Protease Activated Receptors (PARs), a seven domain transmembrane G-protein coupled receptor (GPCRs) has a part in the invasive program. hPar1 over expression correlates directly with the invasion properties of cytotrophoblast as well as of carcinoma cells. Our histological analyses of EVTs indicated a marked nuclear localization of PAR1, especially in the abundant population of cell outgrowth and invading cells after PAR1 activation. In-contrast, cytoplasmic and cell membrane localization of PAR1 were seen in the basal levels of sprouting cells before PAR1 activation or when PAR1 was silenced (siRNA-hPar1). Immmunodetection of PAR1 was noticed in the nuclei of the invading front of the cytotrophoblasts (i.e., intermediate trophoblasts) while very little or none in the nuclei of the villous throphoblast. When double fluorescent staining for PAR1 and nuclear stain (DRAQ5), were performed a marked PAR1 nuclear localization was observed. Nuclear localization of an increasing list of GPCRs is recently emerging with a growing body of evidence. Analysis of nuclear fractions of trophoblastic cell lines (e.g., HTR/8 and JAR) showed distinct and specific expression of PAR1 following 3-8 hrs of PAR1, activation. Leptomycin, induced levels of PAR1 nuclear localization. This effect was seen only in placenta originated cells but not in a variety of epithelial carcinoma cells or tissue biopsy where PAR1 is over-expressed. The nuclear PAR1 localization is mediated most likely after PAR1 internalization, whereby in a yet unknown manner PAR1 forms a complex with importin 1b and enters the cell nuclei. Indeed, the application of siRNA – importin 1b abrogated nuclear localization of PAR1. Nuclear-cytoplasmic shuttling may play important novel roles in the regulation of GPCR cellular function.
Sorina Grisaru-Granovsky MD PhD 1,2, Arun Krumar Kancharla MSc1 , Myriam Maoz MSc 1, Rachel Bar-Shavit MD PhD 1
Dept of Oncology and Tumor Biology Haddasah Medical Center1
Dept of Obstetrics and Gynecology, Shaare Zedek Medical Center2, the Hebrew University, Jerusalem