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Holdsworth, Gill; Osborne, Daniel A.; Pham, TrucChi Thi; Fells, James I.; Hutchinson, Gillian; Milligan, Graeme; Parrill, Abby L.

doi:10.1186/1471-2091-5-12

Cited by 21 publications

(27 citation statements)

References 25 publications

(32 reference statements)

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“…6C). In the LPA 1 /S1P 1 receptor pair and in S1P 4 , the Gln-3.29 residue determines selectivity to LPA over S1P and the Q3.29A mutant showed no ligand binding or activation (19,20). The Q3.29A point mutant of LPA 3 showed a profound 40% reduction in E max and an order of magnitude shift to higher EC 50 for LPA 18:1 compared with the wild type LPA 3 (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Additionally, a cationic residue in the seventh transmembrane domain at position 7.34 in S1P 1-3 , but 7.33 in S1P 4 , is required for ligand binding and activation of S1P 1 but not S1P 4 . Our previous studies succeeded in pinpointing position 3.29, conserved as glutamine in LPA-specific and glutamate in S1P-specific members of the EDG family, as the single locus that determines ligand specificity for S1P versus LPA (19,20). Although residues involved in phosphate recognition are conserved between LPA 1 and LPA 3 receptors, differences in the SAR between the two receptors (14) led us to hypothesize that residues positioned lower in the transmembrane domains might be involved in ligand recognition and selectivity.…”

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confidence: 99%

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Identification of Residues Responsible for Ligand Recognition and Regioisomeric Selectivity of Lysophosphatidic Acid Receptors Expressed in Mammalian Cells

Fujiwara

Sardar

Tokumura

et al. 2005

Journal of Biological Chemistry

115

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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.

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Identification of Residues Responsible for Ligand Recognition and Regioisomeric Selectivity of Lysophosphatidic Acid Receptors Expressed in Mammalian Cells

Fujiwara

Sardar

Tokumura

et al. 2005

Journal of Biological Chemistry

115

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“…The reciprocal mutation in S1P 1 or S1P 4 , E3.29Q, changed receptor specificity from S1P to LPA. Alanine mutants abolished receptor activation by either ligand as measured by GTP␥S binding assays (24,25). In this study, using more sensitive Ca 2ϩ mobilization assays for monitoring receptor activation, we examined activation by S1P in wild type as well as Q3.29E and Q3.29A mutants in all three EDG family LPA receptor subtypes.…”

Section: Discussionmentioning

confidence: 99%

“…Alanine mutation at this position diminished activation by either ligand in both receptors (24). Similarly the E3.29Q mutation in S1P 4 conferred responsiveness to LPA but decreased responsiveness to S1P (25).…”

mentioning

confidence: 99%

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

Valentine

Fells

Perygin

et al. 2008

Journal of Biological Chemistry

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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...

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“…Therefore, knowing the structure of these regions is critical to understanding the mechanism of ligand-protein interaction which, in the future, may allow inhibition or mimicry of the effects of S1P on the cardiovascular and immune systems by rationally designed analogs. We have developed, validated, and published models for human S1P 1 , 10 human S1P 4 , 15 and mouse S1P 4 13 receptors.…”

Section: Introductionmentioning

confidence: 99%

Peptide design and structural characterization of a GPCR loop mimetic

2007

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G protein-coupled receptors (GPCRs) control fundamental aspects of human physiology and behaviors. Knowledge of their structures, especially for the loop regions, is limited and has principally been obtained from homology models, mutagenesis data, low resolution structural studies, and high resolution studies of peptide models of receptor segments. We developed an alternate methodology for structurally characterizing GPCR loops, using the human S1P(4) first extracellular loop (E1) as a model system. This methodology uses computational peptide designs based on transmembrane domain (TM) model structures in combination with CD and NMR spectroscopy. The characterized peptides contain segments that mimic the self-assembling extracellular ends of TM 2 and TM 3 separated by E1, including residues R3.28(121) and E3.29(122) that are required for sphingosine 1-phosphate (S1P) binding and receptor activation in the S1P(4) receptor. The S1P(4) loop mimetic peptide interacted specifically with an S1P headgroup analog, O-phosphoethanolamine (PEA), as evidenced by PEA-induced perturbation of disulfide cross-linked coiled-coil first extracellular loop mimetic (CCE1a) (1)H and (15)N backbone amide chemical shifts. CCE1a was capable of weakly binding PEA near biologically relevant residues R29 and E30, which correspond to R3.28 and E3.29 in the full-length S1P(4) receptor, confirming that it has adopted a biologically relevant conformation. We propose that the combination of coiled-coil TM replacement and conformational stabilization with an interhelical disulfide bond is a general design strategy that promotes native-like structure for loops derived from GPCRs.

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Untitled

Cited by 21 publications

References 25 publications

Identification of Residues Responsible for Ligand Recognition and Regioisomeric Selectivity of Lysophosphatidic Acid Receptors Expressed in Mammalian Cells

Identification of Residues Responsible for Ligand Recognition and Regioisomeric Selectivity of Lysophosphatidic Acid Receptors Expressed in Mammalian Cells

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

Peptide design and structural characterization of a GPCR loop mimetic

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