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• W2016206520 abstract "Lysophosphatidic acid (LPA) is a ligand for three endothelial differentiation gene family G protein-coupled receptors, LPA1–3. We performed computational modeling-guided mutagenesis of conserved residues in transmembrane domains 3, 4, 5, and 7 of LPA1–3 predicted to interact with the glycerophosphate motif of LPA C18:1. The mutants were expressed in RH7777 cells, and the efficacy (Emax) and potency (EC50) of LPA-elicited Ca2+ transients were measured. Mutation to alanine of R3.28 universally decreased both the efficacy and potency in LPA1–3 and eliminated strong ionic interactions in the modeled LPA complexes. The alanine mutation at Q3.29 decreased modeled interactions and activation in LPA1 and LPA2 more than in LPA3. The mutation W4.64A had no effect on activation and modeled LPA interaction of LPA1 and LPA2 but reduced the activation and modeled interactions of LPA3. The R5.38A mutant of LPA2 and R5.38N mutant of LPA3 showed diminished activation by LPA; however, in LPA1 the D5.38A mutation did not, and mutation to arginine enhanced receptor activation. In LPA2, K7.36A decreased the potency of LPA; in LPA1 this same mutation increased the Emax. In LPA3, R7.36A had almost no effect on receptor activation; however, the mutation K7.35A increased the EC50 in response to LPA 10-fold. In LPA1–3, the mutation Q3.29E caused a modest increase in EC50 in response to LPA but caused the LPA receptors to become more responsive to sphingosine 1-phosphate (S1P). Surprisingly micromolar concentrations of S1P activated the wild type LPA2 and LPA3 receptors, indicating that S1P may function as a weak agonist of endothelial differentiation gene family LPA receptors. Lysophosphatidic acid (LPA) is a ligand for three endothelial differentiation gene family G protein-coupled receptors, LPA1–3. We performed computational modeling-guided mutagenesis of conserved residues in transmembrane domains 3, 4, 5, and 7 of LPA1–3 predicted to interact with the glycerophosphate motif of LPA C18:1. The mutants were expressed in RH7777 cells, and the efficacy (Emax) and potency (EC50) of LPA-elicited Ca2+ transients were measured. Mutation to alanine of R3.28 universally decreased both the efficacy and potency in LPA1–3 and eliminated strong ionic interactions in the modeled LPA complexes. The alanine mutation at Q3.29 decreased modeled interactions and activation in LPA1 and LPA2 more than in LPA3. The mutation W4.64A had no effect on activation and modeled LPA interaction of LPA1 and LPA2 but reduced the activation and modeled interactions of LPA3. The R5.38A mutant of LPA2 and R5.38N mutant of LPA3 showed diminished activation by LPA; however, in LPA1 the D5.38A mutation did not, and mutation to arginine enhanced receptor activation. In LPA2, K7.36A decreased the potency of LPA; in LPA1 this same mutation increased the Emax. In LPA3, R7.36A had almost no effect on receptor activation; however, the mutation K7.35A increased the EC50 in response to LPA 10-fold. In LPA1–3, the mutation Q3.29E caused a modest increase in EC50 in response to LPA but caused the LPA receptors to become more responsive to sphingosine 1-phosphate (S1P). Surprisingly micromolar concentrations of S1P activated the wild type LPA2 and LPA3 receptors, indicating that S1P may function as a weak agonist of endothelial differentiation gene family LPA receptors. Lysophosphatidic acid (LPA) 2The abbreviations used are: LPA, lysophosphatidic acid; EDG, endothelial differentiation gene; GPCR, G protein-coupled receptor; S1P, sphingosine 1-phosphate; TM, transmembrane domain; PBS, phosphate-buffered saline; BSA, bovine serum albumin; RH7777, rat hepatoma 7777; DH-S1P, dihydrosphingosine 1-phosphate; GTPγS, guanosine 5′-3-O-(thio) triphosphate. and sphingosine 1-phosphate (S1P) are structurally related lysophospholipid growth factors that mediate a variety of cellular effects, including regulation of cellular proliferation, survival, migration, and morphology (1Ishii I. Fukushima N. Ye X. Chun J. Annu. Rev. Biochem. 2004; 73: 321-354Crossref PubMed Scopus (649) Google Scholar, 2Moolenaar W.H. van Meeteren L.A. Giepmans B.N. BioEssays. 2004; 26: 870-881Crossref PubMed Scopus (489) Google Scholar, 3Radeff-Huang J. Seasholtz T.M. Matteo R.G. Brown J.H. J. Cell. Biochem. 2004; 92: 949-966Crossref PubMed Scopus (171) Google Scholar). LPA has been shown to play an important role in a variety of diseases including ovarian cancer, prostate cancer, breast cancer, and cardiovascular disease (4Xu Y. Fang X.J. Casey G. Mills G.B. Biochem. J. 1995; 309: 933-940Crossref PubMed Scopus (252) Google Scholar, 5Xu Y. Gaudette D.C. Boynton J.D. Frankel A. Fang X.J. Sharma A. Hurteau J. Casey G. Goodbody A. Mellors A. Clin. Cancer Res. 1995; 1: 1223-1232PubMed Google Scholar, 6Xu Y. Shen Z. Wiper D.W. Wu M. Morton R.E. Elson P. Kennedy A.W. Belinson J. Markman M. Casey G. J. Am. Med. Assoc. 1998; 280: 719-723Crossref PubMed Scopus (566) Google Scholar, 7Schwartz B.M. Hong G. Morrison B.H. Wu W. Baudhuin L.M. Xiao Y.J. Mok S.C. Xu Y. Gynecol. Oncol. 2001; 81: 291-300Abstract Full Text PDF PubMed Scopus (105) Google Scholar, 8Sliva D. Mason R. Xiao H. English D. Biochem. Biophys. Res. Commun. 2000; 268: 471-479Crossref PubMed Scopus (50) Google Scholar, 9Goetzl E.J. Dolezalova H. Kong Y. Zeng L. Cancer Res. 1999; 59: 4732-4737PubMed Google Scholar, 10Qi C. Park J.H. Gibbs T.C. Shirley D.W. Bradshaw C.D. Ella K.M. Meier K.E. J. Cell. Physiol. 1998; 174: 261-272Crossref PubMed Scopus (60) Google Scholar, 11Guo R. Kasbohm E.A. Arora P. Sample C.J. Baban B. Sud N. Sivashanmugam P. Moniri N.H. Daaka Y. Endocrinology. 2006; 147: 4883-4892Crossref PubMed Scopus (63) Google Scholar, 12Karliner J.S. Biochim. Biophys. Acta. 2002; 1582: 216-221Crossref PubMed Scopus (77) Google Scholar, 13Durgam G.G. Virag T. Walker M.D. Tsukahara R. Yasuda S. Liliom K. van Meeteren L.A. Moolenaar W.H. Wilke N. Siess W. Tigyi G. Miller D.D. J. Med. Chem. 2005; 48: 4919-4930Crossref PubMed Scopus (99) Google Scholar, 14Zhang C. Baker D.L. Yasuda S. Makarova N. Balazs L. Johnson L.R. Marathe G.K. McIntyre T.M. Xu Y. Prestwich G.D. Byun H.S. Bittman R. Tigyi G. J. Exp. Med. 2004; 199: 763-774Crossref PubMed Scopus (173) Google Scholar). Many of the biological effects of LPA are mediated through cell surface receptors of the endothelial differentiation gene (EDG) family of G protein-coupled receptors (GPCRs). The EDG family of GPCRs includes eight closely related genes that show the conserved GPCR topology of an extracellular amino terminus followed by seven α-helical transmembrane domains (TMs) (15Chun J. Goetzl E.J. Hla T. Igarashi Y. Lynch K.R. Moolenaar W. Pyne S. Tigyi G. Pharmacol. Rev. 2002; 54: 265-269Crossref PubMed Scopus (447) Google Scholar). Three of these genes (LPA1–3) are cellular receptors for LPA and share 55% overall homology in humans. The other five (S1P1–5) are cellular receptors for S1P and share 50% homology in humans. The two subclusters are 35% homologous with each other. The transmembrane domains of human LPA1–3 where ligand binding takes place show 81% homology with each other. LPA has also been shown to elicit cellular responses through binding to three non-EDG family GPCRs, p2y9/LPA4, GPR92/LPA5, and GPR87/LPA6, which are more closely related to the purinoreceptor cluster of GPCRs (16Noguchi K. Ishii S. Shimizu T. J. Biol. Chem. 2003; 278: 25600-25606Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar, 17Kotarsky K. Boketoft A. Bristulf J. Nilsson N.E. Norberg A. Hansson S. Owman C. Sillard R. Leeb-Lundberg L.M. Olde B. J. Pharmacol. Exp. Ther. 2006; 318: 619-628Crossref PubMed Scopus (185) Google Scholar, 18Lee C.W. Rivera R. Gardell S. Dubin A.E. Chun J. J. Biol. Chem. 2006; 281: 23589-23597Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar, 19Tabata K. Baba K. Shiraishi A. Ito M. Fujita N. Biochem. Biophys. Res. Commun. 2007; 363: 861-866Crossref PubMed Scopus (188) Google Scholar). Modeling and mutagenesis studies of S1P receptors in the EDG family have demonstrated that conserved residues can play either conserved or non-conserved roles in different family members. A validated computational model of S1P1 was developed that successfully identified residues in TM3 and TM7 of S1P1 that participated in ligand binding. A critical role for residues R3.28, E3.29, and R7.34 of S1P1 in ligand binding and receptor activation was experimentally confirmed using a sitedirected mutagenesis strategy (20Parrill A.L. Baker D.L. Wang D.A. Fischer D.J. Bautista D.L. Van Brocklyn J. Spiegel S. Tigyi G. Ann. N. Y. Acad. Sci. 2000; 905: 330-339Crossref PubMed Scopus (28) Google Scholar). Later studies determined that in S1P4 the residues R3.28, E3.29, W4.64, and K5.38 were critical for ligand binding and receptor activation (21Inagaki Y. Pham T.T. Fujiwara Y. Kohno T. Osborne D.A. Igarashi Y. Tigyi G. Parrill A.L. Biochem. J. 2005; 389: 187-195Crossref PubMed Scopus (46) Google Scholar), whereas K5.38 was not essential in S1P1 (22Naor M.M. Walker M.D. Van Brocklyn J.R. Tigyi G. Parrill A.L. J. Mol. Graph. Model. 2007; 26: 519-528Crossref PubMed Scopus (18) Google Scholar). Based upon the high sequence homology within the EDG family, the experimentally validated S1P1 model was used as a template to map the ligandbinding pocket of the LPA-specific EDG receptors. Computational modeling predicted that the residues R3.28, Q3.29, R5.38, and K7.35 of LPA3 form critical interactions with the polar head group of LPA, and this was confirmed experimentally (23Fujiwara Y. Sardar V. Tokumura A. Baker D. Murakami-Murofushi K. Parrill A. Tigyi G. J. Biol. Chem. 2005; 280: 35038-35050Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). These previous studies suggest a conserved and essential role for R3.28 in all receptors examined so far but a variable role for K5.38 in two family members. Position 3.29, which is conserved as a glutamine in LPA-specific EDG receptors and a glutamate in S1P-specific EDG receptors, was computationally identified and experimentally validated as a key residue that determines receptor selectivity for LPA or S1P in the S1P1 and LPA1 receptor pair (24Wang D.A. Lorincz Z. Bautista D.L. Liliom K. Tigyi G. Parrill A.L. J. Biol. Chem. 2001; 276: 49213-49220Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). The E3.29Q mutant of S1P1 responded to LPA rather than S1P; the reciprocal Q3.29E mutation in LPA1 showed diminished activation by LPA but was activated by S1P, indicating the involvement of additional residues in ligand recognition. Alanine mutation at this position diminished activation by either ligand in both receptors (24Wang D.A. Lorincz Z. Bautista D.L. Liliom K. Tigyi G. Parrill A.L. J. Biol. Chem. 2001; 276: 49213-49220Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Similarly the E3.29Q mutation in S1P4 conferred responsiveness to LPA but decreased responsiveness to S1P (25Holdsworth G. Osborne D.A. Pham T.T. Fells J.I. Hutchinson G. Milligan G. Parrill A.L. BMC Biochem. 2004; 5: 12Crossref PubMed Scopus (19) Google Scholar). Most cell types express multiple EDG receptor subtypes (26Fischer D.J. Liliom K. Guo Z. Nusser N. Virag T. Murakami-Murofushi K. Kobayashi S. Erickson J.R. Sun G. Miller D.D. Tigyi G. Mol. Pharmacol. 1998; 54: 979-988Crossref PubMed Scopus (108) Google Scholar). The role of the different LPA receptor subtypes in physiological and pathophysiological processes is often difficult to determine because of the lack of LPA receptor subtype-specific reagents. Subtype-specific agonists and antagonists could elucidate the role of the different LPA receptor subtypes in physiological and disease states as well as function as lead compounds in drug development. To aid in the development of subtype-specific reagents, it is important to identify differences between the EDG family LPA receptor subtypes in the ligand-binding pocket. The fundamental assumption underlying homology modeling and comparative sequence analysis is that identical residues fulfill the same role in homologous proteins. Given the very high degree of sequence identity, especially in the transmembrane domains of the EDG receptors, one would hypothesize that the function of those residues validated to play a role in ligand recognition applies universally within the family. The variable importance of K5.38 in the S1P1 and S1P4 receptors, however, suggests that this assumption is not always accurate (21Inagaki Y. Pham T.T. Fujiwara Y. Kohno T. Osborne D.A. Igarashi Y. Tigyi G. Parrill A.L. Biochem. J. 2005; 389: 187-195Crossref PubMed Scopus (46) Google Scholar, 22Naor M.M. Walker M.D. Van Brocklyn J.R. Tigyi G. Parrill A.L. J. Mol. Graph. Model. 2007; 26: 519-528Crossref PubMed Scopus (18) Google Scholar). In the present study we carried out a comparative analysis of the conserved key residues experimentally validated to be involved in ligand recognition in one or another LPA- or S1P-specific EDG family receptor to find that many of these head group-interacting residues play different roles. We extended our analysis to include all three of the LPA-specific EDG receptors and generated mutations at sites that are computationally predicted to impact ligand recognition: R3.28, Q3.29, W4.64, D/R5.38, K7.35, and K/R7.36. We determined the effect of each mutation upon the potency (EC50) and efficacy (Emax) of LPA relative to the activation elicited in the wild type receptors. We also evaluated the impact of some of these mutations on receptor activation by the related lipid mediator S1P. Experimental results were correlated to predictions based upon computational modeling of the wild type and mutant receptors docked with ligand. These studies reveal that major differences exist between the different LPA receptor subtypes in the functional utilization of several conserved residues in the predicted ligand-binding pocket. Only one residue when mutated to alanine identically impacted the three receptor subtypes; the mutation R3.28A universally reduced both the efficacy and potency in LPA1–3 and eliminated strong ionic interactions in the modeled LPA complexes. The different roles that conserved residues can play among the highly homologous members of the EDG family provide insight into nature's diverse answers for high affinity molecular recognition and challenge the concept that automatically assigns identical function to homologous residues. Reagents—All analogs of LPA and S1P were purchased from Avanti Polar Lipids (Alabaster, AL). Lipids were prepared before use asa1mm stock in PBS containing 1 mm charcoalstripped bovine serum albumin (BSA). Alexa Fluor 488-conjugated goat anti-mouse IgG was purchased from Molecular Probes (Eugene, OR). Anti-FLAG M2 monoclonal antibody was purchased from Sigma. Residue Nomenclature—Amino acids in the TMs were assigned index positions by the method of Ballesteros and Weinstein (27Ballesteros J.A., and Weinstein, H. (1995) in Methods in Neurosciences (Conn, P. M., and Sealfon, S. C., eds) pp. 366-428, Academic Press, San Diego, CAGoogle Scholar) based upon homology found in the seven helical TMs of GPCRs. Index positions are in the format X.YY where X refers to the number of the TM in which that residue is found and YY refers to the position within that TM relative to the most highly conserved residue in that TM throughout the GPCR superfamily, which is arbitrarily designated position 50 (27Ballesteros J.A., and Weinstein, H. (1995) in Methods in Neurosciences (Conn, P. M., and Sealfon, S. C., eds) pp. 366-428, Academic Press, San Diego, CAGoogle Scholar). Computational Homology Modeling—Previously developed computational models of LPA1, LPA2, and LPA3 (23Fujiwara Y. Sardar V. Tokumura A. Baker D. Murakami-Murofushi K. Parrill A. Tigyi G. J. Biol. Chem. 2005; 280: 35038-35050Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 28Sardar V.M. Bautista D.L. Fischer D.J. Yokoyama K. Nusser N. Virag T. Wang D.A. Baker D.L. Tigyi G. Parrill A.L. Biochim. Biophys. Acta. 2002; 1582: 309-317Crossref PubMed Scopus (78) Google Scholar) were used for mutation studies. LPA C18:1 was docked into each receptor with a –2 charge because previous quantum mechanical studies suggest that is appropriate for phospholipid binding sites with multiple cationic residues (22Naor M.M. Walker M.D. Van Brocklyn J.R. Tigyi G. Parrill A.L. J. Mol. Graph. Model. 2007; 26: 519-528Crossref PubMed Scopus (18) Google Scholar). Docking studies were done using Autodock 3.0 (29Morris G.M. Goodsell D.S. Halliday R.S. Huey R. Hart W.E. Belew R.K. Olson A.J. J. Comput. Chem. 1998; 19: 1639-1662Crossref Scopus (9208) Google Scholar). Default docking parameters were used except for number of runs (15Chun J. Goetzl E.J. Hla T. Igarashi Y. Lynch K.R. Moolenaar W. Pyne S. Tigyi G. Pharmacol. Rev. 2002; 54: 265-269Crossref PubMed Scopus (447) Google Scholar), energy evaluations (9.0 × 1010), generations (30,000), and local search iterations (3000). The complex with the greatest number of cationic interactions with the LPA phosphate group was chosen and subjected to molecular dynamics simulations using a 1-fs time step at 500 ps. The lowest energy structure from the simulation was geometry-optimized and used as the wild type receptor for mutation studies. Mutation studies were done as described previously (22Naor M.M. Walker M.D. Van Brocklyn J.R. Tigyi G. Parrill A.L. J. Mol. Graph. Model. 2007; 26: 519-528Crossref PubMed Scopus (18) Google Scholar). Mutant models were generated by amino acid side chain replacement. Each mutant was modeled with LPA bound. The models were refined using the MOE (Molecular Operating Environment) software (version 2004.03, Chemical Computing Group, Montreal, Canada). The models were subjected to molecular dynamics and geometry optimization. The MMFF94 force field (30Halgren T.A. J. Comput. Chem. 1996; 17: 490-519Crossref Scopus (4330) Google Scholar) was used for all force field simulations. Default parameters for molecular dynamics simulations were used with the exception of the total simulation length, which was 1 ns. LPA was removed from each mutant receptor and docked back into the receptor using Autodock 3.0. The best LPA complex with each mutant was selected as the one with the most cationic interactions with the phosphate group. Site-directed Mutagenesis—Amino-terminal FLAG epitope-tagged LPA1, LPA2, and LPA3 receptor constructs were subcloned into pcDNA3.1 vector (Invitrogen). Receptor constructs were mutated at residues computationally predicted to participate in ligand recognition using the QuikChange II XL site-directed mutagenesis kit (Stratagene, La Jolla, CA). In some cases, a PCR-based site-directed mutagenesis strategy was used to generate the desired mutation as described previously (23Fujiwara Y. Sardar V. Tokumura A. Baker D. Murakami-Murofushi K. Parrill A. Tigyi G. J. Biol. Chem. 2005; 280: 35038-35050Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). TOP10 competent cells (Invitrogen) were transformed with the mutant constructs, and clones were verified by complete sequencing of the inserts. Cell Culture and Transfection—LPA does not elicit Ca2+ transients in the parental McArtl rat hepatoma 7777 (RH7777) cells (31Fukushima N. Kimura Y. Chun J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6151-6156Crossref PubMed Scopus (251) Google Scholar) (ATCC, Manassas, VA). RH7777 cells and rat hepatoma HTC4 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal bovine serum, 100 units/ml penicillin, 10 μg/ml streptomycin, and 2 mm glutamine. RH7777 cells stably expressing LPA1 and LPA3 receptors have been characterized elsewhere (32Fischer D.J. Nusser N. Virag T. Yokoyama K. Wang D. Baker D.L. Bautista D. Parrill A.L. Tigyi G. Mol. Pharmacol. 2001; 60: 776-784PubMed Google Scholar). RH7777 cells stably expressing LPA2 were a generous gift from Dr. Fumikazu Okajima (Gunma University, Gunma, Japan) and were characterized previously (33Ohta H. Sato K. Murata N. Damirin A. Malchinkhuu E. Kon J. Kimura T. Tobo M. Yamazaki Y. Watanabe T. Yagi M. Sato M. Suzuki R. Murooka H. Sakai T. Nishitoba T. Im D.S. Nochi H. Tamoto K. Tomura H. Okajima F. Mol. Pharmacol. 2003; 64: 994-1005Crossref PubMed Scopus (336) Google Scholar). Stable transfectants were maintained in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal bovine serum, 100 units/ml penicillin, 10 μg/ml streptomycin, 2 mm glutamine, and 250 μg/ml G418. Transient transfections of RH7777 cells and HTC4 cells were performed using Effectene transfection reagent (Qiagen, Valencia, CA). Flow Cytometric Analysis—Expression of all receptor constructs on the cell surface was confirmed by flow cytometric analysis using indirect immunofluorescence staining with anti-FLAG M2 antibody. RH7777 cells were transfected with FLAG epitope-tagged LPA receptor constructs, replated after 16 h, and cultured for an additional 24 h. The culture medium was replaced with Krebs buffer (120 mm NaCl, 5 mm KCl, 0.62 mm MgSO4, 1.8 mm CaCl2, 6mm glucose, 10 mm HEPES, pH 7.4) for 4 h before collection; cells were detached using HyQTase Cell Detachment Solution (Hyclone Laboratories) and collected on ice. Cells were washed with PBS that contained 3% BSA and incubated for 30 min in PBS that contained 5% BSA and 5% normal donkey serum. The cells were washed with PBS that contained 3% BSA, incubated with anti-FLAG M2 monoclonal antibody (1:200) in PBS containing 5% BSA for 1 h followed by two washes in PBS with 3% BSA, and incubated with Alexa Fluor 488-conjugated goat antimouse IgG (1:1000) in PBS that contained 5% BSA for 30 min. Cells were washed two times with PBS that contained 3% BSA and resuspended in PBS that contained 1% BSA. Cells were analyzed using an LSR II flow cytometer (BD Biosciences), and data were analyzed using FlowJo software. Receptor Activation Assays—FLAG-tagged LPA1, LPA2, and LPA3 receptor constructs were transiently expressed in LPA-nonresponsive RH7777 cells using Effectene transfection reagent (Qiagen). Cells were replated in poly-l-lysine-coated 96-well microplates 16 h after transfection at a density of 30,000 cells/well and cultured for 24 h. The culture medium was replaced with Krebs buffer for 4 – 6 h before assays. The transfected cells were loaded with Fura-2/AM in Krebs buffer containing 0.001% pluronic acid for 30 min and rinsed with Krebs buffer, and the Ca2+ response to LPA C18:1 or S1P was measured using a FlexStation II fluorescence plate reader (Molecular Devices, Sunnyvale, CA). The ratio of peak emissions at 510 nm after 2 min of ligand addition was determined for excitation wavelengths of 340/380 nm. All samples were run in triplicate, and assays were performed at least three times for each receptor construct. The responses to LPA by the wild type and mutant receptors were measured and reported in terms of maximal activation (Emax) and efficacy (EC50) ± S.D. Radioligand Binding Assay—HEK293T cells were plated in 24-well plates at 4 × 105/well and the following day transiently transfected with 0.4 μg of receptor constructs using Lipofectamine 2000 (Invitrogen). Two days later cells were washed with ice-cold binding buffer (50 mm Tris, pH 7.4, 150 mm NaCl). Cells were then incubated in binding buffer containing 4 mg/ml fatty acid-free BSA and [32P]S1P ranging from 10 nm to 1 μm in the presence or absence of 10 μm unlabeled S1P as a competitor on ice for 45 min. After washing twice with cold binding buffer containing 0.4 mg/ml BSA, cells were lysed in 0.5% SDS, and binding was quantified by scintillation counting. Triplicate samples were measured for each condition. Receptor Internalization Assays—RH7777 cells were transiently transfected with FLAG-tagged LPA2 using Effectene transfection reagent. Cells were serum-starved for 4 and then incubated with a 10 μm concentration of either LPA, S1P, ATP, or vehicle for 30 min at 37 °C before collection on ice and subsequent anti-FLAG flow cytometric analysis. The assay was repeated three times with similar results. Theoretical Models of LPA1, LPA2, and LPA3: Mutation Site Selection—In LPA1–3, we evaluated the effect of alanine mutation of residues in TM3, TM4, TM5, and TM7 that were computationally predicted to impact binding to LPA C18:1. The summary of the computationally predicted ionic ligand-receptor interactions in the wild type receptors as well as in the mutants is summarized in Table 1. The number of interactions that were predicted to occur over distances of less than 4.5 Å between the polar head group of LPA and charged residues in TM3, TM5, and TM7 varied between the receptors and were two, two, and three for LPA1, LPA2, and LPA3, respectively. Mutation of R3.28 to alanine universally diminished these interactions in each receptor model. Mutation of other residues had variable impact on the number of ionic interactions with the alanine mutants of the five residues R3.28, Q3.29, W4.64, D/R5.38, K7.35, and K7.36 we examined (Table 1).TABLE 1The computationally predicted number of close charge-charge interactions between LPA receptor constructs and LPAConstructLPA1LPA2LPA3Wild type223R3.28A000Q3.29E120Q3.29A003W4.64A120D5.38A2NANAD5.38R2NANAR5.38ANA10R5.38NNANA0K7.36A12NAR7.36ANANA3K7.35ANANA0 Open table in a new tab Mutation and Cell Surface Expression of LPA1, LPA2, and LPA3 Receptor Constructs—To validate and refine our computational models of the ligand head group-binding pocket of the EDG family LPA receptors, we generated alanine point mutants of amino-terminal FLAG epitope-tagged LPA1, LPA2, and LPA3 receptor constructs at residues in TM3, TM4, TM5, and TM7 that were computationally identified to surround the glycerophosphate portion of LPA C18:1 in the wild type LPA1–3 complexes. Additionally in LPA1, LPA2, and LPA3 Q3.29 was also mutated to glutamate, the residue occurring at this position throughout the S1P receptors in the EDG receptor family; previously this mutation was shown to change the ligand specificity of LPA1 from LPA to S1P (24Wang D.A. Lorincz Z. Bautista D.L. Liliom K. Tigyi G. Parrill A.L. J. Biol. Chem. 2001; 276: 49213-49220Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Expression and surface targeting of the receptor constructs was confirmed by indirect immunofluorescence flow cytometric analysis using antibodies directed against the amino-terminal FLAG epitope present in the constructs (Table 2). All receptor constructs showed targeting to the cell surface when transiently transfected into RH7777 cells except for the R5.38A mutant of LPA3, which was not expressed at a detectable level. However, the asparagine mutant R5.38N of LPA3 did show cell surface expression and was used instead of R5.38A in our studies (23Fujiwara Y. Sardar V. Tokumura A. Baker D. Murakami-Murofushi K. Parrill A. Tigyi G. J. Biol. Chem. 2005; 280: 35038-35050Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar).TABLE 2Cell surface expression of wild type and mutant LPA1–3 receptor constructs determined by flow cytometryConstructAnti-FLAG-stained cells, percentage of total cellsLPA1LPA2LPA3%Wild type42.155.218.1R3.28A51.666.139.7Q3.29E48.627.415.0Q3.29A11.920.519.9W4.64A43.847.617.6D5.38A54.7NANAD5.38R53.0NANAR5.38ANA10.76.2R5.38NNANA13.2K7.36A54.741.7NAR7.36ANANA38.3K7.35ANANA32.3 Open table in a new tab Some variability in percentage of cells expressing the receptor mutants on their surface was noted among the FLAG-tagged receptor constructs. Although the LPA1, LPA2, and LPA3 receptor constructs that we used were all subcloned into the pcDNA3.1 vector, surface expression tended to be higher for LPA1 and LPA2 constructs than for LPA3 constructs (Table 2). This variation was possibly due to receptor subtype-specific differences in processing, cell surface targeting, and/or stability; however, these variances in receptor surface expression levels did not correlate with absolute measurements of maximal receptor activation. The absolute values of our ratiometric measurements of LPA-induced calcium mobilization were consistently higher by ∼2-fold for cells transfected with wild type LPA3 than for cells transfected with wild type LPA1 or LPA2 despite the lower surface expression of the LPA3 (Table 2). This lack of correlation between surface expression and maximal receptor activation may reflect an excess of receptor expression relative to the endogenous G proteins that couple to the activated receptors to mediate calcium mobilization as well as different coupling efficiencies among the EDG family receptor subtypes for these G proteins. Indeed in our transient transfection system, heterologous expression of S1P1 is insufficient to cause calcium mobilization in response to S1P unless G16 is cotransfected (data not shown). Variation in surface expression was also noted among several mutant constructs compared with the wild type receptors (Table 2). For LPA1 constructs, cells transfected with Q3.29A showed low (11.9% of cells) surface expression compared with cells transfected with wild type (42.1%) likely due to diminished cell surface targeting or stability of this construct. In the case of LPA2, surface expression levels of two mutants, Q3.29A (20.5%) and R5.38A (10.7%), were less than half of the level measured for the wild type receptor (55.2%). In LPA3, surface expression of R5.38A was not detected above background (6.2% compared with 5.0% for empty vector-transfected cells); the R5.38N mutant did show cell surface expression although somewhat less than the wild type receptor (13.2 versus 18.1% for wild type). Because of the relatively high expression levels of the receptors in our transient transfection system, the variation in cell surface expression of the different constructs should have only minor" @default.
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• W2016206520 creator A5001837381 @default.
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