Helix 8 of leukotriene B4 receptor 1 inhibits ligand-induced internalization.
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.
Department of Medical Biochemistry, Graduate School of Medical Sciences, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan.
Yoshifusa
Y
Aratake
Toshiaki
T
Okuno
Takehiko
T
Matsunobu
Kazuko
K
Saeki
Ryoichi
R
Takayanagi
Sonoko
S
Furuya
Takehiko
T
Yokomizo
D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D006224:Cricetinae; D004705:Endocytosis / Q000235:genetics / Q000502:physiology*; D047468:Immunoprecipitation; D007975:Leukotriene B4 / Q000378:metabolism*; D018613:Microscopy, Confocal; D016253:Microscopy, Immunoelectron; D010766:Phosphorylation; D011485:Protein Binding / Q000235:genetics / Q000502:physiology; D017433:Protein Structure, Secondary; D018102:Receptors, Leukotriene B4 / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship
C513240:LTB4R protein, human; D018102:Receptors, Leukotriene B4; D007975:Leukotriene B4