Bowles C E, Wilkinson I, Smith R A G, Moir A J G, Montgomery H, Ross R J M
University of Sheffield, Room 112 Floor M, Royal Hallamshire Hospital, Glossop Road, Sheffield S10 2JF, United Kingdom.
Endocrinology. 2007 Feb;148(2):824-30. doi: 10.1210/en.2006-1002. Epub 2006 Nov 9.
The actions of GH are mediated through a cell surface cytokine receptor. We previously demonstrated that naturally occurring truncated membrane bound GH receptors (GHRs) can block GH receptor signaling. We have now investigated whether recombinant extracellular GHR can be conjugated to a myristoylated-peptide (mp) tail and inserted into cell membranes to modulate GHR signaling. Recombinant human extracellular domain (1-241) GHR was expressed in Escherichia coli, purified, and refolded from cell lysate. The free C-terminal cysteine was then reduced and conjugated to an activated preformed mp tail. The properties of the purified tailed GHR (GHR-mp) were then compared with those of the untailed purified GHR 1-241. Fluorescence-activated cell sorter analysis and cell surface binding assays demonstrated that GHR-mp inserted into the cell surface membranes of CHO cells, whereas untailed GHR 1-241 showed no insertion. In a cell-based bioassay GHR-mp partially inhibited wild-type GHR signaling, whereas GHR 1-241 had no effect. Truncated extracellular domain GHR can, when specifically modified with a membrane-localizing mp unit, insert into cell surface membranes and modulate GHR signaling.
生长激素（GH）的作用是通过细胞表面细胞因子受体介导的。我们之前证明，天然存在的截短型膜结合生长激素受体（GHRs）可阻断生长激素受体信号传导。我们现在研究了重组细胞外GHR是否可以与肉豆蔻酰化肽（mp）尾缀合，并插入细胞膜以调节GHR信号传导。重组人细胞外结构域（1-241）GHR在大肠杆菌中表达、纯化，并从细胞裂解物中复性。然后将游离的C末端半胱氨酸还原，并与活化的预制mp尾缀合。然后将纯化的带尾GHR（GHR-mp）的特性与未带尾的纯化GHR 1-241的特性进行比较。荧光激活细胞分选分析和细胞表面结合试验表明，GHR-mp插入到CHO细胞的细胞表面膜中，而未带尾的GHR 1-241没有插入。在基于细胞的生物测定中，GHR-mp部分抑制野生型GHR信号传导，而GHR 1-241没有作用。当用膜定位mp单元进行特异性修饰时，截短的细胞外结构域GHR可以插入细胞表面膜并调节GHR信号传导。
