title: Identification of residues responsible for ligand recognition and regioisomeric selectivity of lysophosphatidic acid receptors expressed in mammalian cells.
abstract: The endothelial differentiation gene family encodes three highly homologous G protein-coupled receptors for lysophosphatidic acid (LPA). Based on baculoviral overexpression studies, differences have been proposed in the structure-activity relationship (SAR) of these receptors. We have compared the SAR of the individual receptors either overexpressed transiently at high or at lower levels following stable transfection in LPA-nonresponsive RH7777 cells. The SAR in transfected RH7777 cells was markedly different from that described in insect cells. The LPA(3) receptor has been proposed to be selectively activated by unsaturated LPA species and shows a strong preference for sn-2 versus the sn-1 acyl-LPA regioisomer. Because of the short half-life of sn-2 LPA due to acyl migration under some conditions, we have synthesized acyl migration-resistant analogs using an acetyl group in place of the free hydroxyl group in order to evaluate LPA receptor SAR. Only LPA(1) and LPA(2) showed regioisomeric preference and only for the 18:2 fatty acyl-stabilized LPA sn-1 regioisomer. To identify residues involved in ligand recognition of LPA(3), we developed and validated computational models of LPA(3) complexes with the analogs studied. The models revealed that Arg-3.28 and Gln-3.29 conserved within the LPA-selective endothelial differentiation gene receptors and the more variable Lys-7.35 and Arg-5.38 of LPA(3) form critical interactions with the polar headgroup of LPA. The models identified Leu-2.60 and Val-7.39 of LPA(3) underlying the regioisomer-selective interaction with the acetyl group of the stabilized regioisomers. Mutation of Leu-2.60 to alanine selectively increased the EC(50) of the sn-2 acetyl-LPA regioisomers, whereas alanine replacement of Val-7.39 profoundly affected both regioisomers.
authors.affiliation: Department of Physiology, University of Tennessee Health Science Center, Memphis, Tennessee 38152, USA.
authors.forename: Yuko
authors.initials: Y
authors.lastname: Fujiwara
authors.forename: Vineet
authors.initials: V
authors.lastname: Sardar
authors.forename: Akira
authors.initials: A
authors.lastname: Tokumura
authors.forename: Daniel
authors.initials: D
authors.lastname: Baker
authors.forename: Kimiko
authors.initials: K
authors.lastname: Murakami-Murofushi
authors.forename: Abby
authors.initials: A
authors.lastname: Parrill
authors.forename: Gabor
authors.initials: G
authors.lastname: Tigyi
mesh_terms: D000409:Alanine / Q000737:chemistry; D000818:Animals; D001120:Arginine / Q000737:chemistry; D016367:Baculoviridae / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007313:Insecta; D007700:Kinetics; D008024:Ligands; D008246:Lysophospholipids / Q000378:metabolism; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D051381:Rats; D049368:Receptors, Lysophosphatidic Acid / Q000737:chemistry*; D012984:Software; D013237:Stereoisomerism; D013329:Structure-Activity Relationship; D014162:Transfection; D014633:Valine / Q000737:chemistry
chemical_list: D008024:Ligands; D008246:Lysophospholipids; D049368:Receptors, Lysophosphatidic Acid; D001120:Arginine; D014633:Valine; D000409:Alanine; C032881:lysophosphatidic acid; D002118:Calcium