Subtype-specific Residues Involved in Ligand Activation of the Endothelial Differentiation Gene Family Lysophosphatidic Acid Receptors

Abstract

Lysophosphatidic acid (LPA) is a ligand for three endothelial differentiation gene family G protein-coupled receptors, LPA 1-3 . We performed computational modeling-guided mutagenesis of conserved residues in transmembrane domains 3, 4, 5, and 7 of LPA 1-3 predicted to interact with the glycerophosphate motif of LPA C18:1. The mutants were expressed in RH7777 cells, and the efficacy (E max ) and potency (EC 50 ) of LPA-elicited Ca 2؉ transients were measured. Mutation to alanine of R3.28 universally decreased both the efficacy and potency in LPA 1-3 and eliminated strong ionic interactions in the modeled LPA complexes. The alanine mutation at Q3.29 decreased modeled interactions and activation in LPA 1 and LPA 2 more than in LPA 3 . The mutation W4.64A had no effect on activation and modeled LPA interaction of LPA 1 and LPA 2 but reduced the activation and modeled interactions of LPA 3 . The R5.38A mutant of LPA 2 and R5.38N mutant of LPA 3 showed diminished activation by LPA; however, in LPA 1 the D5.38A mutation did not, and mutation to arginine enhanced receptor activation. In LPA 2 , K7.36A decreased the potency of LPA; in LPA 1 this same mutation increased the E max . In LPA 3 , R7.36A had almost no effect on receptor activation; however, the mutation K7.35A increased the EC 50 in response to LPA 10-fold. In LPA 1-3 , the mutation Q3.29E caused a modest increase in EC 50 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 LPA 2 and LPA 3 receptors, indicating that S1P may function as a weak agonist of endothelial differentiation gene family LPA receptors. Lysophosphatidic acid (LPA)2 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 (1-3). LPA has been shown to play an important role in a variety of diseases including ovarian cancer, prostate cancer, breast cancer, and cardiovascular disease (4 -14). 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 ␣-helical transmembrane domains (TMs) (15). Three of these genes (LPA 1-3 ) are cellular receptors for LPA and share 55% overall homology in humans. The other five (S1P 1-5 ) 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 LPA 1-3 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/LPA 4 , GPR92/LPA 5 , and GPR87/LPA 6 , which are more closely related to the purinoreceptor clus...

Unique Ligand Selectivity of the GPR92/LPA5 Lysophosphatidate Receptor Indicates Role in Human Platelet Activation

“…Computational Model Development and Refinement-A homology model of the LPA 5 receptor was built based on a previously published LPA 1 receptor model (14,16,21,23) with the Molecular Modeling Environment (MOE) software program using the sequence alignment shown in Fig. 1.…”

“…Eight LPA-specific mammalian GPCRs, LPA [1][2][3][4][5][6][7][8] , have been identified to date (10 -12). Among the eight LPA receptors, LPA 1 , LPA 2 , and LPA 3 are members of the endothelial differentiation gene (EDG) family (13), and the transmembrane domains of human LPA [1][2][3] show 81% homology with each other (14). The five other members of the EDG family are specific for the related lysophospholipid sphingosine 1-phosphate (S1P).…”

“…Although EDG-LPA receptors have been well characterized and differences in ligand selectivity among LPA 1-3 have been reported (20,21), the SAR of LPA 5 is presently limited to only a few LPA species (17,18,22). Further complicating this issue is a recent report by Oh da et al (22) that suggests that two other naturally occurring ligands, farnesyl pyrophosphate (FPP) and N-arachidonoylglycine (NAG), are more potent agonists for LPA 5 /GPR92 than LPA 18:1, which necessitates a re-classification of LPA 5 not as an LPA receptor.We have previously generated computational models of each EDG-LPA receptor (14,16,21,23), validated the models with site-directed mutagenesis, identified residues involved in ligand-induced activation of the receptors, and developed pharmacophore models enabling the in silico identification of selective ligands. However, LPA 4 -8 exhibit little sequence identity…”

“…We have previously generated computational models of each EDG-LPA receptor (14,16,21,23), validated the models with site-directed mutagenesis, identified residues involved in ligand-induced activation of the receptors, and developed pharmacophore models enabling the in silico identification of selective ligands. However, LPA 4 -8 exhibit little sequence identity…”

“…We have previously generated computational models of each EDG-LPA receptor (14,16,21,23), validated the models with site-directed mutagenesis, identified residues involved in ligand-induced activation of the receptors, and developed pharmacophore models enabling the in silico identification of selective ligands. However, LPA 4 -8 exhibit little sequence identity with LPA [1][2][3] , and thus we hypothesized that these receptors may have unique properties and recognize LPA by different motifs as well as display SAR distinct from LPA [1][2][3] .…”