Identification of the intracellular region of the leukotriene B4 receptor type 1 that is specifically involved in Gi activation.
Many G-protein-coupled receptors can activate more than one G-protein subfamily member. Leukotriene B4 receptor type 1 (BLT1) is a high affinity G-protein-coupled receptors for leukotriene B4 functioning in host defense, inflammation, and immunity. Previous studies have shown that BLT1 utilizes different G-proteins (the Gi family and G16 G-proteins) in mediating diverse cellular events and that truncation of the cytoplasmic tail of BLT1 does not impair activation of Gi and G16 proteins. To determine responsive regions of BLT1 for G-protein coupling, we performed an extensive mutagenesis study of its intracellular loops. Three intracellular loops (i1, i2, and i3) of BLT1 were found to be important for both Gi and G16 coupling, as judged by Gi-dependent guanosine 5'-(gamma-thio) triphosphate (GTPgammaS) binding and G16-dependent inositol phosphate accumulation assays. The i3-1 mutant, with a mutation at the i3 amino terminus, exhibited greatly reduced GTPgammaS binding but intact inositol phosphate accumulation triggered by leukotriene B4 stimulation. These results suggest that the i3-1 region is required only for Gi activation. Moreover, in the i3-1 mutant, the deficiency in Gi activation was accompanied by a loss of the high affinity leukotriene B4 binding state seen with the wild type receptor. A three-dimensional model of BLT1 constructed based on the structure of bovine rhodopsin suggests that the i3-1 region may consist of the cytoplasmic end of the transmembrane helix V, which protrudes the helix into the cytoplasm. From mutational studies and three-dimensional modeling, we propose that the extended cytoplasmic helix connected to the transmembrane helix V of BLT1 might be a key region for selective activation of Gi proteins.
Kuniyeda
Kanako
K
Department of Biochemistry and Molecular Biology, Faculty of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Okuno
Toshiaki
T
Terawaki
Kan
K
Miyano
Masashi
M
Yokomizo
Takehiko
T
Shimizu
Takao
T
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D006224:Cricetinae; D003412:Cricetulus; D003593:Cytoplasm / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D006801:Humans; D007424:Intracellular Fluid / Q000737:chemistry* / Q000502:physiology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D018102:Receptors, Leukotriene B4 / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D015398:Signal Transduction / Q000235:genetics
C513240:LTB4R protein, human; D018102:Receptors, Leukotriene B4; D019206:GTP-Binding Protein alpha Subunits, Gi-Go