Identification of two functional nuclear localization signals mediating nuclear import of liver receptor homologue-1

Abstract

Liver receptor homologue-1 (LRH-1) is a member of the nuclear receptor superfamily. We characterized two functional nuclear localization signals (NLSs) in LRH-1. NLS1 (residues 117-168) overlaps the second zinc finger in the DNA binding domain. Mutagenesis showed that the zinc finger structure and two basic clusters on either side of the zinc finger loop are critical for nuclear import of NLS1. NLS2 (residues 169-204) is located in the Ftz-F1 box that contains a bipartite signal. In full-length LRH-1, mutation of either NLS1 or NLS2 had no effect on nuclear localization, but disruption of both NLS1 and NLS2 resulted in the cytoplasmic accumulation of LRH-1. Either NLS1 or NLS2 alone was sufficient to target LRH-1 to the nucleus. Both NLS1 and NLS2 mediate nuclear transport by a mechanism involving importin α/β. Finally, we showed that three crucial basic clusters in the NLSs are involved in the DNA binding and transcriptional activities of LRH-1.

Nuclear pore complex evolution: a trypanosome Mlp analogue functions in chromosomal segregation but lacks transcriptional barrier activity

“…, 2001; Lange et al. , 2007; Yang et al. , 2011), which is shared with the TbNup92 kinetoplastid orthologues (Figure 1 and Supplemental Figure S2A).…”

sNASP inhibits TLR signaling to regulate immune response in sepsis

“…IP and IB were performed essentially as described elsewhere (44). Cells were lysed by IP lysis buffer (20 mM Tris-HCl at pH 7.5, 0.5% NP-40, 150 mM NaCl, and protease inhibitors) (catalog P8340, Sigma).…”

Cytoplasmic Localization of Lrh-1 Down-Regulates Ovarian Follicular cyp19a1a Expression in a Teleost, the Orange-Spotted Grouper Epinephelus coioides1

“…Thus, the transition of Lrh-1 cellular localization from the nucleus to the cytoplasm abolishes the activation of cyp19a1a by Lrh-1 and related signals, and may represent an important mechanism for the down-regulation of cyp19a1a expression in the follicular cells of the mature ovary of the orange-spotted grouper and possibly other teleosts as well. It has been shown that disruption of the two nuclear localization signals (NLSs) in rat LRH-1 resulted in the cytoplasmic accumulation of LRH-1 [24] and phosphorylation of Ser72 by Akt within the putative NLS of Skp2 promoted its cytoplasmic translocation [36]. Because putative phosphorylation could be predicted at Tyr48 and Thr61 within NLS1 of the orange-spotted grouper Lrh-1 (with the online software NetPhos 2.0, for example), it is possible that modifications, most likely phosphorylation, of NLSs in the orange-spotted grouper Lrh-1 may also be involved in the cytoplasmic sequestration of Lrh-1 in the follicular cells of the mature ovary.…”

“…The orange-spotted grouper Lrh-1 homologue shared the typical structures of the nuclear receptor of Nr5a subfamily (Supplemental Fig. S1), namely the conserved DNA-binding regions (I and Ftz-f1 box), ligandbinding regions (II and III), the activation function-2 (AF-2) hexamer, and two nuclear localization sequences (amino acid residues 24 to ;75 for NLS1 and 76 to ;111 for NLS2) as in the rat [24]. Phylogenetic analysis categorized the orangespotted grouper Lrh-1 homologue to the Nr5a2 subfamily (Supplemental Fig.…”