Structural Determinants Regulating Expression of the High Affinity Leukotriene B4 Receptor
2004; Elsevier BV; Volume: 279; Issue: 11 Linguagem: Inglês
10.1074/jbc.m309207200
ISSN1083-351X
AutoresRémi Gaudreau, Marie‐Eve Beaulieu, Zhangguo Chen, Christian Le Gouill, Pierre Lavigne, Jana Staňková, Marek Rola‐Pleszczynski,
Tópico(s)Monoclonal and Polyclonal Antibodies Research
ResumoMutational analysis of determinants located in the C-terminal (C) tail of the high affinity leukotriene (LT) B4 receptor, BLT1, was performed to assess their significance in BLT1 trafficking. When expressed in COS-7 cells, a BLT1 deletion mutant lacking the C-tail (G291stop) displayed higher numbers of binding sites and increased signal transduction compared with wild-type (WT) BLT1. Addition of the C-tail from either the platelet-activating factor receptor or the LTD4 receptor, CysLT1, did not restore WT phenotype. Moreover, the number of LTB4 binding sites was higher in the chimeras than in the WT BLT1, suggesting the requirement for specific structural determinants within the BLT1 C-tail. Elimination of a distal C-tail dileucine motif (Leu304-Leu305), but not the proximal (Leu292-Leu293) motif, altered BLT1 pharmacological characteristics and caused a moderate constitutive receptor activation. Surprisingly, all mutant receptors were efficiently delivered to the plasma membrane, but not to a greater extent than WT BLT1, as assessed by flow cytometry. Furthermore, substitution of Leu304-Leu305 prevented LTB4-induced BLT1 internalization. Molecular modeling of BLT1 on the bovine rhodopsin receptor scaffold strongly suggested the involvement of the distal dileucine motif (Leu304-Leu305) in a hydrophobic core, including intrahelical interactions within α-helix VIII and interhelical interactions with residues of helix I. Disruption of this hydrophobic core is proposed to increase the population of receptors in the active form, to restrain their trafficking and to facilitate the activation of BLT1 as indicated by the increased maximal level of binding of the ligand and constitutive activation of the receptor. Mutational analysis of determinants located in the C-terminal (C) tail of the high affinity leukotriene (LT) B4 receptor, BLT1, was performed to assess their significance in BLT1 trafficking. When expressed in COS-7 cells, a BLT1 deletion mutant lacking the C-tail (G291stop) displayed higher numbers of binding sites and increased signal transduction compared with wild-type (WT) BLT1. Addition of the C-tail from either the platelet-activating factor receptor or the LTD4 receptor, CysLT1, did not restore WT phenotype. Moreover, the number of LTB4 binding sites was higher in the chimeras than in the WT BLT1, suggesting the requirement for specific structural determinants within the BLT1 C-tail. Elimination of a distal C-tail dileucine motif (Leu304-Leu305), but not the proximal (Leu292-Leu293) motif, altered BLT1 pharmacological characteristics and caused a moderate constitutive receptor activation. Surprisingly, all mutant receptors were efficiently delivered to the plasma membrane, but not to a greater extent than WT BLT1, as assessed by flow cytometry. Furthermore, substitution of Leu304-Leu305 prevented LTB4-induced BLT1 internalization. Molecular modeling of BLT1 on the bovine rhodopsin receptor scaffold strongly suggested the involvement of the distal dileucine motif (Leu304-Leu305) in a hydrophobic core, including intrahelical interactions within α-helix VIII and interhelical interactions with residues of helix I. Disruption of this hydrophobic core is proposed to increase the population of receptors in the active form, to restrain their trafficking and to facilitate the activation of BLT1 as indicated by the increased maximal level of binding of the ligand and constitutive activation of the receptor. A variety of stimuli, including light, neurotransmitters, hormones, and inflammatory lipid mediators produce their effects via activation of G-protein-coupled receptors (GPCRs). 1The abbreviations used are: GPCRs, G-protein-coupled receptors; BLT1, leukotriene B4 receptor; CMV, cytomegalovirus; G-protein, GTP-binding regulatory protein; GRK, GPCR kinase; HEK, human embryonic kidney; IP, inositol phosphate(s); LTB4, leukotriene B4; PI, phosphoinositide; TM, transmembrane; WT, wild type; FITC, fluorescein isothiocyanate; PAFR, platelet-activated factor receptor. Taking into account the heterogeneity of ligands, it is interesting that all members of this superfamily of receptors share the same topology characterized by the presence of seven transmembrane α-helices. Conserved residues within subfamily A (related to rhodopsin) are localized throughout these helices, serving as reference points for sequence alignments. Biophysical and biochemical evidence indicate that upon photoactivation, relative movements of transmembrane (TM) 3 and TM6 occur in rhodopsin; similarly, rearrangement of the cytoplasmic proximal portion of TM7 relative to TM1 and of TM2 relative to the intracellular helix VIII were also observed (reviewed in Ref. 1Gether U. Asmar F. Meinild A.K. Rasmussen S.G. Pharmacol. Toxicol. 2002; 91: 304-312Crossref PubMed Scopus (76) Google Scholar). Through structural mimicry, despite the incredible diversity of ligands, GPCRs presumably share the core function of activation through similar conformational changes (2Ballesteros J. Shi L. Javitch J.A. Mol. Pharmacol. 2001; 60: 1-19Crossref PubMed Scopus (405) Google Scholar). GPCRs exist in equilibrium between the inactive (R) and active (R*) conformations, as described in the cubic ternary model of GPCR activation (3Weiss J.M. Morgan P.H. Lutz M.W. Kenakin T.P. J. Theor. Biol. 1996; 178: 169-172Crossref Scopus (97) Google Scholar). 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Kumasaka T. Hori T. Behnke C.A. Motoshima H. Fox B.A. Le Trong I. Teller D.C. Okada T. Stenkamp R.E. Yamamoto M. Miyano M. Science. 2000; 289: 739-745Crossref PubMed Scopus (5054) Google Scholar). Post-translational modifications such as phosphorylation and palmitoylation in the C-tail are involved in receptor coupling selectivity (24Kuang Y. Wu Y. Jiang H. Wu D. J. Biol. Chem. 1996; 271: 3975-3978Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar) and G-protein activation (25Okamoto Y. Ninomiya H. Tanioka M. Sakamoto A. Miwa S. Masaki T. J. Biol. Chem. 1997; 272: 21589-21596Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Other motifs are recognized as docking sites for scaffolding proteins (26Bockaert J. Marin P. Dumuis A. Fagni L. FEBS Lett. 2003; 546: 65-72Crossref PubMed Scopus (188) Google Scholar). In the present study, we investigated whether specific determinants located in the C-tail were implicated in the activation and trafficking of the high affinity human leukotriene (LT)B4 receptor (BLT1). LTB4 is a powerful inflammatory mediator derived from lipoxygenation of arachidonic acid, which induces neutrophil chemotaxis, chemokinesis, aggregation, degranulation, cation fluxes, as well as other immunodulatory functions (reviewed in Ref. 27Rola-Pleszczynski M. Immunol. Today. 1985; 10: 302-307Abstract Full Text PDF Scopus (127) Google Scholar) (28Stankova J. Gagnon N. Rola-Pleszczynski M. Immunology. 1992; 76: 258-263PubMed Google Scholar, 29Stankova J. Rola-Pleszczynski M. Biochem. J. 1992; 282: 625-629Crossref PubMed Scopus (49) Google Scholar, 30Rola-Pleszczynski M. Stankova J. Blood. 1992; 80: 1004-1011Crossref PubMed Google Scholar). LTB4 mediates its functions through interaction with two specific plasma membrane receptors showing different affinity for the ligand: a high affinity LTB4 receptor, BLT1 (31Yokomizo T. Izumi T. Chang K. Takuwa Y. Shimizu T. Nature. 1997; 387: 620-624Crossref PubMed Scopus (856) Google Scholar) and low affinity receptor, BLT2 (32Yokomizo T. Kato K. Terawaki K. Izumi T. Shimizu T. J. Exp. Med. 2000; 192: 421-432Crossref PubMed Scopus (472) Google Scholar), which share 45% identity. Several structural determinants purportedly involved in the regulation of transmembrane protein trafficking are present in the BLT1 C-tail and are conserved among species (human, rat, mouse, guinea pig) as well as in the human BLT2 (Fig. 1A). We recently demonstrated that within the BLT1 C-tail reside structural elements involved in its desensitization as well as in arrestin-independent and GRK- and dynamin-dependent BLT1 internalization; both regulatory processes require an intact C-tail.2 (33Gaudreau R. Le Gouill C. Venne M.H. Stankova J. Rola-Pleszczynski M. J. Biol. Chem. 2002; 277: 31567-31576Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar) In addition, preliminary data revealed that a BLT1 deletion mutant (G291stop) lacking the C-tail, transiently expressed in COS-7 cells, showed higher numbers of LTB4 binding sites and increased signal transduction when compared with the WT receptor (33Gaudreau R. Le Gouill C. Venne M.H. Stankova J. Rola-Pleszczynski M. J. Biol. Chem. 2002; 277: 31567-31576Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). These data suggest that specific structural determinants of BLT1 C-tail regulate cell surface expression, yet the motifs involved are still unknown. Within this receptor segment are located most of the multiple potential intracellular phosphorylation sites (Ser/Thr) as well as two dileucine motifs (Fig. 1A). We therefore sought to determine which structural determinants of BLT1 direct its expression. Reagents—cDNA encoding Gα16 was a generous gift from Dr. M. I. Simon (Passadena, CA); cDNA encoding GRK2 was a generous gift from Dr. Jeffrey Benovic (Thomas Jefferson University, Philadelphia, PA). Other materials and their sources were as follow: Geneticin and all culture media from Invitrogen Canada Inc. (Burlington, Ontario, Canada); bovine serum albumin, Hanks' balanced salt solution (HBSS), paraformaldehyde, and poly-l-lysine from Sigma-Aldrich Canada Ltd. (Oakville, Ontario, Canada); fetal bovine serum from BIO MEDIA Canada Inc. (Drummondville, Quebec, Canada); FuGENE 6 transfection reagent, Pwo polymerase, and restriction endonuclease from Roche Applied Science (Mississauga, Ontario, Canada); T4 DNA ligase, anti-mouse antibody conjugated to horseradish peroxidase, 32Pi, [3H]LTB4, and myo-[3H]inositol from Amersham Biosciences (Baie D'Urfé, Quebec, Canada); LTB4 from Cayman Chemical (Ann Arbor, MI); perchloric acid from VWR Canlab (Ville Mont-Royal, Quebec, Canada); fluorescein isothiocyanate-conjugated goat anti-mouse antibody from BIO/CAN Scientific (Mississauga, Ontario, Canada); gentamicin sulfate from Schering Canada Inc. (Pointe-Claire, Quebec, Canada). Construction of Myc-tagged Wild Type (WT) and Mutant Receptors— The cloning of WT BLT1 cDNA was previously described (34Gaudreau R. Le Gouill C. Metaoui S. Lemire S. Stankova J. Rola-Pleszczynski M. Biochem. J. 1998; 335: 15-18Crossref PubMed Scopus (51) Google Scholar). In order to allow rapid assessment of cell surface expression of BLT1, the N-terminal initiator methionine was replaced by the Myc sequence MEQKLISEEDLSRGSPG resulting in Myc epitope-tagged WT and mutant BLT1. The G291stop mutant generation was also described previously (33Gaudreau R. Le Gouill C. Venne M.H. Stankova J. Rola-Pleszczynski M. J. Biol. Chem. 2002; 277: 31567-31576Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). The chimeric mutant receptors were generated by PCR. Primers were designed to link BLT1 core receptor at residue Gly291 (Fig. 1A), immediately after the TM7, in-frame with the C-terminal tail of either PAFR C-tail, from residue Lys298, or CysLT1 C-tail, from residue Gly300. The 4L/A (in which Leu292, Leu293, Leu304, and Leu305 are substituted to alanine) or 2L(304–5)/A and 2L(292–3)/A mutant BLT1 were also constructed by PCR amplification. Each mutant BLT1 is identified by the single letter code of the original amino acid followed by the position number and the single letter code of the exchanged amino acid (Fig. 1B). All constructions were subcloned into pcDNA3 expression vector (Invitrogen). Mutant and chimeric receptor cDNAs were then sequenced (University of Calgary, Alberta, Canada) to confirm substitutions or fusion of the chimeric receptor. The human Gα16 and GRK2 cDNAs were in pCIS, under the control of a CMV promoter, and in pcDNA3, respectively. Cell Culture and Transfections—COS-7 and HEK293 were grown in Dulbecco's modified Eagle's medium with glucose, supplemented with 10% fetal bovine serum and gentamicin sulfate (40 μg/ml). Transient transfections were carried out with FuGENE 6, and experiments were performed 48 h post-transfection. Total transfected cDNA quantities were adjusted for each experiment with the pcDNA3 vector DNA. Radioligand Binding Assay—1.2 × 106 COS-7 cells were transiently transfected with 2 μg of cDNA encoding WT or mutant BLT1. After 48 h, cells were harvested and washed twice in phosphate-buffered saline and twice in Hepes-Tyrode's Buffer containing 0.1% (w/v) bovine serum albumin (35Honda Z. Takano T. Gotoh Y. Nishida E. Ito K. Shimizu T. J. Biol. Chem. 1994; 269: 2307-2315Abstract Full Text PDF PubMed Google Scholar) in which cells were also resuspended for the assay. Competition binding curves were carried out on 2 × 105 cells with 0.25 nm [3H]LTB4 and increasing concentrations of non-radioactive LTB4, for 2 h at 4 °C. For saturation binding curves, 1 × 105 cells were subjected to increasing [3H]LTB4 concentrations (0–16 nm). Nonspecific binding was measured in presence of 2 μm nonradioactive LTB4 and represented less than 10% of total binding. Free radioactivity was separated from cells by centrifugation and a double wash with 1 ml of Hepes-Tyrode's buffer. Radioactivity contained in the cell pellet was counted in liquid scintillation using a β counter. Inositol Phosphate Determination—COS-7 cells were plated in 30-mm dishes (2.0 × 105 cells/dish) and cultured 24 h before transient transfection with cDNAs encoding WT or mutant BLT1 in combination with cDNA of Gα16 subunit. In brief, 24 h post-transfection, cells were labeled and the following day, stimulated with 100 nm LTB4. Inositol phosphates (IP) were then extracted, and radioactivity was counted as described previously (34Gaudreau R. Le Gouill C. Metaoui S. Lemire S. Stankova J. Rola-Pleszczynski M. Biochem. J. 1998; 335: 15-18Crossref PubMed Scopus (51) Google Scholar). Total IP levels represent IP production in response to the agonist, over basal levels (or unstimulated). Constitutive activity was determined by comparing the ratio between the basal IP levels of mutant and WT receptors. Flow Cytometry Studies—COS-7 cells transiently expressing the Myc-tagged WT or mutant BLT1 were subjected to flow cytometry analysis. Cells (2,5 × 105) were labeled as previously described (34Gaudreau R. Le Gouill C. Metaoui S. Lemire S. Stankova J. Rola-Pleszczynski M. Biochem. J. 1998; 335: 15-18Crossref PubMed Scopus (51) Google Scholar) with anti-Myc followed by incubation with fluorescein isothiocyanate-conjugated goat anti-mouse IgG antibody. All measures were performed on a FACScan flow cytometer using CellQuest sofware (BD Biosciences). Internalization Assay—HEK293 grown at 80% confluency in Petri dishes were transfected with 2 μg of cDNA encoding WT or mutant BLT1 with 4 μg of each others cDNA or the pcDNA3 expression vector DNA as indicated in figure legends. Transfected cells cultured for 24 h were transferred to 6-well plates with 1.5 × 106 cells/well and incubated for another 24 h. After washing once with HBSS, medium containing LTB4 (300 nm) or its vehicle was added, and the cells were incubated for 1 h at 37 °C. Incubations were stopped on ice, and cells were washed twice with ice-cold HBSS. Flow cytometry analysis was carried out as described earlier except that antibody labeling was performed at 4 °C while cells were still attached to the plates. Cells were collected and analyzed. Percentage of receptor internalization was calculated based on either the mean fluorescence intensity (MFI) value of cells or on the percentage of positive cells, with comparable results, using LTB4-treated cells compared with cells treated with the vehicle alone. Molecular Modeling—The modeling and rendering of BLT1 were executed using the INSIGHTII suite of programs (Accel Inc., San Diego, CA). All the calculations were performed on an SGI Octane2 workstation (Silicon Graphics Inc., Mountain View, CA), as described previously (36Deraët M. Rihakova L. Boucard A. Pérodin J. Sauvé S. Mathieu A.P. Guillemette G. Leduc R. Lavigne P. Escher E. Can. J. Physiol. Pharmacol. 2002; 80: 418-425Crossref PubMed Scopus (25) Google Scholar). Briefly, a pairwise sequence alignment between the primary structures of BLT1 (SwissProt accession no. GenBank™ accession no. D89078) and the bovine rhodopsin (Protein Data Bank: 1L9H) was performed using the program HOMOLOGY. The sequences of the two proteins were aligned so as to match the positions of the following conserved residues: Asn55-Asn1.5036 (the superscripts represent the residue numbering in rhodopsin structure Protein Data Bank code 1F88, and human BLT1 sequence, respectively, and 1.50 is the numbering in the standardized nomenclature), Asp83-Asp2.5064, Arg135-Arg3.50115, Trp161-Trp4.50142, Pro215-Pro5.50193, Pro267-Pro6.50236 and Pro303-Pro7.50282. The coordinates of the identified structurally conserved regions were then transferred to the sequence of the BLT1. To relieve residual strain resulting from suboptimal positioning of the side chains, the resulting model was subjected to energy minimization using the program DISCOVER with a consistent valence force field (37Dauber-Osguthorpe P. Roberts V.A. Osguthorpe D.J. Wolff J. Genest M. Hagler A.T. Proteins. 1988; 4: 31-47Crossref PubMed Scopus (1949) Google Scholar). During this process, the Cα atoms and the side chains of conserved residues were kept fixed at their positions in the rhodopsin crystal structure. A distance-dependent dielectric constant of 4 was used with simple harmonic potential for bond length energy. No cross-term energies were included, and the peptide bonds were forced to planarity. Statistical Analysis—Data were analyzed for statistical significance using Student's paired t test. Differences were considered significant at p < 0.05. WT and mutant BLT1 were expressed in a mammalian expression system in order to determine and characterize structural elements involved in BLT1 trafficking and receptor activation. Motifs located in the C-terminal tail of the receptor were investigated with regard to receptor expression, targeting to the membrane and functionality. Structural Determinants of BLT1 C-tail Are Essential for Regulation of BLT1 Expression—We previously reported that a complete C-tail-truncated BLT1 (G291stop) showed increased numbers of [3H]LTB4 binding sites and a greater IP production in response to LTB4 stimulation than WT BLT1 (33Gaudreau R. Le Gouill C. Venne M.H. Stankova J. Rola-Pleszczynski M. J. Biol. Chem. 2002; 277: 31567-31576Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). In order to define whether selective BLT1 determinants could regulate its cell surface trafficking, we created two chimeric receptors composed of the main core of BLT1: residues 1–291 at the end of TM7 of BLT1 and the C-tail of either the PAFR or the LTD4 receptor CysLT1 (Fig. 1B). The amino acids present in the C-tail of each receptor are shown in Fig. 1A. Chimeric receptors (BLT1-PAFR and BLT1-CysLT1) were transiently expressed in COS-7 cells and were present at the plasma membrane as assessed by [3H]LTB4 binding (Table I). Nonlinear regression analysis of competition binding curves revealed the presence of one class of binding sites exhibiting high affinity for LTB4 (BLT1-PAFR, Kd = 3.6 ± 0.7 nm; BLT1-CysLT1, Kd = 6.7 ± 1.9 nm). These Kd values were slightly higher than the value for WT BLT1 value (Kd = 1.0 ± 0.5 nm) (Table I).Table IBinding characteristics of WT and modified BLT1ReceptorsDissociation constant, KdnmBLT1 WT0.99 ± 0.46BLT1-PAFR3.59 ± 0.74BLT1-CysLT16.71 ± 1.874L/A10.6 ± 2.402L(304·5)/A12.2 ± 3.002L(292·3)/A4.47 ± 1.47 Open table in a new tab We next investigated whether those chimeric receptors were able to transduce LTB4 signaling. Total IP accumulation was monitored in COS-7 cells coexpressing Gα16 subunit protein and either chimeric or WT receptor. Surprisingly, in response to 100 nm of LTB4, IP production was markedly increased for both BLT1-PAFR (155 ± 16%) and BLT1-CysLT1 (163 ± 18%) in comparison with WT (defined as 100%) (Fig. 2A). Furthermore, the increased ability of the chimeric BLT1 to transduce LTB4 signaling was associated with a higher number of binding sites, as shown by [3H]LTB4 saturation binding curves (Fig. 2B). These results suggest that the C-tail of WT BLT1 contains a signal that is not conserved in PAFR or CysLT1 C-tail, which controls BLT1 trafficking or functional expression at the cell surface. We further sought to define the structural determinants present in the C-tail that could regulate BLT1 expression. Since a partially C-tail-truncated BLT1 mutant (G319stop) had shown binding characteristics similar to WT BLT1 (33Gaudreau R. Le Gouill C. Venne M.H. Stankova J. Rola-Pleszczynski M. J. Biol. Chem. 2002; 277: 31567-31576Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), we investigated the cytoplasmic segment comprising residues 291–319. Two types of structural determinants located within the 291–319 C-tail segment could be involved in receptor trafficking: phosphoacceptor sites (S/T) and dileucine motifs (LL). Notably, the addition of PAFR and CysLT1 C-tails containing potential phosphoacceptor sites did not prevent the increase in binding site numbers observed with the G291stop BLT1. Moreover, several phosphorylation sites (Thr308, Ser310, Ser313, Ser314, Thr315) within this segment had been mutated without any effect on [3H]LTB4 binding (Ref. 33Gaudreau R. Le Gouill C. Venne M.H. Stankova J. Rola-Pleszczynski M. J. Biol. Chem. 2002; 277: 31567-31576Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar and data not shown). Consequently, BLT1 C-tail dileucine motifs were further studied. Characterization of Dileucine Mutant BLT1—Dihydrophobic motifs, such as leucine-valine and leucine-leucine are involved in efficient trafficking of several transmembrane proteins through sorting, localization or internalization signals as observed for the β2-AR and CXCR4 (13Heilker R. Spiess M. Crottet P. Bioessays. 1999; 21: 558-567Crossref PubMed Scopus (121) Google Scholar, 20Gabilondo A.M. Hegler J. Krasel C. Boivin-Jahns V. Hein L. Lohse M.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12285-12290Crossref PubMed Scopus (83) Google Scholar, 21Orsini M.J. Parent J.L. Mundell S.J. Benovic J.L. Marchese A. J. Biol. Chem. 1999; 274: 31076-31086Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). Two dileucine motifs are located between residues 291–319 of the BLT1 C-tail. In order to assess whether the dileucines were essential for the WT phenotype, we used site-directed mutagenesis to disrupt Leu292, Leu293, and Leu304, Leu305 in the full-length BLT1. Leucines were substituted to alanines so as to change each motif alone or in combination, generating three new mutant receptors: 2L(292–3)/A, 2L(304–5)/A, and 4L/A mutant BLT1 (Fig. 1B). Again, mutant receptors displayed a higher Kd for LTB4 (respective Kd values: 4.47 ± 1.47 nm, 12.2 ± 3.0 nm, 10.6 ± 2.4 nm) than the WT BLT1 (Table I). LTB4-induced IP accumulation was increased for the 4L/A mutant receptor (136 ± 17%) when compared with the WT (defined as 100%) (Fig. 3A). Interestingly, mutation of the distal dileucine motif 2L(304–5)/A led to a significant increase in IP production upon LTB4 exposure (177 ± 22%), whereas mutation of the proximal motif 2L(292–3)/A had no detectable effect (108 ± 22%). Similar results were also observed in a time course study of IP production (Fig. 3B). We next addressed whether dileucines might regulate BLT1 expression. Saturation binding curves correlated with the IP production, given that the 4L/A and the 2L(304–5)/A mutant receptors showed higher numbers of binding sites at the plasma membrane, but saturation could not be achieved (Fig. 3C). In contrast, WT and the 2L(292–3)/A BLT1 binding sites for LTB4 could be saturated. Overall, these results suggest that leucines 304 and 305 are responsible for the phenotype observed with the G291stop deletion. We assessed the expression levels of Myc-tagged WT and mutant BLT1 at the plasma membrane by flow cytometry analysis. Surprisingly, the mean fluorescence intensity of COS-7 cells expressing each mutant receptor did not significantly vary from that of WT BLT1 (Fig. 4 and Table II). Similar results were observed when the number of Myc+ cells was evaluated (Table II). Thus, the cell surface expression of the protein was not affected by removal of either one or both of the dileucine motifs and hence, the increased binding capacity of the population of receptors was not the consequence of a higher number of cell surface BLT1. Therefore, it could be hypothesized that the distal dileucine motif regulated in some way LTB4 binding or LTB4 mediated activation of BLT1, by an augmentation of the population of receptors in the active state.Table IIExpression of WT and mutant BLT1ReceptorsPo
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