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

Abstract

Lysophosphatidic acid (LPA) is a ligand for LPA 1-3 of the endothelial differentiation gene family G-protein-coupled receptors, and LPA 4 -8 is related to the purinergic family G-protein-coupled receptor. Because the structure-activity relationship (SAR) of GPR92/LPA 5 is limited and whether LPA is its preferred endogenous ligand has been questioned in the literature, in this study we applied a combination of computational and experimental site-directed mutagenesis of LPA 5 residues predicted to interact with the headgroup of LPA. Four residues involved in ligand recognition in LPA 5 were identified as follows: R2.60N mutant abolished receptor activation, whereas H4.64E, R6.62A, and R7.32A greatly reduced receptor activation. We also investigated the SAR of LPA 5 using LPA analogs and other non-lysophospholipid ligands. SAR revealed that the rank order of agonists is alkyl glycerol phosphate > LPA > farnesyl phosphates Ͼ Ͼ N-arachidonoylglycine. These results confirm LPA 5 to be a bona fide lysophospholipid receptor. We also evaluated several compounds with previously established selectivity for the endothelial differentiation gene receptors and found several that are LPA 5 agonists. A pharmacophore model of LPA 5 binding requirements was developed for in silico screening, which identified two non-lipid LPA 5 antagonists. Because LPA 5 transcripts are abundant in human platelets, we tested its antagonists on platelet activation and found that these non-lipid LPA 5 antagonists inhibit platelet activation. The present results suggest that selective inhibition of LPA 5 may provide a basis for future anti-thrombotic therapies.Lysophosphatidic acid (LPA, 2 1-radyl-2-hydroxy-sn-3-glycero phosphate) specifically interacts with several protein targets that regulate physiological and pathophysiological processes (1-3). LPA targets include specific G-protein-coupled receptors (GPCRs) that mediate a wide variety of biological effects, including cell proliferation (4), cell survival (5), cell migration (6), and platelet aggregation (7,8). GPCRs are the largest family of transmembrane receptors and represent targets of many therapeutics (9). Eight LPA-specific mammalian GPCRs, LPA 1-8 , 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 1-3 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). The structural foundation for LPA selectivity over S1P has been linked to a single amino acid at position 3.29, a conserved glutamine in the LPA-specific and glutamate in the S1P-specific members of the EDG family (14 -16). However, the recently identified non-EDG family LPA receptors, LPA 4 /p2y9 (13), LPA 5 /GPR92 (17, 18), LPA 6 /GPR87 (19), LPA 7 /p2y5 (12), and LPA 8 /p2y10 (10), are more closely related to the purinoreceptor gene cluster and share less than 20% amino acid...

24,25-Dihydroxyvitamin D3 cooperates with a stable, fluoromethylene LPA receptor agonist to secure human (MG63) osteoblast maturation

“…The LPA1/3 receptor antagonist, Ki16425 [29], was a very generous gift from the Kirin Brewery Company Ltd. (Tokyo, Japan) and was reconstituted at 10mM in DMSO. The preferential LPA3 receptor antagonist, diacylglycerol pyrophosphate [30] as the dioctanoyl form (DGPP 8:0, INstruchemie BV, Zwet 26, The Netherlands), was reconstituted in chloroform to a stock concentration of 25mg/ml and stored at -20°C. Within minutes of intended use the DGPP 8:0 was diluted in ethanol to a working stock concentration of 1mM.…”

LPA receptor signaling: pharmacology, physiology, and pathophysiology

“…2, Table 2). Most of these receptor modulators are directed at LPA receptors, particularly against LPA [1][2][3] , although a few compounds have demonstrated some LPA 4 and LPA 5 activity with limited selectivity (182,193,194). Additionally, several compounds target enzymes that regulate the production of LPA, notably ATX and LPPs (195,196).…”

Autotaxin: structure-function and signaling

“…All six LPA receptors can be stimulated by 1-acyl-LPA, albeit with different potencies. Of note, some LPA receptors (LPA 3 and LPA 6 ) prefer unsaturated 2-acyl-LPA as a ligand, while LPA 5 exhibits a strong preference for ether-linked 1-alkyl-LPA species ( 13,33 ). Detailed accounts of LPA receptor signaling pathways, their impact on gene expression, and biological outcomes can be found elsewhere ( 6,12,34 ).…”