Nuclear localization of orphan receptor protein kinase (Ror1) is mediated through the juxtamembrane domain

Abstract

Background: Several receptor tyrosine kinases (RTKs) such as EGFR, FGFR, TRK, and VEGFR are capable of localizing in the cell nucleus in addition to their usual plasma membrane localization. Recent reports also demonstrate that nuclear-localized RTKs have important cellular functions such as transcriptional activation. On the basis of preliminary bioinformatic analysis, additional RTKs, including receptor tyrosine kinase-like orphan receptor 1 (Ror1) were predicted to have the potential for nuclear subcellular localization. Ror1 is a receptor protein tyrosine kinase that modulates neurite growth in the central nervous system. Because the nuclear localization capability of the Ror1 cytoplasmic domain has not been reported, we examined the cellular expression distribution of this region.

Results: The Ror1 cytoplasmic region was amplified and cloned into reporter constructs with fluorescent tags. Following transfection, the nuclear distribution patterns of transiently expressed fusion proteins were observed. Serial deletion constructs were then used to map the juxtamembrane domain of Ror1 (aa_471-513) for this nuclear translocation activity. Further site-directed mutagenesis suggested that a KxxK-16 aa-KxxK sequence at residues 486-509 is responsible for the nuclear translocation interaction. Subsequent immunofluorescence analysis by cotransfection of Ran and Ror1 implied that the nuclear translocation event of Ror1 might be mediated through the Ran pathway.

Conclusions: We have predicted several RTKs that contain the nuclear localization signals. This is the first report to suggest that the juxtamembrane domain of the Ror1 cytoplasmic region mediates the translocation event. Ran GTPase is also implicated in this event. Our study might be beneficial in future research to understand the Ror1 biological signaling pathway.

Figure 1

Ror1/2 mutant with the truncation of the ligand-binding and transmembrane domains are capable of nuclear transport. AGS and HR cells were transfected with the cytoplasmic part of Ror1/2 whose N-terminal was HcRed-tagged or with a vector control, and then analyzed by fluorescence confocal microscopy. The protein size of HcRed only is about 30 kDa; and the size of the HcRed-tagged Ror1/2 is about 85 kDa.

Figure 2

Identification of the minimal Ror1 domain that plays a critical role in nuclear accumulation. Confocal microscopic analysis was used to detect subcellular localization of Ror1 fragments. HR cells were transfected for 24 hours with indicated Ror1 fragments and stained with anti-HA monoclonal antibody (green) followed by a fluorescent (FITC) secondary antibody and with phalloidin rhodamine (red). DAPI staining shows the nucleus. PKM2, a known cytoplasmic protein, served as a negative control.

Figure 3

Quantification of the subcellular distribution ratios of different Ror1 fragments. The mean percentage of FITC fluorescence in the nuclear or cytoplasmic compartments for each construct was determined by analyzing the FITC intensity from 30 randomly selected cells.

Figure 4

Site-directed mutagenesis on Ror1 NLdomain unveils one nuclear localization signal. HR cells were transfected with indicated Ror1 mutants and analyzed using anti-HA monoclonal antibody (green) followed by a fluorescent (FITC) secondary antibody. DAPI staining shows the nucleus.

Figure 5

Confocal microscopy analysis of increased nuclear import of Ror1 by cotransfection with wild-type Ran. The flag-tagged WT Ran or empty vector control (-) was cotransfected into HR cells with the Ror1 constructs. The protein distribution was analyzed by confocal microscopy.

Figure 6

Quantification of Ror1 localization in transfected cells. The localization pattern of Ror1 in cells expressing the indicated Ror1 and Ran constructs was scored for 80 cells. The graph shows the percentage of cells with the indicated Ror1 localization patterns.