Maruyama I N, Mikawa Y G, Maruyama H I
Department of Cell Biology, Scripps Research Institute, La Jolla, CA 92037, USA.
J Mol Biol. 1995 Nov 3;253(4):530-46. doi: 10.1006/jmbi.1995.0571.
Extracellular information is transduced by transmembrane receptors into the inside of the cell across a membrane barrier. To understand the molecular basis of transmembrane signalling, we replaced the transmembrane segment 2 (TM2) of the Escherichia coli aspartate receptor, Tar, with random sequences that are 21 amino acid residues in length and consist of Arg, Gly, Ser, Cys, Val, Leu, Ile and Phe at each position. From this ensemble for recombinant molecules, functional receptors were recovered as clones that could bind aspartate and transmit a signal to the intracellular domain. Restricted average hydrophobicity values were observed for functional transmembrane domains, and support the observation that transmembrane segments typically have hydrophobicity values greater than 1.6. However, non-functional transmembrane domains with greater hydrophobicity than 1.6 indicate that hydrophobicity is not a sole determinant for its function. Fourier transform analysis of the functional TM2 sequences suggests that the transmembrane segment has an alpha-helical structure with three distinct faces. Cross-linking of the faces to transmembrane segment 1 (TM1) mimics the "locked" signalling phenotypes of the wild-type receptor. The results are consistent with a model in which TM2 rotates in the plane of the lipid bilayer, and the rotation becomes locked at one face of the alpha-helix in the presence of attractant and at another face in the presence of repellent. This dynamic movement of the transmembrane domain may be a common signalling mechanism of homologous membrane receptor molecules such as the insulin receptor. Random-cassette mutagenesis and disulfide cross-linking provide powerful strategies for examining the structure and function of transmembrane segments.
细胞外信息通过跨膜受体穿过膜屏障传递到细胞内部。为了理解跨膜信号传导的分子基础，我们用长度为21个氨基酸残基的随机序列替换了大肠杆菌天冬氨酸受体Tar的跨膜片段2（TM2），每个位置由精氨酸、甘氨酸、丝氨酸、半胱氨酸、缬氨酸、亮氨酸、异亮氨酸和苯丙氨酸组成。从这个重组分子集合中，回收了功能性受体克隆，它们能够结合天冬氨酸并将信号传递到细胞内结构域。观察到功能性跨膜结构域的受限平均疏水性值，支持了跨膜片段通常具有大于1.6的疏水性值这一观察结果。然而，疏水性大于1.6的非功能性跨膜结构域表明疏水性不是其功能的唯一决定因素。对功能性TM2序列的傅里叶变换分析表明，跨膜片段具有带有三个不同面的α螺旋结构。这些面与跨膜片段1（TM1）的交联模拟了野生型受体的“锁定”信号表型。结果与一个模型一致，即TM2在脂质双层平面内旋转，并且在存在吸引剂时旋转在α螺旋的一个面上锁定，在存在驱避剂时在另一个面上锁定。跨膜结构域的这种动态运动可能是胰岛素受体等同源膜受体分子的常见信号传导机制。随机盒式诱变和二硫键交联为研究跨膜片段的结构和功能提供了有力策略。
