LRH-1核輸出訊息定位及其特性探討

Abstract

肝受器同源體-1 (Liver receptor homolog-1 ; LRH-1)為核受器 NR5A 家族的成員之一，主要表現於肝腸組織與卵巢。LRH-1為轉錄因子，穩定表現於細胞核。本實驗室先前發現mLRH-1胺基酸片段443-560具有引導GFP綠色螢光蛋白質離開細胞核的能力。為了解mLRH-1是否具有NES，我們建構了幾個 mLRH-1片段與GFP融合蛋白質的表現質體。結果顯示，464-493片段會使GFP融合蛋白質主要分布於細胞質，表示其可能具有NES的特性。在464-493中的兩個區域470-475以及483-488具有leucine-rich序列LXXLXL為典型的NES序列，其分別稱之為NES1與NES2。我們將NES1或NES2的leucine都突變成alanine後發現，NES1或NES2的突變，會大大地降低464-493分布於細胞質的比例，顯示這段NES序列具有核輸出作用。 CRM 1 (Chromosome maintenance region 1, Exportin 1, XPO1)為重要的核輸出因子，加入抑制劑leptomycin B (LMB)會影響CRM1與NES結合，造成蛋白質留在細胞核。我們給予LMB後，並不影響464-493的細胞分布；但對443-560片段則有23%螢光蛋白質聚集於細胞核膜周圍之'perinuclear'區域。以免疫沉澱法分析未偵測443-560之NES與CRM1有交互作用。這些結果說明464-493之NES可能不是藉由CRM1而移出細胞。 mLRH-1的NES1之leucine突變後，使得mLRH-1不再分布於細胞核，並且部分細胞分布於perinuclear區域，此外也降低了mLRH-1對CYP11A1啟動子的轉錄活性調控能力；但對於NES2處突變則無影響。進一步將NES1的三個leucine分別單獨突變成alanine，結果發現NES1的3個leucine都能將大部分(約75%)的mLRH-1留在細胞核內，顯示這3個leucine對於將mLRH-1留在細胞核內都具有重要性。本論文說明了mLRH-1之NES具有核輸出作用，而且由不同長度片段的mLRH-1在細胞質分佈的比例顯示蛋白質結構會影響本身NES的功能。

Liver receptor homologue-1 (LRH-1) is a member of the nuclear receptor NR5A subfamily. LRH-1 is mainly expressed of liver, intestine and ovary. To study the mechanisms of LRH-1 nuclear export, we generated a series of GFP-tagged deletion mutants and assessed their subcellular localization. Our results showed that the fragments 464-493 were predominantly localized in the cytoplasm and contained two clusters of leucine-rich residues 470-475 (NES1) and 483-488 (NES2), typically present in the NES, were identified in this region. Two mutants were generated by simultaneous mutation of three leucine residues into alanine in each cluster. The mutations of NES1 or NES2 significantly abolished the cytoplasmic localization of fragment 464-493. The most thoroughly studied and well characterized nuclear export pathway involves the exportin CRM1. Treatment with exportin CRM1-specific inhibitor leptomycin B resulted no specific effect in fragment 464-493, but there was a perinuclear accumulation of fragment 443-560. The immunoprecipitation assay results also suggested that the major nuclear export pathway utilized by mLRH-1 is CRM1-independent. Unexpectedly, the mutant NES1 translocated to cytoplasm and reduced the transcriptional activity of LRH-1 to the human steroidogenic gene CYP11A1 promoter, but there was no speicifc effect of mutant NES2. Then, single mutation was generated by mutation of one leucine into alanine in NES1. The results indicated that leucine amino acids in NES1 contribute to remain in nucleus. Overall, the identification of NESs in this study implicated that the NESs may sufficient for the nuclear export of mLRH-1, and an alrtnative structural strategy might affect the function of NESs.