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Cysteine (C)-x-C receptor 4 undergoes transportin 1-dependent nuclear localization and remains functional at the nucleus of metastatic prostate cancer cells.

Don-Salu-Hewage AS, Chan SY, McAndrews KM, Chetram MA, Dawson MR, Bethea DA, Hinton CV - PLoS ONE (2013)

Bottom Line: Herein, we demonstrate that CXCR4 associated with the nucleus of malignant prostate cancer tissues.Importantly, Gαi immunoprecipitation and calcium mobilization studies indicated that nuclear CXCR4 was functional and participated in G-protein signaling, revealing that the nuclear pool of CXCR4 retained function.Given the suggestion that functional, nuclear CXCR4 may be a mechanism underlying prostate cancer recurrence, increased metastatic ability and poorer prognosis after tumors have been treated with therapy that targets plasma membrane CXCR4, these studies addresses a novel mechanism of nuclear signaling for CXCR4, a novel mechanism of clinical targeting, and demonstrate an active nuclear pool that provides important new information to illuminate what has been primarily clinical reports of nuclear CXCR4.

View Article: PubMed Central - PubMed

Affiliation: Center for Cancer Research and Therapeutic Development, Clark Atlanta University, Atlanta, GA, USA.

ABSTRACT
The G-protein coupled receptor (GPCR), Cysteine (C)-X-C Receptor 4 (CXCR4), plays an important role in prostate cancer metastasis. CXCR4 is generally regarded as a plasma membrane receptor where it transmits signals that support transformation, progression and eventual metastasis. Due to the central role of CXCR4 in tumorigenesis, therapeutics approaches such as antagonist and monoclonal antibodies have focused on receptors that exist on the plasma membrane. An emerging concept for G-protein coupled receptors is that they may localize to and associate with the nucleus where they retain function and mediate nuclear signaling. Herein, we demonstrate that CXCR4 associated with the nucleus of malignant prostate cancer tissues. Likewise, expression of CXCR4 was detected in nuclear fractions among several prostate cancer cell lines, compared to normal prostate epithelial cells. Our studies identified a nuclear pool of CXCR4 and we defined a nuclear transport pathway for CXCR4. We reveal a putative nuclear localization sequence (NLS), 'RPRK', within CXCR4 that contributed to nuclear localization. Additionally, nuclear CXCR4 interacted with Transportinβ1 and Transportinβ1-binding to CXCR4 promoted its nuclear translocation. Importantly, Gαi immunoprecipitation and calcium mobilization studies indicated that nuclear CXCR4 was functional and participated in G-protein signaling, revealing that the nuclear pool of CXCR4 retained function. Given the suggestion that functional, nuclear CXCR4 may be a mechanism underlying prostate cancer recurrence, increased metastatic ability and poorer prognosis after tumors have been treated with therapy that targets plasma membrane CXCR4, these studies addresses a novel mechanism of nuclear signaling for CXCR4, a novel mechanism of clinical targeting, and demonstrate an active nuclear pool that provides important new information to illuminate what has been primarily clinical reports of nuclear CXCR4.

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A Putative Functional NLS within CXCR4.A, GFP-CXCR4 fusion protein localized similar to endogenous CXCR4. CXCR4-pEGFPN1 transfected PC3 cells were stimulated with SDF1α, fixed with methanol, blocked then incubated with a mouse anti-CXCR4 monoclonal antibody, followed by a Cy3-conjugated anti-mouse secondary antibody. Nuclei were stained with DAPI (blue). Images were taken at 40× maginification using Axiovision software 4.8.2 with a Zeiss Axio Imager.z1 fluorescence microscope at ex = 470 nm for FITC, ex = 358 nm for DAPI and ex = 551 nm for Cy3. Images demonstrate the co-localization (yellow) of endogenous CXCR4 (red) with GFP-tagged CXCR4 (green). B, Localization analysis of wild type CXCR4 (CXCR4-pEGFPN1), NLS-mutant of CXCR4 (pEGFPN1-CXCR4R146A,) and deleted NLS of CXCR4 (CXCR4ΔNLS) by immunocytochemistry in PC3 cells. Nuclei were stained with propidium iodide (red) and CXCR4 was detected as the fusion protein GFP-CXCR4 (green). Imaging was with a Zeiss LSM-510 UV Confocal Microscope using the 63× Plan-Apochromat 63x/1.40 Oil DIC objective at ex = 488 nm for FITC and ex = 543 nm for Cy3. Scale bars represent 50 µm. C, Transfected cells were stimulated with SDF1α prior to subcellular fractionation into non-nuclear and nuclear fractions. Immunoblots were probed with anti-GFP to detect the fusion protein GFP-CXCR4. Anti-CD44 (non-nuclear) and anti-Topoisomerase1 (Topo 1, nuclear) were used as markers for fractionation purity and as loading controls.
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pone-0057194-g003: A Putative Functional NLS within CXCR4.A, GFP-CXCR4 fusion protein localized similar to endogenous CXCR4. CXCR4-pEGFPN1 transfected PC3 cells were stimulated with SDF1α, fixed with methanol, blocked then incubated with a mouse anti-CXCR4 monoclonal antibody, followed by a Cy3-conjugated anti-mouse secondary antibody. Nuclei were stained with DAPI (blue). Images were taken at 40× maginification using Axiovision software 4.8.2 with a Zeiss Axio Imager.z1 fluorescence microscope at ex = 470 nm for FITC, ex = 358 nm for DAPI and ex = 551 nm for Cy3. Images demonstrate the co-localization (yellow) of endogenous CXCR4 (red) with GFP-tagged CXCR4 (green). B, Localization analysis of wild type CXCR4 (CXCR4-pEGFPN1), NLS-mutant of CXCR4 (pEGFPN1-CXCR4R146A,) and deleted NLS of CXCR4 (CXCR4ΔNLS) by immunocytochemistry in PC3 cells. Nuclei were stained with propidium iodide (red) and CXCR4 was detected as the fusion protein GFP-CXCR4 (green). Imaging was with a Zeiss LSM-510 UV Confocal Microscope using the 63× Plan-Apochromat 63x/1.40 Oil DIC objective at ex = 488 nm for FITC and ex = 543 nm for Cy3. Scale bars represent 50 µm. C, Transfected cells were stimulated with SDF1α prior to subcellular fractionation into non-nuclear and nuclear fractions. Immunoblots were probed with anti-GFP to detect the fusion protein GFP-CXCR4. Anti-CD44 (non-nuclear) and anti-Topoisomerase1 (Topo 1, nuclear) were used as markers for fractionation purity and as loading controls.

Mentions: Nuclear-localized PM receptors must contain a nuclear localization sequence (NLS) to permit transport to and/or into the nucleus [80]. To determine whether the PSORT II-predicted NLS, ‘146RPRK149’ was functional and contributed to nuclear expression of CXCR4, we evaluated the intracellular distribution of wildtype GFP-tagged CXCR4 (pEGFPN1-CXCR4, GFP-CXCR4 fusion protein), two mutated fusion proteins in which arginine 146 and 148 were separately mutated to an alanine (CXCR4R146A and CXCR4R148A, respectively), as well as a fusion protein where the NLS was deleted (CXCR4ΔNLS). Plasmids encoding GFP-CXCR4 were transfected into PC3 cells and examined by ICC microscopy (Fig. 3). The localization pattern of GFP-CXCR4 at the plasma membrane and in the cytoplasm was consistent with endogeneous CXCR4 (Fig. 3A). Previous studies have reported an expression pattern for GFP-CXCR4 similar to our observation in other cancer cell lines [81], [82]. Wild-type GFP-tagged CXCR4 was localized predominantly at the PM, with some localization at the nucleus in untreated cells (Fig. 3B). However, an increase in punctate staining was observed at the nucleus/nuclear membrane upon treatment with SDF1α. Interestingly, both CXCR4R146A (Fig. 3B) and CXCR4R148A (data not shown) were detectable at the nucleus, suggesting that neither R146A nor R148A mutations in the NLS were sufficient to inhibit CXCR4 localization to the nucleus. To further examine the requirement of this NLS to localize CXCR4 to the nucleus, we deleted the NLS, ‘146RPRK149’, within pEGFPN1-CXCR4 (CXCR4ΔNLS). We detected CXCR4ΔNLS at the PM and diffusely throughout the cytosol, similar to wild-type GFP-CXCR4, but we did not detect CXCR4ΔNLS at the nucleus (Fig. 3B). To further confirm that CXCR4ΔNLS was excluded from the nucleus, PC3 cells were transiently transfected with wildtype GFP-CXCR4 or CXCR4ΔNLS then fractionated into nuclear and non-nuclear samples for analysis by western blot analysis (Fig. 3C). Consistent with ICC observations, we found that wild type GFP-CXCR4 and CXCR4ΔNLS were both detectable in non-nuclear fractions, while only GFP-CXCR4 was detected in nuclear fractions. Collectively, these data suggest that the ‘RPRK’ motif may be involved in localization of CXCR4 to the nucleus in prostate cancer.


Cysteine (C)-x-C receptor 4 undergoes transportin 1-dependent nuclear localization and remains functional at the nucleus of metastatic prostate cancer cells.

Don-Salu-Hewage AS, Chan SY, McAndrews KM, Chetram MA, Dawson MR, Bethea DA, Hinton CV - PLoS ONE (2013)

A Putative Functional NLS within CXCR4.A, GFP-CXCR4 fusion protein localized similar to endogenous CXCR4. CXCR4-pEGFPN1 transfected PC3 cells were stimulated with SDF1α, fixed with methanol, blocked then incubated with a mouse anti-CXCR4 monoclonal antibody, followed by a Cy3-conjugated anti-mouse secondary antibody. Nuclei were stained with DAPI (blue). Images were taken at 40× maginification using Axiovision software 4.8.2 with a Zeiss Axio Imager.z1 fluorescence microscope at ex = 470 nm for FITC, ex = 358 nm for DAPI and ex = 551 nm for Cy3. Images demonstrate the co-localization (yellow) of endogenous CXCR4 (red) with GFP-tagged CXCR4 (green). B, Localization analysis of wild type CXCR4 (CXCR4-pEGFPN1), NLS-mutant of CXCR4 (pEGFPN1-CXCR4R146A,) and deleted NLS of CXCR4 (CXCR4ΔNLS) by immunocytochemistry in PC3 cells. Nuclei were stained with propidium iodide (red) and CXCR4 was detected as the fusion protein GFP-CXCR4 (green). Imaging was with a Zeiss LSM-510 UV Confocal Microscope using the 63× Plan-Apochromat 63x/1.40 Oil DIC objective at ex = 488 nm for FITC and ex = 543 nm for Cy3. Scale bars represent 50 µm. C, Transfected cells were stimulated with SDF1α prior to subcellular fractionation into non-nuclear and nuclear fractions. Immunoblots were probed with anti-GFP to detect the fusion protein GFP-CXCR4. Anti-CD44 (non-nuclear) and anti-Topoisomerase1 (Topo 1, nuclear) were used as markers for fractionation purity and as loading controls.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585330&req=5

pone-0057194-g003: A Putative Functional NLS within CXCR4.A, GFP-CXCR4 fusion protein localized similar to endogenous CXCR4. CXCR4-pEGFPN1 transfected PC3 cells were stimulated with SDF1α, fixed with methanol, blocked then incubated with a mouse anti-CXCR4 monoclonal antibody, followed by a Cy3-conjugated anti-mouse secondary antibody. Nuclei were stained with DAPI (blue). Images were taken at 40× maginification using Axiovision software 4.8.2 with a Zeiss Axio Imager.z1 fluorescence microscope at ex = 470 nm for FITC, ex = 358 nm for DAPI and ex = 551 nm for Cy3. Images demonstrate the co-localization (yellow) of endogenous CXCR4 (red) with GFP-tagged CXCR4 (green). B, Localization analysis of wild type CXCR4 (CXCR4-pEGFPN1), NLS-mutant of CXCR4 (pEGFPN1-CXCR4R146A,) and deleted NLS of CXCR4 (CXCR4ΔNLS) by immunocytochemistry in PC3 cells. Nuclei were stained with propidium iodide (red) and CXCR4 was detected as the fusion protein GFP-CXCR4 (green). Imaging was with a Zeiss LSM-510 UV Confocal Microscope using the 63× Plan-Apochromat 63x/1.40 Oil DIC objective at ex = 488 nm for FITC and ex = 543 nm for Cy3. Scale bars represent 50 µm. C, Transfected cells were stimulated with SDF1α prior to subcellular fractionation into non-nuclear and nuclear fractions. Immunoblots were probed with anti-GFP to detect the fusion protein GFP-CXCR4. Anti-CD44 (non-nuclear) and anti-Topoisomerase1 (Topo 1, nuclear) were used as markers for fractionation purity and as loading controls.
Mentions: Nuclear-localized PM receptors must contain a nuclear localization sequence (NLS) to permit transport to and/or into the nucleus [80]. To determine whether the PSORT II-predicted NLS, ‘146RPRK149’ was functional and contributed to nuclear expression of CXCR4, we evaluated the intracellular distribution of wildtype GFP-tagged CXCR4 (pEGFPN1-CXCR4, GFP-CXCR4 fusion protein), two mutated fusion proteins in which arginine 146 and 148 were separately mutated to an alanine (CXCR4R146A and CXCR4R148A, respectively), as well as a fusion protein where the NLS was deleted (CXCR4ΔNLS). Plasmids encoding GFP-CXCR4 were transfected into PC3 cells and examined by ICC microscopy (Fig. 3). The localization pattern of GFP-CXCR4 at the plasma membrane and in the cytoplasm was consistent with endogeneous CXCR4 (Fig. 3A). Previous studies have reported an expression pattern for GFP-CXCR4 similar to our observation in other cancer cell lines [81], [82]. Wild-type GFP-tagged CXCR4 was localized predominantly at the PM, with some localization at the nucleus in untreated cells (Fig. 3B). However, an increase in punctate staining was observed at the nucleus/nuclear membrane upon treatment with SDF1α. Interestingly, both CXCR4R146A (Fig. 3B) and CXCR4R148A (data not shown) were detectable at the nucleus, suggesting that neither R146A nor R148A mutations in the NLS were sufficient to inhibit CXCR4 localization to the nucleus. To further examine the requirement of this NLS to localize CXCR4 to the nucleus, we deleted the NLS, ‘146RPRK149’, within pEGFPN1-CXCR4 (CXCR4ΔNLS). We detected CXCR4ΔNLS at the PM and diffusely throughout the cytosol, similar to wild-type GFP-CXCR4, but we did not detect CXCR4ΔNLS at the nucleus (Fig. 3B). To further confirm that CXCR4ΔNLS was excluded from the nucleus, PC3 cells were transiently transfected with wildtype GFP-CXCR4 or CXCR4ΔNLS then fractionated into nuclear and non-nuclear samples for analysis by western blot analysis (Fig. 3C). Consistent with ICC observations, we found that wild type GFP-CXCR4 and CXCR4ΔNLS were both detectable in non-nuclear fractions, while only GFP-CXCR4 was detected in nuclear fractions. Collectively, these data suggest that the ‘RPRK’ motif may be involved in localization of CXCR4 to the nucleus in prostate cancer.

Bottom Line: Herein, we demonstrate that CXCR4 associated with the nucleus of malignant prostate cancer tissues.Importantly, Gαi immunoprecipitation and calcium mobilization studies indicated that nuclear CXCR4 was functional and participated in G-protein signaling, revealing that the nuclear pool of CXCR4 retained function.Given the suggestion that functional, nuclear CXCR4 may be a mechanism underlying prostate cancer recurrence, increased metastatic ability and poorer prognosis after tumors have been treated with therapy that targets plasma membrane CXCR4, these studies addresses a novel mechanism of nuclear signaling for CXCR4, a novel mechanism of clinical targeting, and demonstrate an active nuclear pool that provides important new information to illuminate what has been primarily clinical reports of nuclear CXCR4.

View Article: PubMed Central - PubMed

Affiliation: Center for Cancer Research and Therapeutic Development, Clark Atlanta University, Atlanta, GA, USA.

ABSTRACT
The G-protein coupled receptor (GPCR), Cysteine (C)-X-C Receptor 4 (CXCR4), plays an important role in prostate cancer metastasis. CXCR4 is generally regarded as a plasma membrane receptor where it transmits signals that support transformation, progression and eventual metastasis. Due to the central role of CXCR4 in tumorigenesis, therapeutics approaches such as antagonist and monoclonal antibodies have focused on receptors that exist on the plasma membrane. An emerging concept for G-protein coupled receptors is that they may localize to and associate with the nucleus where they retain function and mediate nuclear signaling. Herein, we demonstrate that CXCR4 associated with the nucleus of malignant prostate cancer tissues. Likewise, expression of CXCR4 was detected in nuclear fractions among several prostate cancer cell lines, compared to normal prostate epithelial cells. Our studies identified a nuclear pool of CXCR4 and we defined a nuclear transport pathway for CXCR4. We reveal a putative nuclear localization sequence (NLS), 'RPRK', within CXCR4 that contributed to nuclear localization. Additionally, nuclear CXCR4 interacted with Transportinβ1 and Transportinβ1-binding to CXCR4 promoted its nuclear translocation. Importantly, Gαi immunoprecipitation and calcium mobilization studies indicated that nuclear CXCR4 was functional and participated in G-protein signaling, revealing that the nuclear pool of CXCR4 retained function. Given the suggestion that functional, nuclear CXCR4 may be a mechanism underlying prostate cancer recurrence, increased metastatic ability and poorer prognosis after tumors have been treated with therapy that targets plasma membrane CXCR4, these studies addresses a novel mechanism of nuclear signaling for CXCR4, a novel mechanism of clinical targeting, and demonstrate an active nuclear pool that provides important new information to illuminate what has been primarily clinical reports of nuclear CXCR4.

Show MeSH
Related in: MedlinePlus