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Lys39-Lysophosphatidate Carbonyl Oxygen Interaction Locks LPA1 N-terminal Cap to the Orthosteric Site and partners Arg124 During Receptor Activation.

Omotuyi OI, Nagai J, Ueda H - Sci Rep (2015)

Bottom Line: In activated state, LPA-type agonists interact with Arg124 (R3.28), Gln125 (Q3.29), Lys294 (K7.36) and a novel N-terminal Lys39.Structurally, LPA-type agonist via Carbonyl-oxygen/Lys39 interaction facilitated the formation of a hypothetical N-terminal cap tightly packed over LPA1 heptahelical bundle.This packing may represent a key mechanism to distinguish an apo-receptor from bound LPA1.

View Article: PubMed Central - PubMed

Affiliation: 1] From the Department of Pharmacology and Therapeutic Innovation, Graduate School of Biomedical Sciences, Nagasaki University, Japan [2] From the Center for Drug Discovery and Therapeutic Innovation, Nagasaki University, Japan.

ABSTRACT
Lysophosphatidic acid (LPA) receptor 1 (LPA1) is a member of the G protein-coupled receptors mediating the biological response to LPA species. Lack of detailed mechanism underlying LPA/LPA1 interaction has hampered the development of specific antagonists. Here, novel N-terminal Lys39 has been identified as a key residue during LPA-type agonist binding and LPA1 activation. Analysis of the molecular dynamics (MD) trajectories showed that LPA-type agonist but not VPC-32183 (antagonist) evolved structures with classical GPCR activation signatures such as reduced cytoplasmic transmembrane (TM) 3/TM6 dynamic network, ruptured ionic lock, and formation of a continuous and highly ordered internal water pathway was also observed. In activated state, LPA-type agonists interact with Arg124 (R3.28), Gln125 (Q3.29), Lys294 (K7.36) and a novel N-terminal Lys39. Site-directed mutagenesis showed complete loss of intracellular calcium mobilization in B103 cells expressing R3.28A and Lys39Ala when treated with LPA-type agonists. Structurally, LPA-type agonist via Carbonyl-oxygen/Lys39 interaction facilitated the formation of a hypothetical N-terminal cap tightly packed over LPA1 heptahelical bundle. This packing may represent a key mechanism to distinguish an apo-receptor from bound LPA1.

No MeSH data available.


Related in: MedlinePlus

Lys39Ala LPA1 mutant failed to mobilize intracellular calcium in response to LPA-type agonists.(a, i–vi) dose-response curves of wildtype, Lys39Ala, R3.28A, K7.35 stimulated by various LPA-type and AGP (18:1) agonists. (vii). Confocal microscopy images in Triton-X100 permeabilized and non-permeabilized samples.
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f3: Lys39Ala LPA1 mutant failed to mobilize intracellular calcium in response to LPA-type agonists.(a, i–vi) dose-response curves of wildtype, Lys39Ala, R3.28A, K7.35 stimulated by various LPA-type and AGP (18:1) agonists. (vii). Confocal microscopy images in Triton-X100 permeabilized and non-permeabilized samples.

Mentions: Till date, the most detailed study on specific residues involved in LPA-type agonist activation of the endothelial differentiation gene (EDG) family LPA receptors was reported by Valentine et al.21. By combining homology modeling, molecular docking and site-directed mutagenesis experiments, the roles of R3.28, Q3.29 were validated as LPA head-group interacting residues. Here, alanine mutant of Lys39 was constructed to validate the role of this residue in LPA1 under cellular condition. When B103 cells expressing normal LPA1, and Lys39Ala, R3.28A or K7.36A mutants of LPA1 were exposed to LPA14:0, intracellular calcium was poorly mobilized (<10% of the maximal response for wildtype) in all the mutants in dose-independent manner up to 100 μM (Fig. 3, i). When the cells were treated with LPA 16:0 and LPA 18:0, as observed for LPA 14:0, Lys39Ala and R3.28A failed to mobilize intracellular calcium up to 100 μM concentration.


Lys39-Lysophosphatidate Carbonyl Oxygen Interaction Locks LPA1 N-terminal Cap to the Orthosteric Site and partners Arg124 During Receptor Activation.

Omotuyi OI, Nagai J, Ueda H - Sci Rep (2015)

Lys39Ala LPA1 mutant failed to mobilize intracellular calcium in response to LPA-type agonists.(a, i–vi) dose-response curves of wildtype, Lys39Ala, R3.28A, K7.35 stimulated by various LPA-type and AGP (18:1) agonists. (vii). Confocal microscopy images in Triton-X100 permeabilized and non-permeabilized samples.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4542628&req=5

f3: Lys39Ala LPA1 mutant failed to mobilize intracellular calcium in response to LPA-type agonists.(a, i–vi) dose-response curves of wildtype, Lys39Ala, R3.28A, K7.35 stimulated by various LPA-type and AGP (18:1) agonists. (vii). Confocal microscopy images in Triton-X100 permeabilized and non-permeabilized samples.
Mentions: Till date, the most detailed study on specific residues involved in LPA-type agonist activation of the endothelial differentiation gene (EDG) family LPA receptors was reported by Valentine et al.21. By combining homology modeling, molecular docking and site-directed mutagenesis experiments, the roles of R3.28, Q3.29 were validated as LPA head-group interacting residues. Here, alanine mutant of Lys39 was constructed to validate the role of this residue in LPA1 under cellular condition. When B103 cells expressing normal LPA1, and Lys39Ala, R3.28A or K7.36A mutants of LPA1 were exposed to LPA14:0, intracellular calcium was poorly mobilized (<10% of the maximal response for wildtype) in all the mutants in dose-independent manner up to 100 μM (Fig. 3, i). When the cells were treated with LPA 16:0 and LPA 18:0, as observed for LPA 14:0, Lys39Ala and R3.28A failed to mobilize intracellular calcium up to 100 μM concentration.

Bottom Line: In activated state, LPA-type agonists interact with Arg124 (R3.28), Gln125 (Q3.29), Lys294 (K7.36) and a novel N-terminal Lys39.Structurally, LPA-type agonist via Carbonyl-oxygen/Lys39 interaction facilitated the formation of a hypothetical N-terminal cap tightly packed over LPA1 heptahelical bundle.This packing may represent a key mechanism to distinguish an apo-receptor from bound LPA1.

View Article: PubMed Central - PubMed

Affiliation: 1] From the Department of Pharmacology and Therapeutic Innovation, Graduate School of Biomedical Sciences, Nagasaki University, Japan [2] From the Center for Drug Discovery and Therapeutic Innovation, Nagasaki University, Japan.

ABSTRACT
Lysophosphatidic acid (LPA) receptor 1 (LPA1) is a member of the G protein-coupled receptors mediating the biological response to LPA species. Lack of detailed mechanism underlying LPA/LPA1 interaction has hampered the development of specific antagonists. Here, novel N-terminal Lys39 has been identified as a key residue during LPA-type agonist binding and LPA1 activation. Analysis of the molecular dynamics (MD) trajectories showed that LPA-type agonist but not VPC-32183 (antagonist) evolved structures with classical GPCR activation signatures such as reduced cytoplasmic transmembrane (TM) 3/TM6 dynamic network, ruptured ionic lock, and formation of a continuous and highly ordered internal water pathway was also observed. In activated state, LPA-type agonists interact with Arg124 (R3.28), Gln125 (Q3.29), Lys294 (K7.36) and a novel N-terminal Lys39. Site-directed mutagenesis showed complete loss of intracellular calcium mobilization in B103 cells expressing R3.28A and Lys39Ala when treated with LPA-type agonists. Structurally, LPA-type agonist via Carbonyl-oxygen/Lys39 interaction facilitated the formation of a hypothetical N-terminal cap tightly packed over LPA1 heptahelical bundle. This packing may represent a key mechanism to distinguish an apo-receptor from bound LPA1.

No MeSH data available.


Related in: MedlinePlus