Regulation of ryanodine receptors by sphingosylphosphorylcholine: Involvement of both calmodulin-dependent and -independent mechanisms

Sphingosylphosphorylcholine (SPC), a lipid mediator with putative second messenger functions, has been reported to regulate ryanodine receptors (RyRs), Ca2+ channels of the sarco/endoplasmic reticulum. RyRs are also regulated by the ubiquitous Ca2+ sensor calmodulin (CaM), and we have previously shown that SPC disrupts the complex of CaM and the peptide corresponding to the CaM-binding domain of the skeletal muscle Ca2+ release channel (RyR1). Here we report that SPC also displaces Ca2+-bound CaM from the intact RyR1, which we hypothesized might lead to channel activation by relieving the negative feedback Ca2+CaM exerts on the channel. We could not demonstrate such channel activation as we have found that SPC has a direct, CaM-independent inhibitory effect on channel activity, confirmed by both single channel measurements and [3H]ryanodine binding assays. In the presence of Ca2+CaM, however, the addition of SPC did not reduce [3H]ryanodine binding, which we could explain by assuming that the direct inhibitory action of the sphingolipid was negated by the simultaneous displacement of inhibitory Ca2+CaM. Additional experiments revealed that RyRs are unlikely to be responsible for SPC-elicited Ca2+ release from brain microsomes, and that SPC does not exert detergent-like effects on sarcoplasmic reticulum vesicles. We conclude that regulation of RyRs by SPC involves both CaM-dependent and -independent mechanisms, thus, the sphingolipid might play a pysiological role in RyR regulation, but channel activation previously attributed to SPC is unlikely

A Single Amino Acid Determines Lysophospholipid Specificity of the S1P 1 (EDG1) and LPA1 (EDG2) Phospholipid Growth Factor Receptors

The phospholipid growth factors sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) are ligands for the related G protein-coupled receptors S1P1/EDG1 and LPA1/EDG2, respectively. We have developed a model of LPA1 that predicts interactions between three polar residues and LPA. One of these, glutamine 125, which is conserved in the LPA receptor subfamily (LPA1/EDG2, LPA2/EDG4, and LPA 3/EDG7), hydrogen bonds with the LPA hydroxyl group. Our previous S1P1 study identified that the corresponding glutamate residue, conserved in all S1P receptors, ion pairs with the S1P ammonium. These two results predict that this residue might influence ligand recognition and specificity. Characterization of glutamate/glutamine interchange point mutants of S1P1 and LPA1 validated this prediction as the presence of glutamate was required for S1P recognition, whereas LPA recognition was possible with either glutamine or glutamate. The most likely explanation for this dual specificity behavior is a shift in the equilibrium between the acid and conjugate base forms of glutamic acid due to other amino acids surrounding that position in LPA1, producing a mixture of receptors including those having an anionic glutamate that recognize S1P and others with a neutral glutamic acid that recognize LPA. Thus, computational modeling of these receptors provided valid information necessary for understanding the molecular pharmacology of these receptors

Stereochemical properties of lysophosphatidic acid receptor activation and metabolism

Ligand recognition by G protein-coupled receptors (GPCR), as well as substrate recognition by enzymes, almost always shows a preference for a naturally occurring enantiomer over the unnatural one. Recognition of lysophosphatidic acid (LPA) by its receptors is an exception, as both the natural L (R) and unnatural D (S) stereoisomers of LPA are equally active in bioassays. In contrast to the enantiomers of LPA, analogs of N-acyl-serine phosphoric acid (NASPA) and N-acyl-ethanolamine phosphoric acid (NAEPA), which contain a serine and an ethanolamine backbone, respectively, in place of glycerol, are recognized in a stereoselective manner. This stereoselective interaction may lead to the development of receptor subtype-selective antagonists. In the present study, we review the stereochemical aspects of LPA pharmacology and describe the chemical synthesis of pure LPA enantiomers together with their ligand-binding properties toward the LPA1, LPA2, and LPA3 receptors and their metabolism by lipid phosphate phosphatase 1 (LPP1). Finally, we evaluate the concept of stereopharmacology in developing novel ligands for LPA receptors. © 2002 Elsevier Science B.V. All rights reserved

Fatty alcohol phosphates are subtype-selective agonists and antagonists of lysophosphatidic acid receptors

A more complete understanding of the physiological and pathological role of lysophosphatidic acid (LPA) requires receptor subtype-specific agonists and antagonists. Here, we report the synthesis and pharmacological characterization of fatty alcohol phosphates (FAP) containing saturated hydrocarbon chains from 4 to 22 carbons in length. Selection of FAP as the lead structure was based on computational modeling as a minimal structure that satisfies the two-point pharmacophore developed earlier for the interaction of LPA with its receptors. Decyl and dodecyl FAPs (FAP-10 and FAP-12) were specific agonists of LPA2 (EC50 = 3.7 ± 0.2 μM and 700 ± 22 nM, respectively), yet selective antagonists of LPA3 (Ki = 90 nM for FAP-12) and FAP-12 was a weak antagonist of LPA1. Neither LPA1 nor LPA3 receptors were activated by FAPs; in contrast, LPA2 was activated by FAPs with carbon chains between 10 and 14. Computational modeling was used to evaluate the interaction between individual FAPs (8 to 18) with LPA2 by docking each compound in the LPA binding site. FAP-12 displayed the lowest docked energy, consistent with its lower observed EC50. The inhibitory effect of FAP showed a strong hydrocarbon chain length dependence with C12 being optimum in the Xenopus laevis oocytes and in LPA3-expressing RH7777 cells. FAP-12 did not activate or interfere with several other G-protein-coupled receptors, including S1P-induced responses through S1P1.2,3.5 receptors. These data suggest that FAPs are ligands of LPA receptors and that FAP-10 and FAP-12 are the first receptor subtype-specific agonists for LPA2

Identification of Edg1 receptor residues that recognize sphingosine 1-Phosphate

Originating from its DNA sequence, a computational model of the Edg1 receptor has been developed that predicts critical interactions with its ligand, sphingosine 1-phosphate. The basic amino acids Arg120 and Arg292 ion pair with the phosphate, whereas the acidic Glu121 residue ion pairs with the ammonium moiety of sphingosine 1-phosphate. The requirement of these interactions for specific ligand recognition has been confirmed through examination of site-directed mutants by radioligand binding, ligand-induced [35S]GTPγS binding, and receptor internalization assays. These ion-pairing interactions explain the ligand specificity of the Edg1 receptor and provide insight into ligand specificity differences within the Edg receptor family. This computational map of the ligand binding pocket provides information necessary for understanding the molecular pharmacology of this receptor, thus underlining the potential of the computational method in predicting ligand-receptor interactions

Identification of the hydrophobic ligand binding pocket of the S1P 1 receptor

Sphingosine 1-phosphate (S1P), a naturally occurring sphingolipid mediator and also a second messenger with growth factor-like actions in almost every cell type, is an endogenous ligand of five G protein-coupled receptors (GPCRs) in the endothelial differentiation gene family. The lack of GPCR crystal structures sets serious limitations to rational drug design and in silico searches for subtype-selective ligands. Here we report on the experimental validation of a computational model of the ligand binding pocket of the S1P1 GPCR surrounding the aliphatic portion of S1P. The extensive mutagenesis-based validation confirmed 18 residues lining the hydrophobic ligand binding pocket, which, combined with the previously validated three head group-interacting residues, now complete the mapping of the S1P ligand recognition site. We identified six mutants (L3.43G/L3.44G, L3.43E/L3.44E, L5.52A, F5.48G, V6.40L, and F6.44G) that maintained wild type [32P]S1P binding with abolished ligand-dependent activation by S1P. These data suggest a role for these amino acids in the conformational transition of S1P1 to its activated state. Three aromatic mutations (F5.48Y, F6.44G, and W6.48A) result in differential activation, by S1P or SEW2871, indicating that structural differences between the two agonists can partially compensate for differences in the amino acid side chain. The now validated ligand binding pocket provided us with a pharmacophore model, which was used for in silico screening of the NCI, National Institutes of Health, Developmental Therapeutics chemical library, leading to the identification of two novel nonlipid agonists of S1P1. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc