Jadot M, Canfield W M, Gregory W, Kornfeld S
Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110.
J Biol Chem. 1992 Jun 5;267(16):11069-77.
The signal for rapid internalization of the mannose 6-phosphate/insulin-like growth factor II receptor has been localized to the sequence Tyr-Lys-Tyr-Ser-Lys-Val in positions 24-29 of its 163-residue cytoplasmic tail. Most of the activity of this signal is mediated by the carboxyl 4 amino acids, especially Tyr26 and Val29 (Canfield, W. M., Johnson, K. F., Ye, R. D., Gregory, W. and Kornfeld, S. (1991) J. Biol. Chem. 266, 5682-5688). In this study, we have tested the effect of a series of mutations on the internalization rate of a mutant receptor that contains a 29-amino acid cytoplasmic tail terminating with the 4-amino acid internalization sequence Tyr-Ser-Lys-Val. Replacement of Tyr26 with Phe or Trp gave rise to mutant receptors that were internalized at 10% the wild-type rate, while receptors with Ala, Leu, Ile, Val, or Asn at this position were totally inactive. Val29 could be replaced by other large hydrophobic residues (Phe, Leu, Ile, or Met) with no loss of activity, but the presence of Ala, Gly, Arg, Gln, or Tyr in this position inactivated the signal. Ser27 could be effectively replaced by many different amino acids, but not by Pro or Gly. However, Gly27 could be tolerated if the residues at positions 28 and 29 were also changed. A change in the 2-residue spacing between Tyr26 and Val29 destroyed the signal. These data show that the essential elements of this signal are an aromatic residue, especially a Tyr in the first position, separated from a large hydrophobic residue in the last position by 2 amino acids. The residues in positions 2 and 3 of the signal may have a modulating effect on its activity. The Tyr-Ser-Lys-Val signal could be moved to a more proximal region of the cytoplasmic tail with only a modest loss of activity. In addition, the signal could be effectively replaced by the putative 4-residue signals of seven other receptors and membrane proteins known to undergo rapid endocytosis, including the Tyr-Thr-Arg-Phe sequence of the transferrin receptor, a Type II membrane protein. These results are compatible with the 4-residue signals of this type being interchangeable, even among Type I and Type II membrane proteins.
甘露糖6 - 磷酸/胰岛素样生长因子II受体快速内化的信号已定位到其163个残基的胞质尾第24 - 29位的Tyr - Lys - Tyr - Ser - Lys - Val序列。该信号的大部分活性由羧基端的4个氨基酸介导，尤其是Tyr26和Val29（坎菲尔德，W.M.，约翰逊，K.F.，叶，R.D.，格雷戈里，W.和科恩菲尔德，S.（1991年）《生物化学杂志》266，5682 - 5688）。在本研究中，我们测试了一系列突变对一种突变受体内化速率的影响，该突变受体含有一个29个氨基酸的胞质尾，末端为4个氨基酸的内化序列Tyr - Ser - Lys - Val。将Tyr26替换为Phe或Trp会产生内化速率仅为野生型10%的突变受体，而该位置为Ala、Leu、Ile、Val或Asn的受体则完全无活性。Val29可被其他大的疏水残基（Phe、Leu、Ile或Met）取代而不丧失活性，但该位置存在Ala、Gly、Arg、Gln或Tyr会使信号失活。Ser27可被许多不同氨基酸有效取代，但不能被Pro或Gly取代。然而，如果第28和29位的残基也发生变化，则Gly27是可以耐受的。Tyr26和Val29之间2个残基间距的改变会破坏信号。这些数据表明，该信号的基本要素是一个芳香族残基，尤其是第一位的Tyr，与最后一位的大疏水残基之间相隔2个氨基酸。信号第2和3位的残基可能对其活性有调节作用。Tyr - Ser - Lys - Val信号可以移至胞质尾更近端的区域，活性仅略有损失。此外，该信号可被已知经历快速内吞作用的其他七种受体和膜蛋白的假定4个残基信号有效取代，包括转铁蛋白受体（一种II型膜蛋白）的Tyr - Thr - Arg - Phe序列。这些结果与这种类型的4个残基信号即使在I型和II型膜蛋白之间也可互换是一致的。
