Department of Medical Biochemistry, Graduate School of Medical Sciences, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan.
FASEB J. 2012 Oct;26(10):4068-78. doi: 10.1096/fj.12-212050. Epub 2012 Jun 15.
Recent crystallographic studies revealed that G-protein-coupled receptors (GPCRs) possess a putative cytoplasmic helical domain, termed helix 8, at the proximal region of the C-terminal tail. However, the significance of helix 8 in GPCR functions and signaling is not fully understood. Helix 8 mutants of leukotriene B4 receptor type 1 (BLT1) exhibit prolonged activation after ligand stimulation, suggesting some regulatory roles of helix 8 in GPCR signaling. Here, we report the inhibitory role of BLT1 helix 8 on ligand-dependent internalization using BLT1 and platelet-activating factor receptor as model GPCRs. Mutating the dileucine motif in helix 8 of BLT1 to alanines (BLT1 LLAA) enhanced LTB4-dependent internalization of BLT1, whereas wild-type (WT) BLT1 exhibited minimal internalization. Mutational studies revealed that phosphorylation of 5 serine/threonine residues between amino acids 308 and 319 of BLT1 was responsible for enhanced ligand-dependent internalization of BLT1 LLAA. BLT1 LLAA showed enhanced basal and ligand-dependent phosphorylation compared to WT BLT1. Taken together, helix 8 of BLT1 inhibits receptor internalization by suppressing the excessive phosphorylation of the C-terminal tail.
最近的晶体学研究表明，G 蛋白偶联受体（GPCR）在 C 端尾部的近端区域具有一个假定的细胞质螺旋域，称为螺旋 8。然而，螺旋 8在 GPCR 功能和信号转导中的意义尚不完全清楚。白三烯 B4 受体 1（BLT1）的螺旋 8 突变体在配体刺激后表现出延长的激活，表明螺旋 8 在 GPCR 信号转导中具有一些调节作用。在这里，我们报告了使用 BLT1 和血小板激活因子受体作为模型 GPCR，BLT1 螺旋 8 在配体依赖性内化中的抑制作用。将 BLT1 螺旋 8 中的双亮氨酸基序突变为丙氨酸（BLT1 LLAA）增强了 LTB4 依赖性 BLT1 内化，而野生型（WT）BLT1 则表现出最小的内化。突变研究表明，BLT1 中氨基酸 308 和 319 之间的 5 个丝氨酸/苏氨酸残基的磷酸化负责增强 BLT1 LLAA 的配体依赖性内化。与 WT BLT1 相比，BLT1 LLAA 显示出增强的基础和配体依赖性磷酸化。总之，BLT1 的螺旋 8 通过抑制 C 端尾部的过度磷酸化来抑制受体内化。
