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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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CXCR4 and TRN1 Demonstrate an Interaction.A, Sixty micrograms of total protein were analyzed for TRN1 expression by western blot analysis using a TRN1 specific antibody. Alpha-tubulin served as a loading control. B, One milligram of PC3 whole cell lysate was immunoprecipitated with anti-CXCR4 and separated by SDS-PAGE. Immunocomplexes were probed with anti-TRN1 or anti-CXCR4 to ensure that CXCR4 interacted with TRN1 and was immunoprecipitated, respectively. Thirty micrograms of whole cell PC3 supernatant, post-immunoprecipitation, were separated by SDS-PAGE and harvested for western blot analysis to assess the efficiency of CXCR4 immunoprecipitation. C and D, Cells were transiently transfected with TRN1-specific siRNA to determine an effective concentration (C), prior to harvesting for immunohistochemistry with anti-Lamin A/C and anti-CXCR4 (D). Images were taken using Zeiss Axio Imager.z1 fluorescence microscope at 40× magnification at excitation 470 nm for FITC and 551 nm for Cy3. Small arrows indicate co-localization of CXCR4 with the nucleus (yellow/orange). Scale bar represents 50 µm.
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pone-0057194-g004: CXCR4 and TRN1 Demonstrate an Interaction.A, Sixty micrograms of total protein were analyzed for TRN1 expression by western blot analysis using a TRN1 specific antibody. Alpha-tubulin served as a loading control. B, One milligram of PC3 whole cell lysate was immunoprecipitated with anti-CXCR4 and separated by SDS-PAGE. Immunocomplexes were probed with anti-TRN1 or anti-CXCR4 to ensure that CXCR4 interacted with TRN1 and was immunoprecipitated, respectively. Thirty micrograms of whole cell PC3 supernatant, post-immunoprecipitation, were separated by SDS-PAGE and harvested for western blot analysis to assess the efficiency of CXCR4 immunoprecipitation. C and D, Cells were transiently transfected with TRN1-specific siRNA to determine an effective concentration (C), prior to harvesting for immunohistochemistry with anti-Lamin A/C and anti-CXCR4 (D). Images were taken using Zeiss Axio Imager.z1 fluorescence microscope at 40× magnification at excitation 470 nm for FITC and 551 nm for Cy3. Small arrows indicate co-localization of CXCR4 with the nucleus (yellow/orange). Scale bar represents 50 µm.

Mentions: We identified a putative NLS motif that could be critical for CXCR4 nuclear localization; however, the motif ‘RPRK’ is not a typical classical NLS [60]. In fact, such sequences can also mediate direct binding to other transport receptors. Among the different molecules that are involved in the transport of various cargos to the nucleus, members of the karyopherin beta (β) family contribute directly or indirectly to the nuclear shuttling of molecules [83]. Transportinβ1 (TRN1), also known as Karyopherinβ2, is a transport molecule of the importin-β family that has been linked to desensitization [63] and nuclear-cytoplasmic shuttling of receptors [84], [85], [86], [87], [88], [89]. To test whether TRN1 was involved in CXCR4 transport to the nucleus, we first established that PC3 cells expressed TRN1 by western blot analysis (Fig. 4A); 293T cells served as a positive control for TRN1 expression. Next, we tested for an interaction between CXCR4 and TRN1. We immunoprecipitated CXCR4 from whole cell lysates and tested for co-purification of TRN1 by western blot analysis. Figure 4B demonstrates that CXCR4 and TRN1 associated in PC3 cell lysates. To test whether the association between CXCR4 and TRN1 led to CXCR4 translocation to the nucleus, we decreased TRN1 protein expression by siRNA (Fig. 4C), then determined CXCR4 localization by ICC. We first confirmed an effective siRNA-mediated depletion of TRN1 by western blot analysis (Fig. 4C). As previously described in control cells, CXCR4 localized to the PM, in the cytoplasm and in distinct foci around the nucleus. When TRN1 expression was diminished, CXCR4 was undetectable around the nucleus, even in the presence of SDF1α (Fig. 4D).


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)

CXCR4 and TRN1 Demonstrate an Interaction.A, Sixty micrograms of total protein were analyzed for TRN1 expression by western blot analysis using a TRN1 specific antibody. Alpha-tubulin served as a loading control. B, One milligram of PC3 whole cell lysate was immunoprecipitated with anti-CXCR4 and separated by SDS-PAGE. Immunocomplexes were probed with anti-TRN1 or anti-CXCR4 to ensure that CXCR4 interacted with TRN1 and was immunoprecipitated, respectively. Thirty micrograms of whole cell PC3 supernatant, post-immunoprecipitation, were separated by SDS-PAGE and harvested for western blot analysis to assess the efficiency of CXCR4 immunoprecipitation. C and D, Cells were transiently transfected with TRN1-specific siRNA to determine an effective concentration (C), prior to harvesting for immunohistochemistry with anti-Lamin A/C and anti-CXCR4 (D). Images were taken using Zeiss Axio Imager.z1 fluorescence microscope at 40× magnification at excitation 470 nm for FITC and 551 nm for Cy3. Small arrows indicate co-localization of CXCR4 with the nucleus (yellow/orange). Scale bar represents 50 µm.
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Related In: Results  -  Collection

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pone-0057194-g004: CXCR4 and TRN1 Demonstrate an Interaction.A, Sixty micrograms of total protein were analyzed for TRN1 expression by western blot analysis using a TRN1 specific antibody. Alpha-tubulin served as a loading control. B, One milligram of PC3 whole cell lysate was immunoprecipitated with anti-CXCR4 and separated by SDS-PAGE. Immunocomplexes were probed with anti-TRN1 or anti-CXCR4 to ensure that CXCR4 interacted with TRN1 and was immunoprecipitated, respectively. Thirty micrograms of whole cell PC3 supernatant, post-immunoprecipitation, were separated by SDS-PAGE and harvested for western blot analysis to assess the efficiency of CXCR4 immunoprecipitation. C and D, Cells were transiently transfected with TRN1-specific siRNA to determine an effective concentration (C), prior to harvesting for immunohistochemistry with anti-Lamin A/C and anti-CXCR4 (D). Images were taken using Zeiss Axio Imager.z1 fluorescence microscope at 40× magnification at excitation 470 nm for FITC and 551 nm for Cy3. Small arrows indicate co-localization of CXCR4 with the nucleus (yellow/orange). Scale bar represents 50 µm.
Mentions: We identified a putative NLS motif that could be critical for CXCR4 nuclear localization; however, the motif ‘RPRK’ is not a typical classical NLS [60]. In fact, such sequences can also mediate direct binding to other transport receptors. Among the different molecules that are involved in the transport of various cargos to the nucleus, members of the karyopherin beta (β) family contribute directly or indirectly to the nuclear shuttling of molecules [83]. Transportinβ1 (TRN1), also known as Karyopherinβ2, is a transport molecule of the importin-β family that has been linked to desensitization [63] and nuclear-cytoplasmic shuttling of receptors [84], [85], [86], [87], [88], [89]. To test whether TRN1 was involved in CXCR4 transport to the nucleus, we first established that PC3 cells expressed TRN1 by western blot analysis (Fig. 4A); 293T cells served as a positive control for TRN1 expression. Next, we tested for an interaction between CXCR4 and TRN1. We immunoprecipitated CXCR4 from whole cell lysates and tested for co-purification of TRN1 by western blot analysis. Figure 4B demonstrates that CXCR4 and TRN1 associated in PC3 cell lysates. To test whether the association between CXCR4 and TRN1 led to CXCR4 translocation to the nucleus, we decreased TRN1 protein expression by siRNA (Fig. 4C), then determined CXCR4 localization by ICC. We first confirmed an effective siRNA-mediated depletion of TRN1 by western blot analysis (Fig. 4C). As previously described in control cells, CXCR4 localized to the PM, in the cytoplasm and in distinct foci around the nucleus. When TRN1 expression was diminished, CXCR4 was undetectable around the nucleus, even in the presence of SDF1α (Fig. 4D).

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