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1.
FIGURE 5.

FIGURE 5. From: Mahogunin Ring Finger-1 (MGRN1) E3 Ubiquitin Ligase Inhibits Signaling from Melanocortin Receptor by Competition with G?s.

MGRN1 does not inhibit adenylyl cyclase activation by forskolin or a constitutively active Gs protein. A, cAMP levels in HEK293T cells expressing MC1R alone (empty bars) or with (+)S (filled bars), following stimulation with forskolin (10−5 m, 30 min). B, cAMP levels in HEK cells expressing a constitutively active Gαs mutant (Gs*) alone or in combination with (+)S MGRN1. Error bars represent S.D., n = 5.

Ana B. Pérez-Oliva, et al. J Biol Chem. 2009 November 13;284(46):31714-31725.
2.
FIGURE 8.

FIGURE 8. From: Mahogunin Ring Finger-1 (MGRN1) E3 Ubiquitin Ligase Inhibits Signaling from Melanocortin Receptor by Competition with G?s.

Nuclear targeting of (+)-MGRN1 isoforms by MC1R and MC4R. A, HEK293T cells were transfected with MC1R, MC4R, or TxA2R and with Myc epitope-labeled MGRN1 isoforms, as indicated. The distribution of the proteins was visualized by confocal microscopy. Receptors are shown in red (left panel of each group). Middle panels, Myc-MGRN1s shown in green. The right columns in each group show the overlays. B, effect of MC1R on the presence of S isoforms in nuclear extracts. HEK cells transfected with Myc-labeled (+)S or (−)S MGRN1, alone or with MC1R, were fractionated into cytosolic and nuclear fractions. The fractions were analyzed for MGRN1 by blotting with αMyc. Also, blots were stripped and stained for β-actin. C, nuclear accumulation of (+)-MGRN1s in human melanoma cells. HBL cells were transfected with HA-labeled MGRN1s, permeabilized, stained with αHA, and analyzed by confocal microscopy.

Ana B. Pérez-Oliva, et al. J Biol Chem. 2009 November 13;284(46):31714-31725.
3.
FIGURE 6.

FIGURE 6. From: Mahogunin Ring Finger-1 (MGRN1) E3 Ubiquitin Ligase Inhibits Signaling from Melanocortin Receptor by Competition with G?s.

Efficient co-immunoprecipitation of MC1R and MC4R and MGRN1 isoforms. A, interaction between FLAG-MC1R and Myc-MGRN1 isoforms. Extracts from HEK cells expressing WT MC1R or the K0-MC1R mutant, alone or in combination with the MGRN1s were immunoprecipitated (IP) for the MGRN1s with αMyc and Western blotted (WB) for MC1R with αFLAG. Extracts from cells expressing MC1R or S(+) alone were used as negative controls. Input controls were performed by Western blotting aliquots of the cell lysates, with detection with αFLAG for MC1R (middle blot) or αMyc for MGRN1 (lower blot). B, same as in A, except that extracts were immunoprecipitated for MC1R with αFLAG and immune pellets were blotted for MGRN1 with αMyc. As a further control for specificity, cells expressing the FLAG epitope-labeled ADP-ribosylation factor ARF6 were also analyzed. An input control was performed by blotting aliquots of cell lysates with αFLAG and αMyc. C, co-immunoprecipitation of MGRN1 and MC4R, but not TxA2R. HEK cells were transfected with HA epitope-labeled MC4R or TxA2R, alone or with (+)S. Cell extracts were immunoprecipitated for the MGRN1 and the pellets blotted for the receptors with an αHA antibody.

Ana B. Pérez-Oliva, et al. J Biol Chem. 2009 November 13;284(46):31714-31725.
4.
FIGURE 4.

FIGURE 4. From: Mahogunin Ring Finger-1 (MGRN1) E3 Ubiquitin Ligase Inhibits Signaling from Melanocortin Receptor by Competition with G?s.

MGRN1 does not ubiquitylate MC1R expressed in HEK293T cells. A, immunoprecipitation of native MC1R by an antibody against Ub. HEK293T cells were transfected with FLAG-tagged WT MC1R or K0-MC1R, alone or with (+)S. Cell lysates were analyzed by immunoprecipitation with an anti-Ub antibody (αUb), followed by immunoblotting for MC1R with anti-FLAG (upper blot). As controls for specificity, cells transfected with empty pcDNA were used, and lysates from cells expressing FLAG-tagged WT MC1R were treated with a preimmune IgG. Untreated aliquots of cell extracts were electrophoresed in parallel and blotted for MC1R with αFLAG as a control for comparable input (lower blot). B, immunoprecipitation of ubiquitylated proteins with an antibody to MC1R. Extracts from HEK293T cells expressing FLAG-tagged WT MC1R or K0-MC1R were immunoprecipitated for MC1R with anti-FLAG and the pellets were Western blotted with αUb. Comparable input was ascertained by Western blot (WB) with αFLAG. C, increased protein ubiquitylation in cells expressing the MGRN1s. HEK293T cells transfected with empty vector or with MGRN1 isoforms were analyzed for ubiquitylated proteins by Western blot with αUb. Comparable loading was verified by reprobing the stripped membrane with αERK2 (lower blot).

Ana B. Pérez-Oliva, et al. J Biol Chem. 2009 November 13;284(46):31714-31725.
5.
FIGURE 2.

FIGURE 2. From: Mahogunin Ring Finger-1 (MGRN1) E3 Ubiquitin Ligase Inhibits Signaling from Melanocortin Receptor by Competition with G?s.

MGRN1 inhibits functional coupling of MC1R and MC4R to the cAMP pathway. A, functional coupling of exogenous MC1R in the presence of MGRN1 isoforms. HEK293T transfected with MC1R and either empty pcDNA or the indicated MGRN1 isoforms were stimulated (10−7 m NDP-MSH, 30 min), lysed, and cAMP was measured. Empty bars correspond to controls and filled bars to stimulated cells. ***, p < 0.001. B, inhibition by MGRN1s of endogenous MC1R in HBL melanoma cells. Cells were transfected with empty vector or with MGRN1 isoforms, treated with vehicle or NDP-MSH as in panel A, and cAMP contents were determined. **, p < 0.01; *, p < 0.1. C, functional coupling of MC4R is inhibited by MGRN1. HEK293T cells transfected with MC4R with or without the MGRN1s were analyzed for functional coupling as in A. D, lack of effect of MGRN1 on signaling from β2-adrenergic receptor (AR). HEK cells were co-transfected with β2-adrenergic receptor and empty vector or (+)S MGRN1, stimulated with isoproterenol (ISO, 50 μm, 30 min), and cAMP was measured. Error bars represent S.D., n > 5.

Ana B. Pérez-Oliva, et al. J Biol Chem. 2009 November 13;284(46):31714-31725.
6.
FIGURE 7.

FIGURE 7. From: Mahogunin Ring Finger-1 (MGRN1) E3 Ubiquitin Ligase Inhibits Signaling from Melanocortin Receptor by Competition with G?s.

Decreased interaction of MGRN1 and MCRs in the presence of excess Gαs. A, expression of exogenous Gαs inhibits co-precipitation of MGRN1s and MC1R. Myc-tagged (+)S was expressed in HEK293T cells alone or in combination with FLAG-MC1R and exogenous Gαs, as indicated. Extracts were immunoprecipitated (IP) for MGRN1 with αMyc, and the immune pellets were analyzed for MC1R by Western blot (WB) with αFLAG. The experiment was performed after stimulation with NDP-MSH to encourage interaction between MC1R and Gs, and was repeated 3 times with similar results. Input controls consisted of blots of suitable aliquots of cell lysates with αMyc (for detection of MGRN1, middle blot) or αFLAG (for detection of MC1R, lower blot). B, expression of (+)S MGRN1 disrupts the interaction of endogenous Gαs and MC1R. Extracts from HEK cells transfected with FLAG-MC1R with or without (+)S MGRN1 were immunoprecipitated for endogenous Gαs and the amount of MC1R associated with the G protein was estimated by Western blotting the pellets with αFLAG. Input controls consisted of blots of suitable aliquots of cell lysates with αFLAG (for detection of MC1R, middle blot) or αGαs (lower blot). C, overexpression of Gαs rescues the MCRs from inhibition by MGRN1s. HEK cells were transfected to express MC1R (left graph) or MC4R (right graph), with or without S MGRN1 isoforms and/or Gαs, as indicated. Cells were stimulated with NDP-MSH, lysed, and cAMP was determined.

Ana B. Pérez-Oliva, et al. J Biol Chem. 2009 November 13;284(46):31714-31725.
7.
FIGURE 1.

FIGURE 1. From: Mahogunin Ring Finger-1 (MGRN1) E3 Ubiquitin Ligase Inhibits Signaling from Melanocortin Receptor by Competition with G?s.

Genomic structure and heterologous expression of human MGRN1 isoforms. A, genomic structure of human MGRN1 indicating exons (numbered boxes) and introns (lines) and scheme of MGRN1 protein isoforms generated by alternative splicing of exons 12 and 17. (+)- and (−)-forms contain or lack exon 12, respectively. L and S isoforms possess a long or shorter exon 17 generated by the use of an alternative exon junction. RF, C3HC4 RING finger domain (residues 278–316 encoded by exon 10). The peptide sequence encoded by exon 12, containing a bipartite nuclear localization signal highlighted with bold characters, is shown. B, Myc epitope-tagged MGRN1 isoforms were transfected in HEK293T cells and HA epitope-labeled forms were used in melanoma cells. MGRN1 expression was compared by Western blot. Cells transfected with empty vector (lanes labeled pcDNA) are shown as controls for specificity. For HEK cells, endogenous Myc provided an internal loading control. For HBL cells, blots were stripped and stained for ERK2 as loading control (not shown). C, confocal microscopy of HEK293T cells expressing Myc epitope-tagged MGRN1 isoforms. Transfected cells were fixed, permeabilized, and stained with αMyc.

Ana B. Pérez-Oliva, et al. J Biol Chem. 2009 November 13;284(46):31714-31725.
8.
FIGURE 3.

FIGURE 3. From: Mahogunin Ring Finger-1 (MGRN1) E3 Ubiquitin Ligase Inhibits Signaling from Melanocortin Receptor by Competition with G?s.

Effects of MGRN1 on plasma membrane availability and intracellular stability of MC1R and inhibition of functional coupling of the K0-MC1R ubiquitylation null MC1R mutant. A, lack of down-regulation of NDP-MSH binding sites by MGRN1. HEK cells were transfected with WT or K0-MC1R and with (+)S MGRN1 (closed bars) or empty pcDNA3 (open bars). Cells were incubated with 10−10 m [125I]NDP-MSH for 1 h, washed, and bound radioactivity was counted. B, competition binding analysis for MC1R in the presence of two MGRN1 variants. Cells expressing MC1R alone or with (+)S or (−)S were incubated with 10−10 m [125I]NDP-MSH and increasing concentrations of unlabeled ligand, as indicated. The specifically bound radioactivity was measured. Results are given as % maximal binding in the absence of competitor. C, effect of MGRN1 isoforms on MC1R internalization. HEK293T cells were transfected with WT or K0-MC1R and the indicated MGRN1 isoforms (closed bars) or empty pcDNA as control (open bars). Transfected cells were incubated with [125I]NDP-MSH for 1.5 h. The amount of internalized radioligand was determined by an acid-wash procedure and an internalization index (percentage of agonist internalized relative to total binding) was calculated. D, effect of (+)S on the intracellular stability of MC1R. HEK293T cells expressing FLAG-tagged WT or K0-MC1R with or without (+)S MGRN1 were treated with 10−4 m cycloheximide and harvested at the times shown. Solubilized cell extracts were electrophoresed and immunoblotted with αFLAG. A representative blot is shown out of three experiments with similar results. E, comparable electrophoretic pattern of WT and K0-MC1R. HEK293T cells transfected to express WT or mutant MC1R with or without Myc epitope-labeled (+)S MGRN1 were analyzed for MC1R by Western blot (left blot, upper). Blots were stripped and probed for ERK2 as a loading control (left blot, middle), and for MGRN1 as a control for comparable expression of (+)S (left blot, lower). As a positive control for recovery and detection of the ubiquitylated species, HEK cells were transfected with empty vector or FLAG-labeled p53 alone or with its E3 Ub ligase Mdm2. Cell extracts were analyzed for p53 to detect ubiquitylated forms (right blot, upper) and for ERK2 (right gel, lower) as a loading control. F, functional coupling of K0-MC1R in the presence or absence of (+)S MGRN1. HEK293T transfected with K0-MC1R and empty vector (pcDNA) or (+)S were stimulated (10−7 m NDP-MSH, 30 min), and cAMP was measured. Empty bars correspond to vehicle-treated controls and filled bars to agonist-stimulated cells. Error bars represent S.D., n > 5.

Ana B. Pérez-Oliva, et al. J Biol Chem. 2009 November 13;284(46):31714-31725.

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