DeCamp D L, Thompson T M, de Sauvage F J, Lerner M R
Departments of Pharmacology and Dermatology, The University of Texas Southwestern Medical Center, Dallas, Texas 75390-9069, USA.
J Biol Chem. 2000 Aug 25;275(34):26322-7. doi: 10.1074/jbc.M004055200.
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 Galpha(i) activation. Smoothened-mediated pigment aggregation was reversed by treatment with pertussis toxin or by co-expression with dominant negative Galpha(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 Galpha(i).
研究了七次跨膜蛋白平滑受体（Smoothened）在非洲爪蟾黑素细胞中作为G蛋白偶联受体发挥作用的能力。将含有人类平滑受体cDNA插入片段的质粒转染到永生化的青蛙色素细胞中。表达该蛋白的细胞表现出持续色素聚集的表型，这是组成型Gα(i)激活的一个标志。用百日咳毒素处理或与显性负性Gα(i)共表达可逆转平滑受体介导的色素聚集。黑素细胞表达功能性平滑受体的能力也通过其与十二次跨膜蛋白帕奇蛋白（Patched）的共表达来确定。帕奇蛋白以剂量依赖的方式阻断平滑受体介导的黑素体聚集，这与其作为平滑受体抑制剂的生理作用一致。通过与激活配体音猬因子（Sonic hedgehog）共转染以及直接应用重组音猬因子蛋白，证明了重组的帕奇蛋白-平滑受体复合物在色素细胞中正常发挥功能。音猬因子逆转了帕奇蛋白介导的对平滑受体的抑制并诱导色素聚集。这些发现表明，人类音猬因子受体复合物可以在黑素细胞中进行功能重建，并且它能够利用内源性Gα(i)进行跨膜信号传导。
