表現選殖小鼠EF-L1可能之受器之研究

Abstract

EF-L1 (又稱為PRAP1) 在小腸表皮細胞表現，為一分泌到胞外之蛋白質。本實驗室先前的研究發現，在EF-L1基因剔除小鼠中，小腸絨毛上已分化的表皮細胞會展現過早的細胞凋亡現象。缺乏EF-L1表現之小腸表皮細胞其PI3K-Akt訊息傳導路徑之活化有缺損；餵食重組之EF-L1蛋白可逆轉缺乏EF-L1小腸表皮細胞之缺陷性狀。這些結果意味著EF-L1可能透過自分泌的機制而影響小腸表皮細胞之存活。此外，EF-L1與小腸表皮細胞的專一性結合之現象也指出小腸表皮細胞表面可能有EF-L1受器的存在。因此我們利用小鼠小腸表皮細胞的mRNA構築一cDNA library，利用表現選殖之方法篩選可能之EF-L1受器。在經過四輪篩選後，我們將候選基因群從每群兩萬五千減少至每群約八十個菌落。在此候選基因群中，我們鑑定了兩個膜蛋白，分別是transmembrane protein 160 (Tmem160)與ABC transporter, Abcb1a (ATP-binding cassette, sub-family B, member 1A)。至於EF-L1是否會與這兩個候選膜蛋白有交互作用則需要更多的實驗來證明。

EF-L1 (epithelial cell produced factor, clone L1; also named proline rich acidic protein 1, PRAP1) is a secreted protein expressed in mouse intestinal epithelial cells (IECs). Our earlier study demonstrated that deficiency of EF-L1 resulted in precocious apoptosis of the differentiated epithelial cells in the villus. Activation of PI3K-Akt pathway was impaired in EF-L1 deficient IECs; oral feeding of recombinant EF-L1 could rescue the phenotype of EF-L1 deficient IECs, suggesting that EF-L1 might function via an autocrine mechanism to affect IECs survival. Moreover, EF-L1 bound to isolated IECs in a specific manner, suggesting that there could be a specific interacting protein or a putative EF-L1 receptor residing on the surface of IECs. We thus constructed a mouse IEC cDNA library and screened for the putative EF-L1 receptor by an expression cloning approach. After quaternary screening, we narrowed down the target pools from ~25000 clones to ~80 clones per pool. Two membrane proteins have been identified in this candidate pool, i.e., transmembrane protein 160 (Tmem160) and the ABC transporter, Abcb1a (ATP-binding cassette, sub-family B, member 1A). Further experiments are needed to confirm the interaction between EF-L1 and the above candidates.