Smoothened activates Gαi-mediated signaling in frog melanophores
Smoothened activates Gαi-mediated signaling in frog melanophores
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DOI：
10.1074/jbc.m004055200
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发表时间：
2000-08-25
影响因子：
4.8
通讯作者：
Lerner, MR
中科院分区：
文献类型：
--
作者：
DeCamp, DL;Thompson, TM;Lerner, MR
研究了7-PASS跨膜蛋白在非洲爪哇黑素载体中作为G蛋白偶联受体的作用。将含有人平滑c DNA插入片段的质粒导入永生化青蛙色素细胞。表达该蛋白的细胞显示出持续性色素聚集的表型，这是结构性G(αI)激活的标志。经百日咳毒素处理或与显性阴性G(αI)共表达可逆转平滑介导的色素聚集。黑素载体表达功能光滑的能力也取决于它与十二次跨膜蛋白Patches的共表达。Patted以剂量依赖的方式阻断Smoothens介导的黑素小体聚集，这与其作为Smoothed抑制剂的生理作用一致。通过与激活配体Sonic hedgehog共转染，以及直接应用重组Sonic hedgehog蛋白，证明重组补丁-光滑受体复合体在色素细胞中功能正常。Sonic Hedgehog逆转了贴片介导的光滑和诱导色素聚集的抑制。这些发现表明，人的Sonic hedgehog受体复合体可以在黑素载体中进行功能性重组，并且它能够利用内源性G(αI)进行跨膜信号传递。
The 7-pass transmembrane protein Smoothened was investigated for its ability to act as a G-protein-coupled receptor in Xenopus laevis melanophores. A plasmid containing the human Smoothened cDNA insert was transfected into immortalized frog pigment cells. Cells expressing the protein showed a phenotype of persistent pigment aggregation, a hallmark of constitutive G(alpha i) activation. Smoothened-mediated pigment aggregation was reversed by treatment with pertussis toxin or by co-expression with dominant negative G(alpha i). The ability of melanophores to express functional Smoothened was also determined by its co-expression with the twelve-pass transmembrane protein, Patched. Patched blocked Smoothened-mediated melanosome aggregation in a dose-dependent manner, consistent with its physiological role as an inhibitor of Smoothened. That the reconstituted Patched-Smoothened receptor complex functions normally in pigment cells was demonstrated by co-transfection with the activating ligand, Sonic hedgehog, as well as by direct application of the recombinant Sonic hedgehog protein. Sonic hedgehog reversed Patched-mediated inhibition of Smoothened and induced pigment aggregation. The findings demonstrate that the human Sonic hedgehog receptor complex can be functionally reconstituted in melanophores and that it is capable of transmembrane signaling by utilizing endogenous G(alpha i).