of meiotic prophase arrest in mouse oocytes by GPR3, a constitutive activator of the Gs G protein

The arrest of meiotic prophase in mouse oocytes within antral follicles requires the G protein Gs and an orphan member of the G protein–coupled receptor family, GPR3. To determine whether GPR3 activates Gs, the localization of Gαs in follicle-enclosed oocytes from Gpr3+/+ and Gpr3−/− mice was compared by using immunofluorescence and GαsGFP. GPR3 decreased the ratio of Gαs in the oocyte plasma membrane versus the cytoplasm and also decreased the amount of Gαs in the oocyte. Both of these properties indicate that GPR3 activates Gs. The follicle cells around the oocyte are also necessary to keep the oocyte in prophase, suggesting that they might activate GPR3. However, GPR3-dependent Gs activity was similar in follicle-enclosed and follicle-free oocytes. Thus, the maintenance of prophase arrest depends on the constitutive activity of GPR3 in the oocyte, and the follicle cell signal acts by a means other than increasing GPR3 activity

Inhibition of G-protein βγ signaling enhances T cell receptor-stimulated interleukin 2 transcription in CD4+ T helper cells.

G-protein-coupled receptor (GPCR) signaling modulates the expression of cytokines that are drug targets for immune disorders. However, although GPCRs are common targets for other diseases, there are few GPCR-based pharmaceuticals for inflammation. The purpose of this study was to determine whether targeting G-protein βγ (Gβγ) complexes could provide a useful new approach for modulating interleukin 2 (IL-2) levels in CD4+ T helper cells. Gallein, a small molecule inhibitor of Gβγ, increased levels of T cell receptor (TCR)-stimulated IL-2 mRNA in primary human naïve and memory CD4+ T helper cells and in Jurkat human CD4+ leukemia T cells. Gβ1 and Gβ2 mRNA accounted for >99% of Gβ mRNA, and small interfering RNA (siRNA)-mediated silencing of Gβ1 but not Gβ2 enhanced TCR-stimulated IL-2 mRNA increases. Blocking Gβγ enhanced TCR-stimulated increases in IL-2 transcription without affecting IL-2 mRNA stability. Blocking Gβγ also enhanced TCR-stimulated increases in nuclear localization of nuclear factor of activated T cells 1 (NFAT1), NFAT transcriptional activity, and levels of intracellular Ca2+. Potentiation of IL-2 transcription required continuous Gβγ inhibition during at least two days of TCR stimulation, suggesting that induction or repression of additional signaling proteins during T cell activation and differentiation might be involved. The potentiation of TCR-stimulated IL-2 transcription that results from blocking Gβγ in CD4+ T helper cells could have applications for autoimmune diseases

Gallein potentiates TCR-stimulated increases in nuclear localization of NFAT1 and intracellular Ca²⁺.

<p>(A) Quantitation of the ratio of nuclear to cytoplasmic NFAT1-GFP and GFP-NFAT2 in basal and stimulated Jurkat cells in the presence or absence of gallein. Cells were stimulated with plate-bound anti-CD3 and soluble anti-CD28 for three days. Data represent means ± SE from 130–162 cells for each condition. ***, <i>p</i> < 0.001. (B) Representative images of NFAT1-GFP in basal and stimulated cells in the presence or absence of gallein. In the lower of the two rows of stimulated cells the nuclear borders are outlined in white. (C) Gallein potentiates TCR-stimulated increases in intracellular Ca²⁺ after three days of TCR stimulation. Relative Ca²⁺ levels were determined using R-GECO-mCerulean as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116575#sec002" target="_blank">Materials and Methods</a>. Data represent the means ± SE from > 320 cells for each stimulated condition and > 200 cells for each unstimulated condition. **, <i>p</i> < 0.01.</p

Gallein enhances TCR-stimulated transcriptional activity of NFAT.

<p>(A) Major TCR-stimulated pathways leading to IL-2 transcription that could be inhibited by Gβγ. Interactions between the TCR and peptide-major histocompatibility complex (MHC) lead to recruitment of CD4 and its associated kinase, p56-Lck, which phosphorylates tyrosine residues in the cytoplasmic tails of the TCR subunits, leading to recruitment and phosphorylation of the tyrosine kinase, ZAP-70. CD28 costimulation provides an additional signal that is needed for complete T cell activation and regulation of IL-2 production [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116575#pone.0116575.ref088" target="_blank">88</a>]. ZAP-70 and p56-Lck then phosphorylate and activate numerous downstream target proteins, including phospholipase C-γ (PLC-γ), leading to Ras activation, Ca²⁺ increases, cytoskeletal rearrangements, and ultimately, activation of transcription factors that bind to the IL-2 promoter and increase IL-2 transcription. (B-C) Gallein increases transcriptional activity of NFAT, but not AP-1 or NFκB. Jurkat cells expressing reporter plasmids for AP-1, NFAT, or NFκB, were stimulated with plate-bound anti-CD3 and soluble anti-CD28 in the presence or absence of gallein for three days. (B) Data from TCR-stimulated cells expressing the indicated reporter plasmids represent means ± SE from 7 experiments. ***, <i>p</i> < 0.001. (C) Data from cells expressing empty vector (pGL3) or the NFAT reporter plasmid represent means ± SD from triplicate determinations in a single experiment representative of 7 experiments. (D) Gallein does not affect mRNA levels of NFAT1, NFAT2, or NFAT4. Portions of the Jurkat cells used for the luciferase assays that measured activity at the NFAT ARRE-2 site were used to measure mRNA levels of NFAT1, NFAT2, and NFAT4 by qPCR. Data represent means ± SE from 7 experiments. (E) Gallein does not cause detectable changes in protein levels of NFAT1 or NFAT2. Jurkat cells were stimulated or not with plate-bound anti-CD3 and soluble anti-CD28 in the absence or presence of gallein or fluorescein. The blot of NFAT1 used lysates from cells stimulated for three days and the blot of NFAT2 used lysates from cells stimulated for two days. Similar results were obtained from cells stimulated for one, two, or three days, and in a second independent experiment.</p

siRNA directed at Gβ₁ but not Gβ₂ enhances TCR-stimulated IL-2 mRNA increases in Jurkat cells.

<p>Expression of Gβ mRNAs in primary naïve (A) and memory (B) human CD4⁺ T cells grown in TH1 or TH2-promoting conditions, and Jurkat T cells (C) treated with Gβ₁ siRNA (si β₁) or NT siRNA (si NT). The primary cells and, where indicated, the Jurkat cells, were stimulated with plate-bound anti-CD3 and soluble anti-CD28 for three days in the presence of the indicated siRNAs. Values represent means ± SE (N = 3). (D) Representative immunoblot (left) and quantification (right) of the effects of Gβ₁ and Gβ₂ siRNAs on the protein levels of Gβ₁ and Gβ₂ in Jurkat cells. Jurkat cells were treated with the indicated siRNAs for 3 days. Values represent means ± SE (N = 3). (E) Gβ₁ siRNA but not Gβ₂ siRNA potentiated TCR-stimulated IL-2 mRNA increases in Jurkat cells. Jurkat cells were stimulated with plate-bound anti-CD3 and soluble anti-CD28 for 3 days in the presence of the indicated siRNAs. Values represent the means ± SE from 17 experiments. The data were normalized to the values for TCR-stimulated cells treated with NT siRNA. (F and G) A second Gβ₁ siRNA also potentiated TCR-stimulated IL-2 mRNA increases. Values represent the means ± SE from 7 experiments. The data were normalized to the values for TCR-stimulated cells treated with NT siRNA. (F) Gβ₁ mRNA was measured in stimulated cells. All mRNA levels were determined by qPCR. **, <i>p</i> < 0.01; ***, <i>p</i> < 0.001.</p

Potentiation of IL-2 mRNA and Ca²⁺ increases by gallein requires two days of TCR stimulation.

<p>(A) IL-2 levels peaked within 24 hours of TCR stimulation and then decreased over the next 48 hours. Jurkat cells were stimulated with plate-bound anti-CD3 and soluble anti-CD28 antibodies and IL-2 mRNA levels were determined by qPCR at the indicated times. Data represent the means ± SD from a single experiment that is representative of three such experiments. (B) TCR-stimulated IL-2 mRNA increases and (C) activity at the minimal 300-bp IL-2 promoter were not potentiated by gallein until after 2–3 days of TCR stimulation. IL-2 promoter activity in (C) was determined in luciferase assays using the same cells in which IL-2 mRNA was measured in (B). Jurkat cells were stimulated with plate-bound anti-CD3 and soluble anti-CD28 antibodies in the presence or absence of gallein for the indicated times. Data points represent the means ± SE of 8 experiments. *, <i>p</i> < 0.05; **, <i>p</i> < 0.01; ***, <i>p</i> < 0.001. (D) IL-2 secretion was not increased by gallein after one day of TCR stimulation. Jurkat cells were stimulated with plate-bound anti-CD3 and soluble anti-CD28 antibodies for one day in the absence or presence of gallein or fluorescein, and IL-2 secreted into the media was quantified by ELISA. Data points represent the means ± SE of 6 experiments. (E) Intracellular Ca²⁺ was not increased by gallein after one day of TCR stimulation. Relative Ca²⁺ levels were determined using R-GECO-mCerulean as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116575#sec002" target="_blank">Materials and Methods</a>. Data represent the means ± SE from > 330 cells for each condition.</p