RGS4 binds to membranes through an amphipathic α-helix
RGS4 binds to membranes through an amphipathic α-helix
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DOI：
10.1074/jbc.m000618200
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发表时间：
2000-06-16
影响因子：
4.8
通讯作者：
Linder, ME
中科院分区：
文献类型：
--
作者：
Bernstein, LS;Grillo, AA;Linder, ME
RGS4 是 G 蛋白 α 亚基的哺乳动物 GTP 酶激活蛋白，需要其 N 端 33 个氨基酸来实现质膜定位和生物活性（Srinivasa, S. P.、Bernstein, L. S.、Blumer, K. J, 和 Linder, M. E. (1998) Proc. Natl. Acad Sci U. S. A. 95， 5584-5589）。在这项研究中，我们测试了 N 末端结构域通过形成两亲性 α 螺旋介导膜结合的假设。 RGS4 以依赖于前 33 个氨基酸的方式与含有阴离子磷脂的脂质体结合。对应于 RGS4 氨基酸 1-31 的肽的圆二色光谱表明，该肽在阴离子磷脂存在的情况下采用 ct 螺旋构象，中和模型螺旋亲水面上的正电荷或取代模型螺旋疏水面上的极性残基的点突变破坏了质膜靶向和生物活性。 RGS4 在酵母中表达。重组突变蛋白在溶液中具有 GTP 酶激活蛋白的活性，但与阴离子脂质体的结合减弱。通过圆二色光谱测量，对应于具有最显着表型的突变体的肽在形成α-螺旋方面也存在缺陷。这些结果支持 RGS4 通过 N 端 α 螺旋的疏水性和静电相互作用与膜直接相互作用的模型。
RGS4 a mammalian GTPase-activating protein for G protein alpha subunits, requires its N-terminal 33 amino acids for plasma membrane localization and biological activity (Srinivasa, S. P., Bernstein, L. S., Blumer, K. J,, and Linder, M. E. (1998) Proc. Natl. Acad Sci U. S. A. 95, 5584-5589). In this study, we tested the hypothesis that the N-terminal domain mediates membrane binding by forming an amphipathic alpha-helix. RGS4 bound to liposomes containing anionic phospholipids in a manner dependent on the first 33 amino acids. Circular dichroism spectroscopy of a peptide corresponding to amino acids 1-31 of RGS4 revealed that the peptide adopted an ct-helical conformation in the presence of anionic phospholipids, Point mutations that either neutralized positive charges on the hydrophilic face or substituted polar residues on the hydrophobic face of the model helix disrupted plasma membrane targeting and biological activity of RGS4 expressed in yeast. Recombinant mutant proteins were active as GTPase-activating proteins in solution but exhibited diminished binding to anionic liposomes. Peptides corresponding to mutants with the most pronounced phenotypes were also defective in forming an a-helix as measured by circular dichroism spectroscopy. These results support a model for direct interaction of RGS4 with membranes through hydrophobic and electrostatic interactions of an N-terminal alpha-helix.