Title Cysteine (C)-X-C Receptor 4 Undergoes Transportin 1- Dependent Nuclear Localization and Remains Functional at the Nucleus of Metastatic Prostate Cancer Cells

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 Transportinb1 and Transportinb1-binding to CXCR4 promoted its nuclear translocation. Importantly, Gai 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. Citation: Don-Salu-Hewage AS, Chan SY, McAndrews KM, Chetram MA, Dawson MR, et al. (2013) Cysteine (C)-X-C Receptor 4 Undergoes Transportin 1Dependent Nuclear Localization and Remains Functional at the Nucleus of Metastatic Prostate Cancer Cells. PLoS ONE 8(2): e57194. doi:10.1371/ journal.pone. 0057194 Editor: Natasha Kyprianou, University of Kentucky College of Medicine, United States of America Received November 20, 2012; Accepted January 18, 2013; Published February 28, 2013 Copyright: 2013 Don-Salu-Hewage et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported, in part, by National Institutes of Health grants 2R25GM060414, P201MD002285 (CVH), F31CA153908 (MAC), 2G12RR00306222 (CVH) and AAAS Women’s International Research Collaboration (WIRC) for Minority Serving Institutions (MSI), a National Science Foundation grant (CVH). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: chinton@cau.edu