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J Cell Sci
2018 May 23;13110:. doi: 10.1242/jcs.212522.
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The VLDL receptor regulates membrane progesterone receptor trafficking and non-genomic signaling.
Nader N
,
Dib M
,
Courjaret R
,
Hodeify R
,
Machaca R
,
Graumann J
,
Machaca K
.
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Progesterone mediates its physiological functions through activation of both transcription-coupled nuclear receptors and seven-pass-transmembrane progesterone receptors (mPRs), which transduce the rapid non-genomic actions of progesterone by coupling to various signaling modules. However, the immediate mechanisms of action downstream of mPRs remain in question. Herein, we use an untargeted quantitative proteomics approach to identify mPR interactors to better define progesterone non-genomic signaling. Surprisingly, we identify the very-low-density lipoprotein receptor (VLDLR) as an mPRβ (PAQR8) partner that is required for mPRβ plasma membrane localization. Knocking down VLDLR abolishes non-genomic progesterone signaling, which is rescued by overexpressing VLDLR. Mechanistically, we show that VLDLR is required for mPR trafficking from the endoplasmic reticulum to the Golgi. Taken together, our data define a novel function for the VLDLR as a trafficking chaperone required for the mPR subcellular localization and, as such, non-genomic progesterone-dependent signaling.This article has an associated First Person interview with the first author of the paper.
Figure 1. Functional characterization of GFP-tagged mPR. (A,B) Effect of mPR on oocyte maturation. Oocytes were injected with RNA coding for GFP, wild-type mPR (untagged), mPR–GFP (C-terminally tagged) or GFP–mPR (N-terminally tagged), and after 48 h, were treated with progesterone (P4) overnight at suboptimal (3×10−8 M) (A), or optimal (3×10−7 M) (B) concentrations. Oocyte maturation was scored ∼16 h after P4 treatment by the appearance of a white spot on the oocyte animal hemisphere, which is indicative of germinal vesicle breakdown (GVBD). (C) Time needed for 50% of the oocytes to reach GVBD after adding P4 (3×10−7 M) in the presence of overexpressed GFP, mPR, mPR–GFP or GFP–mPR as indicated. (D) Representative GVBD time courses in response to P4 (3×10−7 M) in oocytes overexpressing GFP, mPR, mPR–GFP or GFP–mPR as indicated. The data are normalized to the values in GFP-injected cells and a non-linear curve was fitted to the data. (E) Representative orthogonal sections from a confocal stack of images (see Fig. S1) taken 48 h after injecting RNAs encoding mPR–GFP or GFP–mPR along with the PM marker, TMEM–mCherry. Scale bar: 2 µm. (F) Histogram showing the percentage of mPR–GFP or GFP–mPR at the PM. Quantitative results in A–C and F are mean±s.e.m. for three or more experiments. *P<0.05; ***P<0.001.
Figure 2. Identification of VLDLR as an mPR-interacting protein. (A) Illustration of the experimental design to identify proteins that selectively interact with the functional mPR–GFP construct. (B) Oocytes were injected with mPR–GFP or GFP–mPR RNAs and, 48 h later, lysates immunoprecipitated with anti-GFP magnetic microbeads. Whole-cell lysates (input) and eluates from the anti-GFP beads (IP) were examined by western blotting using an anti-GFP antibody. (C) Plot of the heavy:light ratios (mPR–GFP/GFP–mPR) from three separate mass spectrometry experiments showing the consistent enrichment of VLDLR in its selective interaction with mPR–GFP. (D) Oocytes were either uninjected (Naïve) or injected with RNA coding for the C-terminally tagged mPR–GFP alone (injected) or co-injected with either the N- (Ch–VLDLR) or C-terminally (VLDLR–Ch) mCherry-tagged VLDLR and allowed to express proteins for 48 h. After cross-linking, mPR–GFP was immunoprecipitated using anti-GFP magnetic microbeads. Fractions from the whole lysates (input) and the GFP-binding eluate (IP) were examined by western blotting using anti-GFP and anti-mCherry/anti-RFP antibodies. (E) Representative orthogonal sections of an oocyte stained with WGA and overexpressing VLDLR–mCherry or mCherry–VLDLR as indicated. Scale bar: 2 µm. (F) Histogram showing the percentage of VLDLR–mCherry or mCherry–VLDLR at the PM (mean±s.e.m. of 29 oocytes/condition). *P<0.05.
Figure 3. VLDLR is required for the release of oocyte meiotic arrest. (A) Knockdown of VLDLR expression. Oocytes were injected with VLDLR sense oligonucleotides, as a control, or the corresponding antisense oligonucleotides to knockdown VLDLR expression. RNA was prepared 48 h later and analyzed by qRT-PCR to determine the efficacy of the knockdown on VLDLR and mPR expression. Data are expressed as relative levels of VLDLR and mPR mRNA transcripts after normalizing to the levels of Xenopus ornithine decarboxylase (xODC) mRNA. Naïve, uninjected oocytes. (B) Naïve or VLDLR–mCherry-overexpressing oocytes were injected with VLDLR sense or antisense oligonucleotides and cell extracts were analyzed by western blotting using anti-mCherry antibodies 48 h later. Tubulin is shown as a loading control. (C) VLDLR is required for P4-dependent oocyte maturation. Oocytes were injected with VLDLR sense or antisense oligonucleotides and, 48 h later, incubated in P4-containing solution overnight. The percentage of oocytes that had undergone GVBD normalized to the naïve treatment is shown. (D) Western blot assessing the MAPKERK1/2 and Cdc2 phosphorylation state for the different treatments as indicated. Ooc. refers to immature oocytes before progesterone treatment and Egg to mature eggs. Tubulin is shown as a loading control. (E) VLDLR knockdown rescue. Oocytes were injected with VLDLR sense or antisense oligonucleotides in the presence or absence of untagged VLDLR (10 ng RNA/oocyte) or untagged mPR (10 ng RNA/oocyte) as indicated. P4 was added 48 h later and the percentage of oocytes undergoing GVBD was normalized to the GVDB recorded with VLDLR sense-injected oocytes. Quantitative results are mean±s.e.m. for three or more experiments. *P<0.05; **P<0.01; ***P<0.001.
Figure 2. VLDLR is essential for mPR localization to the plasma. (A,B) Effect of VLDLR knockdown on endogenous mPR trafficking. Oocytes were left untreated (Naïve) or injected with mPR RNA or VLDLR sense or antisense oligonucleotides and stained with P4–BSA–Fluorescein 48 h later to quantify the levels of endogenous mPR at the plasma membrane. (A) Low-magnification confocal images showing P4–BSA–Fluorescein staining for the different treatments as indicated. The control treatment shows background staining in the absence of P4–BSA–FITC. Scale bar: 50 µm. (B) Quantification of the P4–BSA–FITC staining from ImageJ in the different treatments normalized to the average in control uninjected oocytes (Naïve). (C–E) Effect of VLDLR knockdown on trafficking of overexpressed mPR. Oocytes were co-injected with RNAs expressing mPR and the PM marker TMEM–mCherry in the presence of VLDLR sense or antisense oligonucleotides. Confocal z-stacks were acquired 48 h later with the pinhole at 1 airy unit. ImageJ was used to quantify the fluorescence in a specific ROI. (C) Representative orthogonal sections of the two individual oocytes highlighted in green in E. Scale bar: 2 µm. (D) GFP and mCherry fluorescence intensities along the z-stack section from the two individual oocytes highlighted in green in E. (E) Quantification of the percentage of overexpressed mPR–GFP localized at the PM following injection of VLDLR sense or antisense oligonucleotides. Data were normalized to the average of mPR percentage at the PM from VLDLR sense-injected oocytes. (F) VLDLR-knockdown rescue experiment. Oocytes were uninjected (Naïve) or injected with VLDLR antisense oligonucleotides with or without mPR RNA. Endogenous mPR at the PM was quantified 48 h later through P4–BSA–FITC staining. P4–BSA–FITC fluorescence in a specific ROI was quantified using ImageJ and the data normalized to the average P4–BSA–FITC fluorescence from naïve oocytes. (G) Oocytes were either uninjected (Naïve) or injected with VLDLR sense or antisense oligonucleotides as indicated, and the endogenous Ca2+-activated Cl currents, as a measure of SOCE, were recorded 48 h later as described in the Materials and Methods section. Quantitative results are mean±s.e.m. for three or more experiments or as indicated by individual data points. **P<0.01; ***P<0.001.
Figure 5. VLDLR is required for mPR trafficking. (A,B) Representative focal plane images of WGA-stained oocytes expressing mPR–GFP with mCherry–VLDLR (A) or VLDLR–mCherry (B). (C) Representative intracellular focal plane and orthogonal sections of oocytes overexpressing GFP–mPR with VLDLR tagged at its N- or C-terminus. The typical reticular ER structure surrounding pigment granules, which are indicated by stars in panels A–C, and the VLDLR–Ch- or mPR–GFP-positive puncta representative of the Golgi are indicated by arrowheads. Scale bars: 2 µm. (D) Higher resolution view of the box indicated in the merge image in C to better highlight the VLDLR-positive Golgi structures and the reticular ER appearance indicated by the GFP–mPR staining. A cartoon rendering showing the ER and Golgi (labeled G) is shown in the bottom-right image. (E) Lack of physical interaction between the N-terminally tagged GFP–mPR and mCherry-tagged VLDLR. Oocytes were injected with RNA coding for GFP–mPR along with N- (Ch–VDLDR) or C-terminally (VLDLR–Ch) tagged VLDLR and allowed to express for 48 h. This was followed by crosslinking, lysing and immunoprecipitation using anti-GFP magnetic microbeads. Fractions from the whole lysates (input) and eluates from the anti-GFP beads (IP) were examined by western blotting using anti-GFP, anti-mCherry and anti-RFP antibodies. (F) Quantification of endogenous mPR PM residence in the presence of overexpressed VLDLR. Oocytes were injected with VLDLR–Ch or Ch–VLDLR RNA and stained with P4–BSA–FITC 48 h later. Data were normalized to the average P4–BSA–FITC fluorescence in naïve (uninjected) oocytes. (G) Quantification of mPR-GFP at the PM following expression of mPR-GFP alone or with mCherry-VLDLR or VLDLR-mCherry as indicated. Oocytes were stained with WGA and confocal z-stacks taken 48 h later. Quantitative results are mean±s.e.m. for three or more experiments or as indicated by individual data points. ***P<0.001.
Figure 6. VLDLR is essential for mPR trafficking from the ER to the Golgi. (A) Representative intracellular focal images of individual oocytes co-injected with the Golgi marker GalNac–GFP and the ER marker KDEL–mCherry, or GalNac–GFP and VLDLR–Ch, or mPR–GFP and VLDLR–Ch as indicated. The arrowheads point to representative Golgi puncta. (B) Effect of VLDLR knockdown on mPR–GFP trafficking from the ER to the Golgi. Oocytes were co-injected with RNAs coding for mPR–GFP and the ER marker KDEL–mCherry, with VLDLR sense or antisense oligonucleotides. Confocal z-stacks were acquired 48 h later. The mPR-positive puncta represent the Golgi. Scale bars: 2 µm. (C) Quantification of the number of mPR–GFP-positive Golgi following injection of VLDLR sense or antisense oligonucleotides from images similar to the one in B. (D) Quantification of the number of Golgi (GalNac-GFP positive) in sense and antisense VLDLR-injected oocytes from images similar to those shown in A. Quantitative results are mean±s.e.m. for three or more experiments. ***P<0.001; ns, not significant.
Supplemental Figure 1. Subcellular localization of N- (GFP-mPR) and C-terminally (mPRGFP)-tagged mPR. Representative focal plane images at the plasma membrane or deep within the cell of oocytes 48 hrs after injecting RNAs expressing the PM marker TMEM-mCherry along with mPR-GFP (A) or GFP-mPR (B).
Supplemental Figure 2. (A) Representative orthogonal section and three different focal plane images taken along the Z stacks 48 hrs after injecting RNAs expressing GFP alone (upper panel) or mCherry alone (lower panel). (B) Representative focal plane images deep within the cell of an immature oocyte and a mature egg expressing for 48 hours the Golgi marker GalNac-GFP along with the ER marker KDEL-mCherry
