Research ArticleGPCR SIGNALING

The protein arginine methyltransferase PRMT5 promotes D2-like dopamine receptor signaling

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Science Signaling  10 Nov 2015:
Vol. 8, Issue 402, pp. ra115
DOI: 10.1126/scisignal.aad0872
  • Fig. 1 Predicated methylation motifs within the intracellular domains of GPCRs.

    (A) Plot indicating the conservation of arginine methylation motifs across species. Among 737 annotated human GPCRs in the NCBI HomoloGene database, 300 contain a total of 583 predicted arginine methylation motifs (RGG or RXR). The height of each bar represents the percentage conservation of these sites between the human receptors and their homologs in the indicated species, and the number above each bar indicates the total number of predicted motifs conserved in each species. Dots indicate the percentage of motifs that are located within the third intracellular loop (ICL). (B) Alignments indicating conservation of the seven human and C. elegans GPCRs that contain conserved predicted arginine methylation motifs, as generated with the MUSCLE (multiple sequence comparison by log-expectation) algorithm (48). The human GPCR sequences are on top, and the corresponding C. elegans receptor sequences are aligned below. Amino acid positions are indicated after the receptor names. Black bars highlight the conserved arginine residues. (C) Alignment indicating conservation of residues among the D2 receptor homologs. The Arg217 and Arg219 residues of the human D2 receptor and their counterparts in other species lie within a highly conserved RXR motif, in which X can be any amino acid residue. These arginines are conserved from the C. elegans D2-like dopamine receptor DOP-3 to the human D2 receptor. The gray shading indicates the end of transmembrane domain five (TM V).

  • Fig. 2 PRMT5 methylates the human D2 receptor in vitro.

    The indicated GST-tagged wild-type (WT) and mutant recombinant fragments of the third intracellular loop (amino acid residues 211 to 241) of the human D2 receptor were used in an in vitro methylation assay with active recombinant human PRMT5 as described in Materials and Methods. The PRMT5 substrate GST-SmB′ was used as a positive control, whereas GST alone served as a negative control. Top: Fluorograph shows that the WT GST-D2211–241 fragment was methylated in a PRMT5-dependent manner. The degrees of methylation of GST-D2211–241(R217–220A, R222A) and GST-D2211–241(R217A/R219A) were 10 and 24%, respectively, of that of the WT fragment. Ponceau S staining of the polyvinylidene difluoride (PVDF) membrane was performed to demonstrate equivalent loading of receptor fragments. Molecular mass markers (kD) are indicated on the left. Bottom: Quantification of the degree of methylation of the receptor fragments based on densitometric analysis of fluorographs. Data are means ± SEM of three independent experiments. *P = 0.000001.

  • Fig. 3 Conserved arginines contribute to the function of D2 receptors in cultured human cells.

    (A) HEK 293T cell lines stably expressing N-terminal HA-tagged WT D2 (top) or D2(R217A/R219A) mutant (bottom) receptors were analyzed by flow cytometry with an anti-HA antibody. Histograms show the analysis of 50,000 cells per sample and are representative of three independent experiments. Shaded gray histograms show background fluorescence of cells stably transfected with empty plasmid. Inset: Cell lines were analyzed by immunofluorescence microscopy with an anti-HA antibody. Images are representative of three experiments. Scale bars, 10 μm. (B) HEK 293T cells stably expressing empty plasmid (gray), N-terminal HA-tagged WT D2 receptors (black), or D2(R217A/R219A) mutant receptors (red) were treated for 30 min with the indicated concentrations of quinpirole in the presence of 20 μM forskolin. The amount of cAMP in each sample was measured using the HitHunter competitive immunoassay (DiscoveRx). Data are means ± SEM of four independent experiments, each performed in triplicate. Log IC50 (half maximal inhibitory concentration) values for each biological replicate are plotted on the x axis, together with the mean ± SEM of the log IC50 for each receptor variant. HA-D2(R217A/R219A) (log IC50 = −8.27 ± 0.10) shows a decrease in quinpirole-mediated inhibition of forskolin-stimulated cAMP accumulation when compared to HA-D2 (log IC50 = −9.11 ± 0.15) (P = 0.0048). MFI, mean fluorescence intensity.

  • Fig. 4 PRMT5 methylates C. elegans DOP-3 in vitro.

    The indicated GST-tagged WT and mutant recombinant fragments of the third intracellular loop (amino acid residues 202 to 232) of the C. elegans DOP-3 receptor were used in an in vitro methylation assay with active, recombinant human PRMT5 as described in Materials and Methods. The PRMT5 substrate GST-SmB′ was used as a positive control, whereas GST alone served as a negative control. Top: Fluorograph shows that the WT GST–DOP-3202–232 fragment was methylated in a PRMT5-dependent manner. The degree of methylation of GST–DOP-3202–232(R208A/R210A) was 26% of that of the WT fragment. Ponceau S staining of the PVDF membrane was performed to demonstrate equivalent loading of receptor fragments. Molecular mass markers (kD) are indicated on the left. Bottom: Quantification of the degree of methylation of the receptor fragments based on densitometric analysis of fluorographs. Data are means ± SEM of three independent experiments. *P = 0.000055.

  • Fig. 5 Loss of C. elegans PRMT-5 function results in enhanced octanol avoidance.

    (A) Effect of prmt-5 mutation on the timing of the octanol avoidance response. The times that it took WT, dop-3 mutant, and prmt-5 mutant C. elegans to respond to the presence of the indicated concentrations of octanol were determined as described in Materials and Methods. Animals were assayed 10 to 20 min after transfer to “off food” plates lacking OP50 Escherichia coli. Loss of prmt-5 function phenocopies loss of dop-3 at each concentration tested. P = 0.17 for 100% octanol, P = 0.84 for 30% octanol, and P = 0.13 for 10% octanol when comparing the responses of dop-3 and prmt-5 mutant animals. (B) Effect on octanol sensitivity of the expression of PRMT-5 in the ASH sensory neurons. WT and prmt-5 mutant animals were compared to prmt-5 mutant animals expressing prmt-5 driven by the osm promoter in ASH neurons in the time taken to respond (s) to the presence of 30% octanol. The combined data from three transgenic lines are shown. All data are means ± SEM. P = 0.7 when comparing WT animals to prmt-5 mutant animals expressing prmt-5 in the ASH neurons. Allele used: prmt-5(gk357). WT: the N2 WT strain. n ≥ 40 animals tested for each strain over at least three independent experiments.

  • Fig. 6 C. elegans PRMT-5 is required for efficacious dopamine-mediated paralysis and locomotor behavior.

    (A) Effect of prmt-5 mutation on the dopamine-induced paralysis phenotype. The percentages of animals that were moving 20 min after transfer to plates containing the indicated concentrations of dopamine are shown for each genotype. Data are means ± SEM of ≥88 animals of each genotype. P > 0.1 when comparing prmt-5 and dop-3 mutant animals in terms of their responses to concentrations of dopamine from 15 to 30 mM. (B) Effect of prmt-5 mutation and its rescue in cholinergic motor neurons on the basal slowing response. Locomotion rates of the indicated C. elegans strains in the absence (white bars) and presence of HB101 E. coli (black bars) are given as the number of body bends per animal per 20-s time period. Animals lacking PRMT-5 function have a diminished basal slowing response. P = 0.00001 when comparing WT and prmt-5 mutant animals. P = 0.09 when comparing WT animals to prmt-5 mutant animals expressing prmt-5 in cholinergic motor neurons under the control of the acr-2 promoter. n ≥ 9 animals, 5 consecutive intervals each, for a total of ≥45 separate measurements. For the rescue experiment, the combined data of three independent transgenic lines are shown. n = 12 transgenic animals, 5 consecutive intervals each, for a total of 60 separate measurements under each condition. Alleles used: dop-3(vs106) and prmt-5(gk357). WT: the N2 WT strain.

  • Fig. 7 DOP-3 residues Arg208 and Arg210 are required for a maximum basal slowing response.

    Effect of mutating Arg208 and Arg210 to alanines or lysines on the basal slowing response. Locomotion rates of the indicated C. elegans strains in the absence (white bars) and presence of HB101 E. coli (black bars) are given as the number of body bends per animal per 20-s time period. Restoring WT DOP-3 fully rescued basal slowing. P = 0.4 when comparing WT animals to dop-3 mutant animals expressing dop-3 in cholinergic motor neurons under the control of the acr-2 promoter. dop-3 animals expressing DOP-3(R208A/R210A) or DOP-3(R208K/R210K) displayed a partially defective basal slowing response, similar to prmt-5 mutant animals. P = 0.3 and P = 0.2 when comparing prmt-5 mutant animals to dop-3 animals expressing DOP-3(R208A/R210A) or DOP-3(R208K/R210K), respectively. Alleles used: dop-3(vs106) and prmt-5(gk357). WT: the N2 WT strain. n ≥ 6 animals, 5 consecutive intervals each, for a total of ≥30 separate measurements. For the rescue experiments, the combined data for three or more independent transgenic lines are shown. n ≥ 12 transgenic animals, 5 consecutive intervals each, for a total of ≥60 separate measurements for each condition.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/8/402/ra115/DC1

    Materials and Methods

    Fig. S1. Overexpression of PRMT5 promotes D2 receptor function in cultured human cells.

    Table S1. Bioinformatics summary.

    Table S2. Predicted arginine methylation motifs in the intracellular domains of human GPCRs and their conservation in GPCRs of other species, as identified by HomoloGene.

    Table S3. Predicted arginine methylation motifs in the third intracellular domain of human GPCRs and their conservation in GPCRs of other species, as identified by HomoloGene.

    Table S4. Predicted arginine methylation motifs in the intracellular domains of GPCRs in the human–C. elegans data set generated by GO analysis.

    Table S5. Predicted arginine methylation motifs in the third intracellular domain of GPCRs in the human–C. elegans data set generated by GO analysis.

    References (5154)

  • Supplementary Materials for:

    The protein arginine methyltransferase PRMT5 promotes D2-like dopamine receptor signaling

    Neah Likhite, Christopher A. Jackson, Mao-Shih Liang, Michelle C. Krzyzanowski, Pedro Lei, Jordan F. Wood, Barbara Birkaya, Kerry L. Michaels, Stelios T. Andreadis, Stewart D. Clark, Michael C. Yu,* Denise M. Ferkey*

    *Corresponding author. E-mail: dmferkey{at}buffalo.edu (D.M.F.); mcyu{at}buffalo.edu (M.C.Y.)

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Overexpression of PRMT5 promotes D2 receptor function in cultured human cells.
    • References (5154)

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    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Bioinformatics summary.
    • Table S2 (Microsoft Excel format). Predicted arginine methylation motifs in the intracellular domains of human GPCRs and their conservation in GPCRs of other species, as identified by HomoloGene.
    • Table S3 (Microsoft Excel format). Predicted arginine methylation motifs in the third intracellular domain of human GPCRs and their conservation in GPCRs of other species, as identified by HomoloGene.
    • Table S4 (Microsoft Excel format). Predicted arginine methylation motifs in the intracellular domains of GPCRs in the human–C. elegans data set generated by GO analysis.
    • Table S5 (Microsoft Excel format). Predicted arginine methylation motifs in the third intracellular domain of GPCRs in the human–C. elegans data set generated by GO analysis.

    [Download Tables S1 to S5]


    Citation: N. Likhite, C. A. Jackson, M.-S. Liang, M. C. Krzyzanowski, P. Lei, J. F. Wood, B. Birkaya, K. L. Michaels, S. T. Andreadis, S. D. Clark, M. C. Yu, D. M. Ferkey, The protein arginine methyltransferase PRMT5 promotes D2-like dopamine receptor signaling. Sci. Signal. 8, ra115 (2015).

    © 2015 American Association for the Advancement of Science

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