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首页> 外文期刊>Biological chemistry >C-terminal fusion of eGFP to the bradykinin B-2 receptor strongly affects down-regulation but not receptor internalization or signaling
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C-terminal fusion of eGFP to the bradykinin B-2 receptor strongly affects down-regulation but not receptor internalization or signaling

机译：eGFP与缓激肽B-2受体的C末端融合强烈影响下调，但不影响受体内在化或信号传导

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A functional comparison was made between the wildtype bradykinin B, receptor (B(2)wt) and the chimera B(2)eGFP (enhanced green-fluorescent protein fused to the C-terminus of B(2)Wt), both stably expressed in HEK 293 cells. There was almost no difference in terms of ligand-inducible receptor phosphorylation and internalization, signal transduction (accumulation of inositol phosphates) or expression and affinity. However, stimulation for up to 8 h with 10 mu M bradykinin (BK) resulted in a strong decrease in surface receptors (by 60% within 5 h) in B(2)Wt, but not in B(2)eGFP. When the expression levels of both constructs where comparably reduced using a weaker promoter, long-term stimulation resulted in a reduction in surface receptors for B(2)wt(low) to less than 20% within 1 h, whereas the chimera B(2)eGFP(low) still displayed 50% binding activity after 2 h. A 1-h incubation in the absence of BK resulted in a recovery of 60% of the binding in B(2)wt(low) after 1-h stimulation with BK, but of only 20% after 7-h stimulation. In contrast, B(2)eGFP(low) levels were restored to more than 70%, even after 7-h stimulation. These data indicate that although the fusion of eGFP to B(2)wt does not affect its ligand-induced internalization, it strongly reduces the down-regulation, most likely by promoting receptor recycling over degradation.
机译：在野生型缓激肽B，受体（B（2）wt）和嵌合体B（2）eGFP（增强的绿色荧光蛋白与B（2）Wt的C末端融合）之间进行了功能比较在HEK 293细胞中在配体诱导的受体磷酸化和内在化，信号转导（肌醇磷酸的积累）或表达和亲和力方面几乎没有差异。但是，用10μM缓激肽（BK）刺激长达8小时会导致B（2）Wt中表面受体的强烈降低（5小时内降低60％），而B（2）eGFP中却没有。当使用弱启动子使两种构建体的表达水平相对降低时，长期刺激导致B（2）wt（low）的表面受体在1小时内减少至小于20％，而嵌合体B（2 2小时后，eGFP（低）仍显示出50％的结合活性。在没有BK的情况下进行1小时孵育会在用BK刺激1小时后恢复B（2）wt（low）中60％的结合，而在7小时刺激后仅恢复20％。相比之下，即使经过7小时刺激，B（2）eGFP（低）水平也可以恢复到70％以上。这些数据表明，尽管eGFP与B（2）wt的融合不会影响其配体诱导的内在化，但它极大地降低了下调，这很可能是通过促进受体循环而不是降解来实现的。

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