Extracellular cysteines of the corticotropin-releasing factor receptor are critical for ligand interaction.
Extracellular cysteines of the corticotropin-releasing factor receptor are critical for ligand interaction.
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DOI：
10.1021/bi970997r
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发表时间：
1997-10
期刊：
影响因子：
2.9
通讯作者：
L. Qi;A. Leung;Y. Xiong;K. Marx;A. Abou-Samra
中科院分区：
文献类型：
--
作者：
L. Qi;A. Leung;Y. Xiong;K. Marx;A. Abou-Samra
促肾上腺皮质激素释放因子受体（CRF-R）在其氨基末端结构域中含有六个保守的半胱氨酸（C30、C44、C54、C68、C87和C102），并且在其第一和第二胞外环中含有一个半胱氨酸（分别为C188和C258）。此外，几个其他半胱氨酸位于跨膜结构域（C128、C211、C233和C364）和第一胞内环（C150）中。还原二硫键DTT减少CRF结合洗涤剂溶解的膜，这表明二硫键在配体识别中的重要作用。因此，使用定点诱变来引入单个和成对的Cys（C）至Ser（S）或Ala（A）突变。在小鼠CRF-R的氨基末端引入来自c myc的沉默的9个氨基酸标签。除了C258 S和C188 S/C258 S突变之外，所有C至S或至A受体突变体具有良好的表面表达，其为对照的至少52.5%。C30 S、C54 S和C30 S/C54 S突变具有良好的CRF结合和CRF刺激的cAMP积累。对于C44 S、C68 S、C87 S、C102 S、C188 S、C258 S、C30 S/C44 S、C30 S/C68 S、C54 S/C68 S、C87 S/C102 S和C188 S/C258 S突变体，未检测到CRF结合，而CRF刺激的cAMP积累具有高EC 50值。特别地，与携带相应的单半胱氨酸突变的受体相比，携带双突变C44 S/C102 S和C68 S/C87 S的受体具有改善的信号传导性质。这些数据以及DTT对CRF结合的影响表明，二硫键对受体功能很重要。来自单个和成对半胱氨酸突变的功能数据表明C44和C102、C68和C87以及C188和C258之间的潜在配对对于配体-受体相互作用至关重要。
The corticotropin-releasing factor receptor (CRF-R) contains six conserved cysteines in its amino-terminal domain (C30, C44, C54, C68, C87, and C102) and one cysteine in its first and second extracellular loops (C188 and C258, respectively). Additionally, several other cysteines are located in the transmembrane domains (C128, C211, C233, and C364) and first intracellular loop (C150). Reduction of disulfide bonds with DTT decreased CRF binding to detergent-solubilized membranes, suggesting an important role for disulfide bonds in ligand recognition. Therefore, site-directed mutagenesis was used to introduce single and paired Cys (C) to Ser (S) or Ala (A) mutations. A silent nine amino acid tag from c myc was introduced in the amino terminus of the mouse CRF-R. With the exception of C258S and C188S/C258S mutations, all C to S or to A receptor mutants had good surface expression that was at least 52.5% of control. C30S, C54S, and C30S/C54S mutations had good CRF binding and CRF-stimulated cAMP accumulation. No CRF binding was detected for the C44S, C68S, C87S, C102S, C188S, C258S, C30S/C44S, C30S/C68S, C54S/C68S, C87S/C102S, and C188S/C258S mutants, while CRF-stimulated cAMP accumulation occurred with high EC50 values. In particular, receptors carrying double mutations, C44S/C102S and C68S/C87S, had an improved signaling property as compared to receptors carrying the respective single cysteine mutations. These data, together with the effects of DTT on CRF binding, indicate that disulfide bridges are important for receptor functions. Functional data from single and paired cysteine mutations suggest potential pairings between C44 and C102, C68 and C87, and C188 and C258 that are critical for ligand-receptor interactions.