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′-(γ-thio) triphosphate (GTPγS) binding and G16-dependent inositol phosphate accumulation assays. The i3-1 mutant, with a mutation at the i3 amino terminus, exhibited greatly reduced GTPγS 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. 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′-(γ-thio) triphosphate (GTPγS) binding and G16-dependent inositol phosphate accumulation assays. The i3-1 mutant, with a mutation at the i3 amino terminus, exhibited greatly reduced GTPγS 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. Leukotriene B4 (LTB4) 3The abbreviations used are: LTB4, leukotriene B4; GPCR, G-protein-coupled receptor; BLT1, leukotriene B4 receptor type 1; GTPγS, guanosine 5′-(γ-thio) triphosphate; IP, inositol phosphate; HA, hemagglutinin; HEK293, human embryonic kidney 293 cell line; CHO, Chinese hamster ovary cell line; BSA, bovine serum albumin; PTX, pertussis toxin; WT, wild type. is an inflammatory lipid mediator biosynthesized from membrane phospholipids by sequential enzymatic reactions (1Samuelsson B. Funk C.D. J. Biol. Chem. 1989; 264: 19469-19472Abstract Full Text PDF PubMed Google Scholar, 2Shimizu T. Wolfe L.S. J. Neurochem. 1990; 55: 1-15Crossref PubMed Scopus (534) Google Scholar, 3Zeldin D.C. J. Biol. Chem. 2001; 276: 36059-36062Abstract Full Text Full Text PDF PubMed Scopus (543) Google Scholar, 4Haeggstrom J.Z. J. Biol. Chem. 2004; 279: 50639-50642Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). 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 (5Luster A.D. Tager A.M. Nat. Rev. Immunol. 2004; 4: 711-724Crossref PubMed Scopus (175) Google Scholar, 6Jala V.R. Haribabu B. Trends Immunol. 2004; 25: 315-322Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). These actions are mediated by two types of G-protein-coupled receptors (GPCRs). One is the LTB4 receptor type-1 (BLT1), a high affinity LTB4 receptor highly expressed in leukocytes (7Yokomizo T. Izumi T. Chang K. Takuwa Y. Shimizu T. Nature. 1997; 387: 620-624Crossref PubMed Scopus (853) Google Scholar). The other, BLT2, is a low affinity LTB4 receptor expressed more ubiquitously than BLT1 in human tissues (8Tryselius Y. Nilsson N.E. Kotarsky K. Olde B. Owman C. Biochem. Biophys. Res. Commun. 2000; 274: 377-382Crossref PubMed Scopus (56) Google Scholar, 9Kamohara M. Takasaki J. Matsumoto M. Saito T. Ohishi T. Ishii H. Furuichi K. J. Biol. Chem. 2000; 275: 27000-27004Abstract Full Text Full Text PDF PubMed Google Scholar, 10Wang S. Gustafson E. Pang L. Qiao X. Behan J. Maguire M. Bayne M. Laz T. J. Biol. Chem. 2000; 275: 40686-40694Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 11Yokomizo T. Kato K. Terawaki K. Izumi T. Shimizu T. J. Exp. Med. 2000; 192: 421-432Crossref PubMed Scopus (470) Google Scholar). Many GPCRs can activate more than one G-protein subfamily member. For example, the prostaglandin E2 receptor EP3D couples to Gs and Go (12Negishi M. Namba T. Sugimoto Y. Irie A. Katada T. Narumiya S. Ichikawa A. J. Biol. Chem. 1993; 268: 26067-26070Abstract Full Text PDF PubMed Google Scholar), platelet-activating factor receptor activates Gi and Gq (13Shi L.C. Wang H.Y. Horwitz J. Friedman E. J. Neurochem. 1996; 67: 1478-1484Crossref PubMed Scopus (21) Google Scholar, 14Ishii S. Shimizu T. Prog. Lipid Res. 2000; 39: 41-82Crossref PubMed Scopus (329) Google Scholar), and the sphingosine 1-phosphate receptors S1P1 and S1P2 activate Gi and G12/13 (15Sugimoto N. Takuwa N. Okamoto H. Sakurada S. Takuwa Y. Mol. Cell Biol. 2003; 23: 1534-1545Crossref PubMed Scopus (234) Google Scholar). Previous reports suggest that in mediating different cellular events, BLT1 also utilizes different G-proteins, namely pertussis toxin (PTX)-sensitive Gi/o and PTX-resistant Gq subfamilies of G-proteins (7Yokomizo T. Izumi T. Chang K. Takuwa Y. Shimizu T. Nature. 1997; 387: 620-624Crossref PubMed Scopus (853) Google Scholar, 16Miki I. Watanabe T. Nakamura M. Seyama Y. Ui M. Sato F. Shimizu T. Biochem. Biophys. Res. Commun. 1990; 166: 342-348Crossref PubMed Scopus (22) Google Scholar). Promiscuous G-protein coupling enables GPCRs to mediate diverse signals. The molecular determinants of many receptors participating in G-protein coupling and activation have been investigated. With some exceptions, most of these studies emphasize the role of membrane-proximal regions of the second and third intracellular loops and of the cytoplasmic tail of the receptor (17Hermans E. Pharmacol. Ther. 2003; 99: 25-44Crossref PubMed Scopus (255) Google Scholar). However, no consensus sequences for G-protein coupling have been clearly identified. We and others have reported that truncation of its cytoplasmic tail does not impair the ability of BLT1 to activate G-proteins (18Gaudreau R. Beaulieu M.E. Chen Z. Le Gouill C. Lavigne P. Stankova J. Rola-Pleszczynski M. J. Biol. Chem. 2004; 279: 10338-10345Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 19Okuno T. Ago H. Terawaki K. Miyano M. Shimizu T. Yokomizo T. J. Biol. Chem. 2003; 278: 41500-41509Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). To identify regions of BLT1 responsible for G-protein coupling, we performed an extensive mutagenesis study of its intracellular domains. Here, we show that BLT1 distinguishes Gi from G16 at the amino terminus of the third intracellular region (the i3-1 region). Furthermore, Scatchard analyses of the i3-1 mutant revealed that BLT1 requires Gi coupling to maintain a high affinity state for LTB4. A three-dimensional 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. Materials—cDNAs encoding Gαi1, Gαi2, Gαi3, Gαq, Gα11, Gα14, and Gα16 were obtained from the Guthrie cDNA Resource Center. LTB4 was purchased from Cayman Chemical (Ann Arbor, MI). [3H]LTB4 and [35S]GTPγS were from PerkinElmer Life Sciences. myo-[3H]Inositol was from Amersham Biosciences. Anti-hemagglutinin (HA) antibody (clone 3F10) was from Roche Applied Science (Penzberg, Germany), polyclonal anti-BLT1 was from Cayman, goat anti-rat IgG-horseradish peroxidase was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and ECL™ anti-rabbit IgG horseradish peroxidase-linked whole antibody was from Amersham Biosciences. Mutagenesis—Using pcDNA3-HA-tagged human BLT1 (19Okuno T. Ago H. Terawaki K. Miyano M. Shimizu T. Yokomizo T. J. Biol. Chem. 2003; 278: 41500-41509Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) as a template, tandem alanine BLT1 mutants were generated using the QuikChange® site-directed mutagenesis kit (Stratagene, La Jolla, CA). The following primers were used for the mutagenesis: i1-1S (5′-ctttgtggtgtggagtgccgcggcagcgatgcagaagcgctc-3′), i1-1AS (5′-gagcgcttctgcatcgctgccgcggcactccacaccacaaag-3′), i1-2S (5′-gtatcctgaaaagggcggcggcggcctctgtcactgccctg-3′), i1-2AS (5′-cagggcagtgacagaggccgccgccgcccttttcaggatac-3′), i1-3S (5′-ggatgcagaagcgcgctgccgctgccctgatggtgctgaac-3′), i1-3AS (5′-gttcagcaccatcagggcagcggcagcgcgcttctgcatcc-3′), i2-1S (5′-ggccatgagtctagccgccg-3′), i2-1AS (5′-cgggccaccgccgctgcggcggct-3′), i2-2S (5′-ctcactggcggcggccgccccctttgtgtc-3′), i2-2AS (5′-gacacaaagggggcggccgccgccagtgag-3′), i2-3S (5′-gcggtggcccgcgccgctgcggcccagaagctacgc-3′), i2-3AS (5′-gcgtagcttctgggccgcagcggcgcgggccaccgc-3′), i2-4S (5′-cgcccctttgtgtccgcggcggcagccaccaaggcgatggcc-3′), i2-4AS (5′-ggccatcgccttggtggctgccgccgcggacacaaaggggcg-3′), i2-5S (5′-cagaagctacgcgccgcggcggcggcccggcgggtg-3′), i2-5AS (5′-cacccgccgggccgccgccgcggcgcgtagcttctg-3′), i3-0S (5′-gctgtggtggccgccgccgccgacatagggcgt-3′), i3-0A (5′-acgccctatgtcggcggcggcggccaccacagc-3′), i3-1S (5′-ggccagctactcggccgcagcggctcggctacaggcc-3′), i3-1AS (5′-ggcctgtagccgagccgctgcggccgagtagctggcc-3′), i3-2S (5′-ggacatagggcgtgcggcagcggcccggcgcttccgcc-3′), i3-2AS (5′-ggcggaagcgccgggccgctgccgcacgccctatgtcc-3′), i3-3S (5′-cgtcggctacaggccgcggccgccgcccgcagccgccgcacc-3′), i3-3AS (5′-ggtgcggcggctgcgggcggcggccgcggcctgtagccgacg-3′), i3-4S (5′-gcccggcgcttccgcgccgccgccgccaccggccgcctggtg-3′), i3-4AS Cell and embryonic kidney 293 and were in and Chinese hamster ovary were in and at in These were with a wild type BLT1 using to the For were with antibody in at for by with anti-rat IgG antibody at for highly and mutant BLT1 were as polyclonal by cell using an and in cell were by the was used for and were to were with some were with for were in a and at for at were at for 1 at and the were in and used as the membrane The in was by the were on and to a with in were with the antibody for 1 The membrane was with and with horseradish antibody for 1 and the was using an membrane of was in of LTB4 binding and bovine serum and of To determine of LTB4 at than were were for 1 at by and with of LTB4 binding was using a GTPγS membrane of was in of GTPγS binding and [35S]GTPγS with of LTB4 for at To determine GTPγS was to the binding to a of [35S]GTPγS was from by and with of and was as were at in a the following were with A with A for at and with a and of LTB4 for at The was by the of in the were by an kit Life were at 1 in a and with BLT1 G-protein on the following was with 1 by for were and for in and by with of LTB4 for was by the with and on for was by and was by R. Le Gouill C. S. S. Stankova J. Rola-Pleszczynski M. Biochem. J. PubMed Scopus Google Scholar, K. Ishii S. K. Yokomizo T. T. Shimizu T. K. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). with were with in for The was in a LTB4 in the BLT1 were at in the at in for on the were with and using a kit The of the was of the of to the was by at using a were for using of using CA). were at as BLT1 on human BLT1 model was constructed based on that of bovine rhodopsin with as a rhodopsin subfamily of GPCRs K. T. T. H. Le I. D.C. T. M. Miyano M. 2000; PubMed Scopus Google Scholar). The amino sequences of transmembrane were to the transmembrane in the F. Res. 2001; PubMed Scopus Google Scholar). S. J. PubMed Scopus Google Scholar) was used to amino to the from bovine rhodopsin to BLT1 on the model of the bovine rhodopsin K. T. T. H. Le I. D.C. T. M. Miyano M. 2000; PubMed Scopus Google Scholar). with molecular using P. J. M. T. Biol. PubMed Scopus Google Scholar) was to the BLT1 The BLT1 model constructed was by were using CA). determine regions of human BLT1 responsible for Gi and Gq we performed extensive mutagenesis on the intracellular loops. Three to tandem amino in regions (i1, i2, and i3) were with by using human BLT1 as a We constructed mutants to to and i3-1 to A and of these receptors on the cell were by as with BLT1 mutants were with antibody to the and BLT1 and mutant receptors were expressed on the cell from the of of the BLT1 and mutants with the determine which Gi to BLT1, we of by a GTPγS binding using membrane of BLT1 and G-protein of BLT1 G-proteins only a in GTPγS binding of LTB4 BLT1 was with of the Gi family of G-protein GTPγS binding were of Gα16 not show such an not a in GTPγS Moreover, GTPγS binding was by the with to membrane These results suggest that GTPγS binding is an to activation of Gi by i3-1 region only in Gi and not G16 binding was performed with a membrane from and BLT1 the i3-1 mutant accumulation was performed using Gα16 and mutant shown are of at two with The alanine mutants were expressed in with and for of by the GTPγS binding mutants GTPγS binding than WT, but the and mutants exhibited reduced GTPγS binding These results that i2, and are for Gi to determine activation of the Gq family of G-proteins by mutant receptors is we accumulation in both BLT1 and reported by R. Le Gouill C. S. S. Stankova J. Rola-Pleszczynski M. Biochem. J. PubMed Scopus Google Scholar), both BLT1 and Gα16 show accumulation than BLT1 BLT1 mutants were in with Gα16 and for ability to activate Gα16 by the accumulation the mutants that GTPγS only the i3-1 mutant accumulation The and mutants exhibited accumulation than these results suggested that the i3-1 region is responsible for we constructed mutant in the more membrane-proximal region of the i3 with the i3-1 mutant, the mutant BLT1 accumulation but reduced GTPγS binding LTB4 and These results show that the amino terminus of the i3 is important for activation. of the i3-1 on and G16 an in GTPγS binding in a in with BLT1 and Gαi2, but no were seen with the i3-1 mutant in GTPγS binding mediated by LTB4 following of with These results suggest that the i3-1 region in activation. However, in were BLT1 and the i3-1 mutant in the of Gα16 in and not accumulation These results the that the i3-1 region of BLT1 to in activation PTX-resistant G16 To into the role of the i3-1 region in we polyclonal BLT1 the i3-1 highly the i3-1 mutant receptor were by cell The of i3-1 mutant was to that of BLT1, as judged from and analyses A and the of GTPγS binding with the i3-1 mutant was only one that of BLT1, and the i3-1 mutant GTPγS binding in a The of LTB4 with the i3-1 mutant was to the from that seen with BLT1 and of in the i3-1 mutant and BLT1 receptors were and been reported that requires activation of Gi and a of H. S. A. 1997; PubMed Scopus Google Scholar) and that is (7Yokomizo T. Izumi T. Chang K. Takuwa Y. Shimizu T. Nature. 1997; 387: 620-624Crossref PubMed Scopus (853) Google Scholar). both and the i3-1 mutant BLT1 to LTB4 with the i3-1 mutant to low of LTB4 However, accumulation in and the i3-1 mutant BLT1 with Gα16 were These results the of both Gi activation and G16 activation by the i3-1 mutant in the of the i3-1 on [3H]LTB4 the of the i3-1 mutation on we performed [3H]LTB4 binding of from BLT1 the i3-1 mutant were with of [3H]LTB4 From Scatchard BLT1 high affinity LTB4 binding with a of and of the i3-1 mutant low affinity LTB4 binding with a of and a of To Gi coupling is required for high affinity LTB4 we LTB4 binding of membrane of with only low affinity LTB4 binding to the i3-1 mutant Cell the the of with the i3-1 mutant was than that in with BLT1 To that the in Gi in the i3-1 mutant were not to of the mutant we cell of BLT1 and and the i3-1 mutant and and and shown in A and the LTB4 binding revealed that for and of and The were and of the i3-1 mutants and were to the by 1 order of with BLT1 and the i3-1 mutant was expressed than the BLT1 and the of Gi-dependent in the i3-1 mutant were than that in BLT1 and However, a G16-dependent accumulation was mutant receptors These results the Gi activation and G16 activation. mutagenesis we show that intracellular loops of BLT1 in G-protein activation and these we the i3-1 region as a selective activation for Gi The i3-1 mutant Gi activation but G16 activation and The i3-1 region is at the amino terminus of the third intracellular of Previous reports suggest that the i3 region of GPCRs in G-protein activation M. M. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar, A. Wang L. PubMed Scopus Google Scholar, T. K. Y. Y. S. M. H. T. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). show that the i3-1 region of BLT1 is required for Gi activation. We used different cell for the different in the of mutant We used for GTPγS binding BLT1 GTPγS binding than We used for the accumulation R. Le Gouill C. S. S. Stankova J. Rola-Pleszczynski M. Biochem. J. PubMed Scopus Google Scholar) reported that with BLT1 and Gα16 are used for are in intracellular and cellular events, such as and these we that the i3-1 mutant BLT1 exhibited Gi activation but intact G16 activation. activation as by GTPγS binding was in the i3-1 mutant in and and and of and was also in the i3-1 mutant in and However, accumulation in not and the i3-1 mutant was These results show that Gi activation by the i3-1 mutant is not cell We also the the i3-1 mutant BLT1 and performed of were to the in both high and low of the i3-1 mutant with BLT1 However, intact in the We that Gi activation by the i3-1 mutant is not on the of the receptor. The binding studies that BLT1 exhibited high affinity for LTB4 and A and The i3-1 mutants and BLT1 only a low affinity binding with reported that LTB4 binding was by with T. Yokomizo T. Y. Shimizu T. Izumi T. J. Biochem. PubMed Scopus Google Scholar). with LTB4 binding most J. J. Mol. Biol. 2003; PubMed Scopus (255) Google Scholar) that in the of BLT1 a high affinity binding using in E. K. H. T. C. K. R. Yokomizo T. Shimizu T. T. T. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar) reported that BLT1 exhibited high affinity LTB4 BLT1 exhibited low affinity binding in a on These results and suggest that Gi coupling enables BLT1 to maintain a high affinity BLT1 to G16 a but only a low affinity studies suggest the of high and low affinity binding for LTB4 in and J. Immunol. Full Text PDF PubMed Scopus Google Scholar, R. J. Pharmacol. Exp. Ther. Google Scholar, R. K. J. Pharmacol. 1996; PubMed Scopus Google Scholar). of low affinity binding be by the of BLT2, which a low affinity LTB4 receptor (8Tryselius Y. Nilsson N.E. Kotarsky K. Olde B. Owman C. Biochem. Biophys. Res. Commun. 2000; 274: 377-382Crossref PubMed Scopus (56) Google Scholar, 9Kamohara M. Takasaki J. Matsumoto M. Saito T. Ohishi T. Ishii H. Furuichi K. J. Biol. Chem. 2000; 275: 27000-27004Abstract Full Text Full Text PDF PubMed Google Scholar, 10Wang S. Gustafson E. Pang L. Qiao X. Behan J. Maguire M. Bayne M. Laz T. J. Biol. Chem. 2000; 275: 40686-40694Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 11Yokomizo T. Kato K. Terawaki K. Izumi T. Shimizu T. J. Exp. Med. 2000; 192: 421-432Crossref PubMed Scopus (470) Google Scholar), human not not BLT1 might be responsible for low affinity binding of LTB4 in The of the of low and high affinity binding in BLT1 to be and J. J. Mol. Biol. 2003; PubMed Scopus (255) Google Scholar) clearly that BLT1 was in a low affinity binding state in a of the of G-protein binding a high affinity binding also that BLT1 with G-protein and by using from the in that the i3-1 mutant might be to form a more studies be required to in To into the molecular the i3-1 region of BLT1 Gi we constructed a BLT1 three-dimensional model based on the structure of bovine rhodopsin (19Okuno T. Ago H. Terawaki K. Miyano M. Shimizu T. Yokomizo T. J. Biol. Chem. 2003; 278: 41500-41509Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, K. T. T. H. Le I. D.C. T. M. Miyano M. 2000; PubMed Scopus Google Scholar). This model suggests that the i3-1 region might localize at the cytoplasmic end of helix V, not in the i3 of based on The structure of bovine rhodopsin that the cytoplasmic of helix V and are extended the membrane in two different and these extended regions are highly to at more than K. T. T. H. Le I. D.C. T. M. Miyano M. 2000; PubMed Scopus Google Scholar, J. M. C. J. Mol. Biol. 2004; PubMed Scopus Google Scholar). also been suggested that region of helix V and the membrane-proximal region of helix in activation of a region of M. M. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar, K. C. 1996; PubMed Scopus Google Scholar, J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar). We propose that the BLT1 i3-1 region with Gi may consist of the end of helix V in the alanine in the which amino of the i3-1 in Gi activation but intact G16 as was seen with the i3-1 mutant and of the to terminus of the i3 region of the receptor 1 suggested of an in the with an for activation of by the receptor A. Wang L. PubMed Scopus Google Scholar). on the structure of H. K. Sato K. T. T. PubMed Scopus Google Scholar) and M. Full Text Full Text PDF PubMed Scopus Google Scholar, Z. R. J. Full Text Full Text PDF PubMed Scopus Google Scholar), might a of by which a for the i3-1 region of BLT1 and and with an with and for the coupling to G-protein M. Full Text Full Text PDF PubMed Scopus Google Scholar). the region also two and The of GPCRs based on bovine rhodopsin have been for and into molecular of structure of GPCRs is not of model and have the molecular of structure such as binding and of helix (18Gaudreau R. Beaulieu M.E. Chen Z. Le Gouill C. Lavigne P. Stankova J. Rola-Pleszczynski M. J. Biol. Chem. 2004; 279: 10338-10345Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 19Okuno T. Ago H. Terawaki K. Miyano M. Shimizu T. Yokomizo T. J. Biol. Chem. 2003; 278: 41500-41509Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, S. T. Y. M. K. J. 25: PubMed Scopus Google Scholar). The i3 regions of BLT1 and bovine rhodopsin can be using the C. J. J. Mol. Biol. 2000; PubMed Scopus Google Scholar). The i3 region of BLT1 is than of bovine the of modeling, the model of region is to than that of the the three-dimensional model of the i3 region of BLT1 to some more are required to the in the GPCRs and G-proteins using BLT1 as a These suggest that BLT1 couples to both Gi and G16 using intracellular region but that the i3-1 region only in Gi coupling, not in G16 The i3-1 mutant G16 to Gi and a low affinity LTB4 binding state to in Gi This mutant receptor be in mediated by G16 BLT1 and the of low and high affinity LTB4 binding to We propose that the helical structure of GPCRs as a for activation of Gi proteins. of the roles of the cytoplasmic end of helix V of GPCRs than BLT1 in for coupling to G-proteins be We S. M. Y. H. and for and with