Abstract
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.
Highlights
Leukotriene B4 (LTB4)3 is an inflammatory lipid mediator biosynthesized from membrane phospholipids by sequential enzymatic reactions [1,2,3,4]
wild type (WT) BLT1 and mutant receptors were expressed on the cell surface, judging from the increased means of fluorescence intensity of the WT BLT1 and mutants compared with the mock transfectant (Fig. 1C)
BLT1/G␣ Coupling—To determine which Gi protein subtypes couple to BLT1, we examined LTB4-induced GDP-GTP exchange of G␣ by a guanosine 5-(␥-thio)triphosphate (GTP␥S) binding assay using membrane fractions of HEK293 cells transiently expressing BLT1 and various G-protein ␣ subunits
Summary
Leukotriene B4 (LTB4) is an inflammatory lipid mediator biosynthesized from membrane phospholipids by sequential enzymatic reactions [1,2,3,4]. LTB4 recruits and activates leukocytes, such as granulocytes, macrophages, monocytes, and T-cells, and plays pivotal roles in host defense and the pathogenesis of inflammatory and immune diseases [5, 6]. These actions are mediated by two types of G-protein-coupled receptors (GPCRs). A Region of BLT1 Responsible for Gi Activation sional model of BLT1 suggested that the i3-1 region might localize to the cytoplasmic end of the transmembrane helix V. This region is suggested to form an extended helical structure proximate to the membrane, which projects toward the cytoplasm in order to interact with Gi
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
